Loss of HD-PTP function results in lipodystrophy, defective cellular signaling and altered lipid homeostasis

Destiny F Schultz; Brian A Davies; Johanna A Payne; Cole P Martin; Annabel Y Minard; Bennett G Childs; Cheng Zhang; Karthik B Jeganathan; Ines Sturmlechner; Thomas A White; Alain de Bruin; Liesbeth Harkema; Huiqin Chen; Michael A Davies; Sarah Jachim; Nathan K LeBrasseur; Robert C Piper; Hu Li; Darren J Baker; Jan van Deursen; Daniel D Billadeau; David J Katzmann

doi:10.1242/jcs.262032

. 2024 Sep 27;137(18):jcs262032. doi: 10.1242/jcs.262032

Loss of HD-PTP function results in lipodystrophy, defective cellular signaling and altered lipid homeostasis

Destiny F Schultz ^1,^2,^3,^*, Brian A Davies ^1,^*, Johanna A Payne ¹, Cole P Martin ¹, Annabel Y Minard ⁴, Bennett G Childs ⁵, Cheng Zhang ⁶, Karthik B Jeganathan ⁵, Ines Sturmlechner ^5,⁷, Thomas A White ⁸, Alain de Bruin ^7,⁹, Liesbeth Harkema ⁹, Huiqin Chen ¹⁰, Michael A Davies ¹¹, Sarah Jachim ¹, Nathan K LeBrasseur ⁸, Robert C Piper ⁴, Hu Li ⁶, Darren J Baker ^1,⁵, Jan van Deursen ^1,⁵, Daniel D Billadeau ³, David J Katzmann ^1,^*,^‡

PMCID: PMC11449442 PMID: 39155850

ABSTRACT

His domain protein tyrosine phosphatase (HD-PTP; also known as PTPN23) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, phosphoinositide 3-kinase (PI3K)/AKT and receptor tyrosine kinases (RTKs), such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. The ESCRT components Vps4 and Hrs have previously been implicated in cholesterol homeostasis; thus, these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.

Keywords: HD-PTP, PTPN23, ESCRT, Lipid homeostasis, Receptor signaling

Summary: Perturbation of ESCRT-associated HD-PTP results in cell-type-specific phenotypes. Loss of HD-PTP in muscle results in canonical ESCRT defects, whereas loss in adipose results in decreased signaling.

INTRODUCTION

The endosomal sorting complexes required for transport (ESCRTs) are evolutionarily conserved machinery that participates in reverse topology membrane budding and scission processes including viral budding, exosome formation, cytokinesis, autophagy, membrane repair and multivesicular body (MVB) formation (Christ et al., 2017). In the context of MVB sorting, ESCRTs actively recognize ubiquitylated cargoes and incorporate them into intralumenal vesicles (ILVs) (Wollert and Hurley, 2010; Katzmann et al., 2001; Babst et al., 2002a,b; Shih et al., 2002). Activated receptors are a cargo whose incorporation into ILVs terminates signaling by sequestering receptor tails from cytosolic signaling effectors (Lloyd et al., 2002). That ESCRTs can act as negative regulators of receptor signaling and tumor suppressors is supported by multiple examples wherein their loss correlates with prolonged signaling and tumorigenesis (Babst et al., 2000; Thompson et al., 2005; Moberg et al., 2005; Vaccari and Bilder, 2005).

The essential nature of ESCRTs in mammals and their involvement in many key cellular processes has presented challenges to understanding specific ESCRT-mediated processes as contributors to normal physiology. Disrupted ESCRT function through knockout of ESCRT-0 (Hrs) (Komada and Soriano, 1999), ESCRT-I (Tsg101) (Wagner et al., 2003), ESCRT-III (Chmp5) (Shim et al., 2006) or the ESCRT-associated factor HD-PTP (Ptpn23) (Gingras et al., 2009a) result in embryonic lethality in mouse models. Moreover, pathogenic gene variants that alter ESCRT function have been linked to congenital disorders including spastic paraplegia (SP80, UBAP1; SP53, VPS37A) (Farazi Fard et al., 2019; Lin et al., 2019; Zivony-Elboum et al., 2012), childhood cataracts (CTRCT31, CHMP4B) (Shiels et al., 2007; Yamada et al., 2000), frontotemporal dementia (FTD or ALS17; CHMP2B) (Cox et al., 2010; Parkinson et al., 2006; Skibinski et al., 2005; van der Zee et al., 2008), pontocerebellar hypoplasia (PCH8, CHMP1A) (Mochida et al., 2012), cerebellar hypoplasia, cataracts, impaired intellectual development, congenital microcephaly, dystonia, dyserythropoietic anemia and growth retardation (CIMDAG, VPS4A) (Lunati et al., 2021; Rodger et al., 2020; Seu et al., 2020), and neurodevelopmental disorder with structural brain anomalies, seizures and spasticity (NEDBASS; HD-PTP, PTPN23) (Bend et al., 2020; Khalaf-Nazzal et al., 2021; Seo et al., 2022; Smigiel et al., 2018; Sowada et al., 2017). Although these studies highlight the importance of ESCRT function in maintaining normal physiology, they have not advanced the understanding of which defective aspect(s) of ESCRT function are the major drivers of disease states. One way to dissect the contributions of ESCRT-mediated processes to mammalian physiology is through ESCRT-associated Bro1 family members. The mammalian Bro1 homologs HD-PTP (Toyooka et al., 2000) and ALIX (also known as PDCD6IP) (Katoh et al., 2003) participate in distinct ESCRT-mediated processes – whereas ALIX has been implicated in exosome formation (Larios et al., 2020; Baietti et al., 2012), viral budding (Dowlatshahi et al., 2012; Carlton et al., 2008) and cytokinesis (Morita et al., 2007; Christ et al., 2016), HD-PTP has been found to be crucial for MVB formation (Ali et al., 2013; Tabernero and Woodman, 2018; Doyotte et al., 2008). Thus, HD-PTP presents an avenue to study the impact of MVB formation on mammalian physiology.

Heterotypic fusion of the MVB and lysosome delivers protein and lipid cargoes to the hydrolytic lumen of the lysosome for processing and utilization by the cell (Futter et al., 1996; Mullock et al., 1998; Wurmser and Emr, 1998). Cholesterol transporters, such as NPC1, reside in the lysosomal membrane and transfer lumenal cholesterol to other organelles through inter-organelle contact sites (Kwon et al., 2009; Infante et al., 2008; Lim et al., 2019), allowing for distribution of lipids throughout cellular membranes to maintain homeostasis. Disruption of cholesterol transporters results in lysosomal storage disorders, where cholesterol accumulates within the lysosome and cannot be utilized by other organelles (Schulze and Sandhoff, 2011; Simons and Gruenberg, 2000). Delivery of cholesterol to the plasma membrane is crucial for lipid microdomains, which serve as signaling platforms for receptor activation (Puri et al., 2005; Orr et al., 2005). Lipid droplet formation also relies upon transfer of cholesterol from the lysosome to the endoplasmic reticulum (ER) (Du et al., 2011; Cianciola et al., 2013; Byers et al., 1989). Perturbation of ESCRT components Hrs and Vps4 (herein referring to Vps4A and Vps4B) in vitro leads to lysosomal accumulation of cholesterol (Bishop and Woodman, 2000; Du et al., 2012, 2013), suggesting that MVB sorting and endosomal maturation play a crucial role in maintaining lipid homeostasis.

Homozygous deletion of Ptpn23 is embryonic lethal in mice (Gingras et al., 2009a), and Ptpn23^+/− heterozygous mice develop spontaneous tumors at 15–18 months (Manteghi et al., 2016). HD-PTP has been proposed to act as a tumor suppressor through negative regulation of receptor signaling (Manteghi et al., 2016; Gingras et al., 2009b, 2017; Zhang et al., 2017). To further understand the contributions of HD-PTP and MVB sorting to mammalian physiology, an allelic series of mice including wild-type (WT), null and hypomorphic alleles was generated. Ptpn23 homozygous hypomorphic mice (Ptpn23^H/H), which express <50% of normal Ptpn23 levels, represent the lowest gene dosage within this series compatible with survival. These mice fail to develop tumors but exhibit severe reduction in white adipose tissue (WAT; lipodystrophy) and decreased receptor tyrosine kinase (RTK) signaling. In vitro characterization revealed that HD-PTP plays a role in trafficking of cholesterol through the endolysosomal system, thus contributing to increased lipid order within the plasma membrane for receptor activation. These results highlight a function of HD-PTP in activation of receptor signaling through cholesterol homeostasis, in addition to its role as a negative regulator of receptor signaling via the MVB pathway.

RESULTS

HD-PTP hypomorphic mice display lipodystrophy

To assess the role of HD-PTP in maintenance of organismal physiology, an allelic series of mice with varied Ptpn23 gene dosages was generated. A hypomorphic allele (Ptpn23^H) was generated by insertion of a neomycin resistance cassette containing a cryptic splice acceptor and donor sequences between exons 6 and 7 (Fig. 1A; strategy based on Baker et al., 2004). Upon incorporation of the cassette, HD-PTP is truncated within the Bro1 domain by a premature stop codon, thus lowering the numbers of normal mRNA transcripts and reducing overall protein expression of HD-PTP in neonates by more than 50% (Fig. 1B). This cassette is flanked by FRT sites and Flp-mediated recombination reverts the hypomorphic allele to an inducible knockout (Ptpn23^flox). Further, CRE-mediated recombination of LoxP sites excises exons 5 and 6 to generate a functionally null allele (Ptpn23⁻). Heterozygous animals were crossed and surviving progeny genotyped (Fig. S1A). HD-PTP expression decreased accordingly with each gene dosage – Ptpn23^−/+ displayed a 50% decrease, Ptpn23^H/H a greater than 50% decrease, and Ptpn23^H/− a 75% decrease in HD-PTP (Fig. 1C; Fig. S1B). Of this allelic series, Ptpn23^−/− mice were unable to survive embryogenesis, as previously observed (Gingras et al., 2009a). Ptpn23^H/− mice did not survive past one day postnatally (data not shown). However, Ptpn23^H/H mice were viable into adulthood and represent the lowest viable gene dosage within this series, despite exhibiting decreased survival relative to Ptpn23^−/+, Ptpn23^H/+ and Ptpn23^+/+ (Fig. 1D).

Both male and female Ptpn23^H/H mice present with decreased size and mass throughout their lives relative to Ptpn23^+/+ littermates (Fig. 1E,F). The reduced body weight was partially accounted for by lower absolute and relative total body fat (Fig. 1G), and lower masses of individual WAT depots (Fig. 1H) throughout the lifespan of Ptpn23^H/H relative to Ptpn23^+/+. No differences were observed in the weights of brown adipose tissue or other organs, such as lung, heart, liver and muscle, in Ptpn23^H/H mice (Fig. 1I). Behavioral changes do not account for the observed reduction in adipose tissue, as male Ptpn23^H/H mice exhibited no significant differences in food intake (Fig. S1C) or physical activity (Fig. S1D). Further characterization of Ptpn23^H/H WAT revealed a decrease in adipocyte size of the inguinal adipose tissue (IAT) (Fig. 1J,K) and the thickness of the subdermal adipose tissue was also decreased (Fig. S1E). Thus, Ptpn23^H/H mice display decreased white adipose mass and reduced adipocyte size relative to that in Ptpn23^+/+ mice. Cumulatively, these analyses uncovered an unexpected phenotype in Ptpn23^H/H mice but did not provide an explanation for the lipodystrophy.

Insulin and RTK signaling is reduced in adipose tissue of HD-PTP hypomorphic mice

To assess alterations in signaling pathways that could be contributing to the observed lipodystrophy in Ptpn23^H/H mice, a reverse phase protein array (RPPA) analysis of the IAT from nine adult age-matched mice was performed. 183 of 377 queried protein species displayed statistically significant alterations, with 91 factors increased and 92 factors decreased in Ptpn23^H/H IAT (Fig. 2A; Table S2). A significant increase in the levels of lipid synthesis enzymes acetyl-CoA synthase, acetyl-CoA carboxylase and fatty acid synthase were observed (Fig. 2B), suggesting that lipid synthesis is intact and pointing toward a defect in lipid storage.

Fig. 2. — *Ptpn23^H/H* adipose tissue exhibits decreased insulin signaling. (A) Volcano plot of adult male IAT RPPA analysis (n=9 mice per genotype). 183 proteins display statistically significant alterations relative to *Ptpn23^+/+*. The dashed line represents the 0.05 P-value. (B) Heat map of significantly altered lipid metabolism factors from male IAT RPPA (two-tailed unpaired t-test). Factors elevated are colored red and factors decreased colored blue. (C) Pathway analysis of adult male IAT RPPA results indicates pathways significantly impacted in *Ptpn23*^H/H, colored blue for a decrease or red for an increase as assessed by z-score. (D) Heat map of significantly altered signaling components in adult male IAT RPPA (two-tailed unpaired t-test). Factors elevated colored red and factors decreased colored blue. (E) Representative western blot and quantification of IAT insulin receptor β protein expression. n=9 male mice per genotype. **P<0.01 (Welch's t-test). (F) Circulating insulin (mg/dl) in adult female (n=8–14 mice per genotype) and male (n=7 mice per genotype) mice. *P<0.05 (assessed within each sex by a Mann–Whitney test). (G) Quantification of levels of Akt phosphorylated T308 normalized to total Akt levels [p/t-Akt (T308)] following 100 ng/ml insulin stimulation of suspended IAT. Lysates generated at 1, 2 and 3 h post stimulation. n=3 experimental replicates. (+/+ insulin AUC 1303±204.9, H/H insulin AUC 955.3±107.3; mean±s.e.m.). (H) Quantification of levels of Akt phosphorylated T308 normalized to total Akt levels [p/t-Akt (T308)] following 100 ng/ml insulin stimulation of *in vitro* differentiated adipocytes. Lysates collected at 5, 10, 15, 30 and 60 min after stimulation (n=2). All error bars presented as mean±s.e.m.

Insulin signaling is a major regulator of lipid storage in IAT (Blüher et al., 2002). RPPA pathway analysis revealed that Ptpn23^H/H IAT exhibits decreased signaling through RTK, RAS/MAPK and phosphoinositide 3-kinase (PI3K)/AKT pathways (Fig. 2C), which can be observed as reduced protein levels and phosphorylation states of multiple signaling pathway components (Fig. 2D). Consistent with the RPPA pathway analysis, western blotting revealed reduced phosphorylation of mTOR, ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively) and Akt family proteins at S473 or equivalent (Fig. S2A), as well as increased levels of insulin receptor in Ptpn23^H/H IAT (Fig. 2E). These results raise the possibility that reduced fat mass in Ptpn23^H/H mice is a consequence of perturbed insulin signaling disrupting lipid storage.

Changes in IAT insulin signaling could result from cell-extrinsic or cell-intrinsic changes within Ptpn23^H/H mice, and our analyses suggest both modes are relevant. Reduced non-fasted circulating insulin concentrations (Fig. 2F) and increased sensitivity to exogenous insulin were observed in Ptpn23^H/H mice (Fig. S2B). Moreover, Ptpn23^H/H muscle exhibited a significantly greater increase in Akt family protein activation in response to insulin compared to Ptpn23^+/+ muscle (Fig. S2C). These data were in contrast with the RPPA analysis that demonstrated reduced insulin signaling in IAT and suggested a level of organ or cell type specificity for the signaling defect in Ptpn23^H/H mice.

To further separate intrinsic from extrinsic contributions to IAT signaling, both ex vivo and in vitro insulin stimulations were completed. IAT isolated from Ptpn23^H/H mice and stimulated ex vivo displayed less Akt family protein phosphorylation than Ptpn23^+/+ IAT (Fig. 2G), revealing that the signaling defect is still present under conditions of equivalent insulin, despite the observed increase in insulin receptor expression (Fig. 2E). Furthermore, Ptpn23^fl/fl primary preadipocytes were utilized to generate Ptpn23^−/− adipocytes in vitro, which display decreased adipogenesis markers, such as PPARγ, FASN and perilipin 1 (Fig. S2D,E). Insulin stimulation of in vitro differentiated adipocytes again revealed decreased Akt family protein phosphorylation upon loss of HD-PTP (Fig. 2H), thus highlighting potential contributions of perturbed adipogenesis to defective signaling.

RNAseq and Ingenuity Pathway Analysis (IPA) on 14-day-old Ptpn23^H/H IAT identified similar alterations in the adipogenesis pathway, with observed decreases in nine markers of adipogenesis and an increase in the preadipocyte marker Dlk1 (Fig. S2F). Consistent with reduced adipogenesis, reductions in lipid synthesis components were observed in 14-day RPPA (Fig. S2G) despite these factors being elevated in the adult RPPA (Fig. 2B), consistent with the conclusion that adipogenesis might be delayed in Ptpn23^H/H mice. However, in both 14-day (Fig. S2G) and adult RPPA (Fig. 2B), decreased insulin signaling was still present, highlighting contributions to signaling aside from adipogenesis. Thus, the presence of signaling defects in ex vivo and in vitro stimulation conditions suggest cell-intrinsic contributions to Ptpn23^H/H lipodystrophy, warranting further investigation.

HD-PTP impacts cellular cholesterol distribution to promote EGFR phosphorylation

To facilitate in vitro assessment of defective RTK signaling upon loss of HD-PTP, immortalized mouse embryonic fibroblasts (iMEFs) were generated for each experiment from doxycycline-inducible knockout (KO) Ptpn23^flox/flox animals (Fig. S3A). Because MEFs respond poorly to insulin (Dinchuk et al., 2010), and epidermal growth factor receptor (EGFR) also displayed reduced phosphorylation in Ptpn23^H/H IAT (Fig. 2D), we utilized EGFR and its ligand EGF to probe RTK signaling defects in Ptpn23 KO iMEFs. In vitro EGF treatment of iMEFs revealed reduced EGFR and ERK1/2 phosphorylation (Fig. 3A–C; Fig. S3B,C). These results suggest that loss of HD-PTP function negatively impacts EGFR-mediated signaling at the level of receptor phosphorylation. However, both EGFR plasma membrane (PM) receptor levels and EGF ligand binding were indistinguishable between WT and Ptpn23 KO cells (Fig. 3D,E). Therefore, in spite of comparable PM levels and ligand binding, EGFR activation is defective with loss of HD-PTP function. These findings are consistent with reduced insulin signaling in adipose tissue (Fig. 2G) despite increased insulin receptor expression.

Fig. 3. — **EGFR activation is impacted at the level of receptor phosphorylation upon loss of HD-PTP.** (A) iMEFs were serum starved 2 h and treated with 100 ng/ml EGF for 0 or 10 min. Cells were then stained for pERK1/2 (pERK) and assessed by flow cytometry as in the Materials and Methods. Each dot represents average of n=3 technical replicates, n=3 experimental replicates. *P<0.05 (two-tailed paired t-test). (B) Representative blots of pEGFR, EGFR, pERK and ERK following 0-, 5-, 10-, 15-, 30- and 45-min EGF stimulation. Blots were analyzed with FIJI software and the ratio of phospho/total (p/t) calculated for the proteins. (C) p/t-EGFR following EGF stimulation normalized to WT 0 min. Representative experimental replicate with n=3 technical replicates. (D) Following starvation as above, iMEFs were incubated on ice with an extracellular EGFR antibody and stained for flow cytometry as in the Materials and Methods. Histograms are an example of a single technical replicate, with ‘secondary only’ having no EGFR antibody. Each dot represents average of n=3 technical replicates, n=4 experimental replicates. ns, not significant (two-tailed paired t-test). (E) Following starvation, iMEFs were incubated with 100 ng/ml Alexa Fluor 488 (A488)–EGF on ice and fixed for assessment by flow cytometry. Saturated samples were pre-treated with 1000 ng/ml non-labelled EGF on ice. Histogram represents single technical replicate and bar graph shows fold increase in fluorescence from saturated to the A488-labelled samples. Each dot represents average of n=3 technical replicates, n=3 experimental replicates. ns, not significant (unpaired two-tailed t-test). All error bars presented as mean±s.e.m.

Incorporation of EGFR into ordered microdomains within the PM is thought to be a crucial step in the initiation of signal transduction by promoting quick receptor movement for dimerization (Orr et al., 2005; Hofman et al., 2009) and providing a site for signaling effectors, such as Ras, to localize in proximity to the receptor (Puri et al., 2005; Irwin et al., 2011; Parpal et al., 2001). Ptpn23 KO iMEFs displayed reduced PM Cholera Toxin B staining compared to WT iMEFs (Fig. 4A), suggesting reduced PM levels of the ganglioside GM1 and thereby a defect in glycosphingolipid content. Membrane order was directly assessed using Di-4-ANEPPDHQ staining and revealed decreased order within the Ptpn23 KO iMEFs relative to WT (Fig. 4B; Fig. S4A,B), consistent with lipid changes in the PM.

Fig. 4. — **Cellular cholesterol is redistributed from the plasma membrane to intracellular compartments upon loss of HD-PTP.** (A) iMEFs were incubated on ice with Alexa Fluor 488-conjugated CTxB and then assessed by flow cytometry as in the Materials and Methods. Histograms represent a single technical replicate, and depict gates used for Hi and Lo levels of plasma membrane CTxB staining. Quantification is given as percentage of cells within each gate. Each dot represents average of n=3 replicates. ***P<0.005 (multiple two-tailed unpaired t-tests). (B) iMEFs were incubated with Di-4-ANEPPDHQ for 45 min at 37°C, then immediately run on a flow cytometer. Shown is the Generalized Polarization (GP) value for membrane order calculated as in Waddington et al. (2019). Each dot represents average of n=3 technical replicates, n=4 experimental replicates. Significance assessed by paired t-test. (C) iMEFs were lysed in RPPA lysis buffer and samples tested for total cholesterol by Amplex Red cholesterol assay and normalized to lysate protein content. Each dot represents average of n=3 technical replicates, n=7 experimental replicates. *P<0.05 (unpaired two-tailed t-test). (D) Representative micrographs of iMEFs stained with Filipin (green) and LAMP1 (magenta). n=3 experimental replicates, with 20 images per replicate quantified in Fig. S4C,D. The line through a lysosome (in inset) was used to assess intensity of Filipin and LAMP1, with intensity values shown below the micrographs, in 4DI and 4DII. (E) iMEFs were lysed in ionic lysis buffer and run on Optiprep gradient. Each fraction was assessed by Amplex Red assay for total cholesterol and normalized to protein content. n=3 experimental replicates, denoted by distinct shapes. (F) Yeast containing an intracellular D4H–GFP probe were assessed for ergosterol distribution. Images processed in FIJI software for plasma membrane or intracellular ergosterol and quantified as percentage of cells within each group. n=398 *bro1*Δ and n=450 Wt, across n=4 experimental replicates. (G) Following overnight treatment with soluble cholesterol, iMEFs were serum starved for 2 h and incubated with MβCD during the final 30 min. Changes in pERK from 0–10 min EGF stimulation measured by flow cytometry. *P<0.05; **P<0.01; ****P<0.0001 (two-way ANOVA followed by Tukey test). (H) Representative micrographs of iMEFs incubated overnight with TopFluor-cholesterol (green) and stained for LAMP1 (magenta), with * denoting nucleus in zoom images. Cell outline (dashed white line) generated using phalloidin staining. n=60 micrographs across n=3 experimental replicates, quantified in Fig. S5C. All error bars presented as mean±s.e.m. Scale bars: 10 μm.

Cholesterol is a major component of the PM and is associated with increased order (Brown and Rose, 1992; Ahmed et al., 1997); thus, we next assessed whether the changes observed to membrane state involved cholesterol. Total cellular cholesterol remained unchanged in Ptpn23 KO iMEFs relative to WT (Fig. 4C); however, filipin staining revealed decreased PM cholesterol and increased intracellular cholesterol fluorescence with LAMP1 (Fig. 4D; Fig. S4C,D) in Ptpn23 KO iMEFs compared to WT iMEFs. Membrane density fractionation revealed decreased cholesterol in fractions 1–8 of Ptpn23 KO iMEFs, which by this method have been termed PM lipid microdomains (Fig. 4E) (MacDonald and Pike, 2005). Regardless of changes to cholesterol, EGFR content within these fractions was unchanged relative to that in WT iMEFs (Fig. S4E,F), consistent with analysis of PM EGFR by flow cytometry (Fig. 3D). Similar changes in distribution away from the PM were observed for yeast ergosterol upon loss of the HD-PTP homolog Bro1 (Fig. 4F), thus highlighting evolutionary conservation of the Bro1 family in sterol homeostasis. Taken together, these data suggest that HD-PTP contributes to cellular cholesterol distribution to promote increased lipid order at the PM and concomitant RTK signaling.

To assess the relationship between PM cholesterol depletion and signaling, EGF signaling was assessed with pharmacological manipulation of cholesterol. As highlighted previously, WT iMEFs displayed increased basal ERK1/2 phosphorylation compared to that in Ptpn23 KO iMEFs (Fig. 4G). Depletion of cholesterol with methyl-β-cyclodextran (MβCD) in WT iMEFs phenocopied basal Ptpn23 KO iMEF ERK1/2 activation. Conversely, addition of cholesterol to Ptpn23 KO iMEFs was unable to suppress the defect in ERK1/2 activation (Fig. 4G) despite improving cellular lipid order (Fig. S4G). BODIPY-cholesterol was used to gauge whether cholesterol treatment achieved PM cholesterol enrichment as intended in Ptpn23 KO iMEFs. However, BODIPY-cholesterol accumulated within LAMP1⁺ vesicles in Ptpn23 KO iMEF (Fig. 4H; Fig. S5A–C), similar to endogenous cholesterol (Fig. 4D), suggesting cholesterol addition was unable to restore PM cholesterol content and therefore unable to complement signaling. Together these findings suggest HD-PTP promotes cholesterol trafficking and PM cholesterol homeostasis to ensure appropriate functions, including RTK signaling.

Loss of HD-PTP negatively impacts lysosome–ER contact sites and cholesterol transport

Cholesterol delivery to the PM or lipid droplets requires prior transport from the lysosome to ER (Lys to ER) for esterification (Chang et al., 1997). This process is disrupted in lysosomal storage disorders, such as Niemann–Pick disease type C (Pentchev et al., 1994; Kwon et al., 2009), resulting in decreased esterified cholesterol (Höglinger et al., 2019) and similar lysosomal cholesterol accumulations to those observed in Ptpn23 KO iMEFs. Decreased esterified cholesterol was observed in Ptpn23 KO iMEFs (Fig. 5A), consistent with a defect in cholesterol transport to the ER. As cholesterol esterification and transport to the ER is important for the biogenesis of lipid droplets, oleate-induced lipid droplet formation in Ptpn23 KO iMEFs was performed. Compared to WT iMEFs, Ptpn23 KO iMEFS displayed decreased size and number of oleate-induced lipid droplets (Fig. 5B–D; Fig. S6A); this phenotype is consistent with the reduced adipocyte volume in Ptpn23^H/H adipose tissue (Fig. 1J,K; Fig. S1E).

Fig. 5. — **HD-PTP is necessary for localization of cholesterol transport proteins and lysosome–ER contact site formation.** (A) Lysates were assessed by an Amplex Red cholesterol assay with or without a cholesterol esterase reagent. Esterified cholesterol was calculated by subtracting the without esterase value from the with esterase value. Each dot represents average of n=3 technical replicates, n=3 experimental replicates. *P<0.05 (two-tailed unpaired t-test). (B) Following plating on coverslips, iMEFs were loaded overnight with 500 μM oleate and then stained for lipid droplets utilizing Oil Red O staining (magenta), as described in the Materials and Methods. Shown are example micrographs. (C,D) Oil Red O micrographs quantified for size (C) and number of lipid droplets (D) in FIJI software. n=60 cells across n=3 experimental replicates, each experimental replicate matched by color, with experimental mean outlined in black. *P<0.05 (paired two-tailed t-test on experimental means). (E) iMEFs lysed with RPPA and shown as representative immunoblot of cholesterol transfer proteins ORP1, StARD3, NPC1, NPC2 and Actin. n=3 experimental replicates (quantified in Fig. S6B). (F) Example micrographs of cells processed for PLA (magenta) between Rab7 and VapA, as described in the Materials and Methods. (G) Quantification of PLA puncta/cell done in FIJI software. n=60 cells across n=3 experimental replicates, each experimental replicate matched by color, with experimental mean outlined in black. *P<0.05 (paired two-tailed t-test on experimental means). (H) Example micrographs of iMEFs plated on coverslips and processed for immunofluorescence, as described in the Materials and Methods, for NPC1 (green) and LAMP1 (magenta). (I) Quantification of NPC1 overlap with LAMP1 processed in FIJI software. Each dot represents a single cell z-stack, n=60 cells across n=3 experimental replicates, each experimental replicate matched by color, with experimental mean outlined in black. *P<0.05 (paired two-tailed t-test on experimental means). All error bars presented as mean±s.e.m. Scale bars: 10 μm (B,H); 5 μm (F).

We suspected that expression of Lys to ER cholesterol transporters or cholesterol synthesis enzymes might contribute to these phenotypes; however, the expression of the transporters NPC1, NPC2, StARD3, ORP1, ABCA1 and ABCG1 and the cholesterol synthesis factors SQLE, HMGCR and IDI1 were unchanged in Ptpn23 KO iMEFs as assessed by western blotting or quantitative (q)PCR (Fig. 5E; Fig. S6B,C). A lack of gross alterations in cholesterol transport protein expression begged an examination of inter-organellar contact site formation and localization of the Lys to ER cholesterol transport proteins, such as NPC1. To assess the formation of Lys–ER contact sites, a proximity ligation assay (PLA) was performed using the late endosome and lysosome protein Rab7 (herein referring to Rab7a and Rab7b collectively) and the ER protein VapA (Yun et al., 2023; Atakpa et al., 2018). Compared to WT, Ptpn23 KO iMEFs exhibited decreased numbers of Rab7–VapA PLA puncta (Fig. 5F,G) indicating defective Lys–ER contact site formation. PLA controls lacking any single component displayed <10 puncta/cell and iMEFs with ∼50% VapA also displayed a reduction in the number of puncta per cell (Fig. S6D–G). Moreover, immunofluorescence for NPC1 and LAMP1 revealed a decrease in co-incidence of these proteins in Ptpn23 KO iMEFs (Fig. 5H,I), indicating that cholesterol transfer protein distribution is perturbed when HD-PTP function is perturbed. This observation was further supported by lysosome fractionation, which revealed decreased NPC1 relative to LAMP1 in Ptpn23 KO iMEFs (Fig. S6H,I). Thus, we suggest that perturbed cholesterol transport protein localization combines with reduced Lys–ER contact site formation to disrupt Lys to ER cholesterol trafficking in the absence of HD-PTP.

Inhibition of Rab5 suppresses EGFR signaling defect in Ptpn23 KO

HD-PTP contributes to MVB formation (Doyotte et al., 2008), and silencing HD-PTP leads to accumulation of ubiquitylated cargoes and endosomal Rab5 (herein referring to Rab5a–Rab5c collectively) (Parkinson et al., 2021), suggesting that loss of HD-PTP function confers a defect in endosomal maturation. Ptpn23 KO iMEFs displayed an accumulation of ubiquitylated cargoes and membrane-bound Rab5 (Fig. 6A–D). Maturation from early to late endosomes involves exchange of Rab5 (early) for Rab7 (late) (Rink et al., 2005; Poteryaev et al., 2010). Late endosomes are where NPC1-mediated cholesterol transport to the ER occurs (van den Boomen et al., 2020). We hypothesized that Ptpn23 KO iMEF Rab5 accumulation contributes to the cholesterol trafficking phenotype by disrupting the conversion of early endosomes into Rab7-positive late endosomes, thereby decreasing Lys–ER contact site formation and disrupting localization of Lys to ER cholesterol transporter(s). Thus, we tested the ability of the Rab5 inhibitor neoandrographolide (NAP) (Zhang et al., 2020) to suppress the cellular defects of Ptpn23 KO iMEFs. Treatment with NAP restored the coincidence of NPC1 with LAMP1 in Ptpn23 KO iMEFs but had no impact on coincidence in WT cells (Fig. 6E,F), thus suggesting an ability to suppress defects associated with cholesterol transport in Ptpn23 KO iMEFs. Treatment with NAP also increased ERK1/2 phosphorylation in Ptpn23 KO iMEFs to the level seen in basal WT iMEFs (Fig. 6G), suggesting an ability to suppress the signaling defect. The observed suppression of signaling in Ptpn23 KO iMEFs upon NAP treatment might be the result of restored cholesterol transfer, whereas the increase in WT signaling cannot be attributed to changes in cholesterol transporter localization and is unlikely to result from aberrant endosomal signaling owing to the short timepoint assessed. Cumulatively, these data support a model whereby the role of HD-PTP in endosomal maturation contributes to trafficking of cholesterol from the Lys to the ER for proper PM cholesterol content and receptor activation (Fig. 7).

Fig. 6. — **Pharmacologic inhibition of Rab5 restores cellular phenotypes associated with loss of HD-PTP.** Representative micrographs of WT and HD-PTP KO iMEFs stained for (A) ubiquitin and (B) Rab5 following the immunofluorescence protocol with digitonin permeabilization. Dashed line represents cell outline, generated using phalloidin staining. iMEFs in suspension, stained following same protocol as for the micrographs, were quantified by flow cytometry for (C) ubiquitin and (D) Rab5. Each dot represents average of n=3 technical replicates, n=3 experimental replicates. *P<0.05; **P<0.01 (two-tailed paired t-test). (E) Example micrographs of iMEFs stained by immunofluorescence for LAMP1 (magenta) and NPC1 (green) following overnight incubation with 40 μM NAP. Dashed line represents cell outline, generated using phalloidin staining. (F) Quantification of NPC1 overlap with LAMP1, done in FIJI software, following NAP incubation. n=60 cells across n=3 experimental replicates matched by color, with experimental mean outlined in black. A two-way ANOVA on experimental means revealed no significant differences. (G) iMEFs incubated with either 0 or 40 μM NAP overnight and serum starved for 2 h prior to treatment with 100 ng/ml EGF for 0 and 10 min. Cells then stained for pERK and assessed by flow cytometry as in the Materials and Methods. Bars represent fold increase in pERK from 0 to 10 min. Each dot represents average of n=3 technical replicates, n=3 experimental replicates. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001; ns, not significant (two-way ANOVA followed by Tukey test). All error bars presented as mean±s.e.m. Scale bars: 10 μm.

Fig. 7. — **Proposed model of HD-PTP in adipose tissue homeostasis.** *Ptpn23^H/H* mice display lipodystrophy, defined as a decrease in mass of all WAT, and characterized by decreased RTK and insulin receptor signaling. *In vitro*, *Ptpn23* KO iMEFs display decreased EGFR activation and an imbalance in cellular cholesterol distribution towards endosomal compartments. We propose this is due to a decrease in Lys–ER contact site formation and mislocalization of cholesterol transport proteins away from the lysosome, therefore limiting cholesterol transfer to the ER for redistribution through the cell. Accumulation of endosomal Rab5 may be upstream of the cholesterol transport defect, as inhibition of Rab5 by NAP rescues HD-PTP phenotypes. Figure created with BioRender.com.

DISCUSSION

Herein, we propose a model whereby HD-PTP plays a role in cellular cholesterol homeostasis through Lys–ER contact site formation, thereby impacting plasma membrane cholesterol content necessary for signal initiation (Fig. 7). This creates a paradigm by which HD-PTP can play a positive role in signal activation through cellular cholesterol homeostasis, in addition to its canonical role in signal termination through receptor downregulation (Manteghi et al., 2016; Gingras et al., 2009b). Decreased RTK signaling is observed in both WAT of Ptpn23^H/H mice and Ptpn23 KO iMEF models, contrasting with much of the past ESCRT and HD-PTP literature in which depletion enhances cell signaling, leading to their classification as tumor suppressors (Babst et al., 2000; Thompson et al., 2005; Moberg et al., 2005; Vaccari and Bilder, 2005; Tabernero and Woodman, 2018). One exception in the literature to the role of ESCRTs in terminating signaling exists in Drosophila. Both the HD-PTP homolog Mop and the ESCRT-0 component Hrs promote EGFR signaling in Drosophila (Miura et al., 2008), in addition to Mop playing a role in promoting Toll signaling (Huang et al., 2010). However, the necessity of ESCRT components for signal promotion in Drosophila might be attributable to differences in the signaling pathways, which seem to require trafficking to an endosomal compartment for maximal signaling to occur (Miura et al., 2008). Drosophila Vps4 has also been found to play a cell-type-specific role in activation of Notch signaling (Legent et al., 2015), similar to the differences we observe between muscle and adipose in Ptpn23^H/H mice. Thus, the cell type used to assess ESCRT function elicits differential outcomes for receptor signaling. These cell-type-specific differences might be attributable to cellular demands on lipid trafficking, with adipose cells likely requiring more Lys to ER lipid transfer for lipid droplet formation and adipocyte growth, as compared to muscle or tumor cells. Fitting with the tendency of mammals to exhibit enhanced signaling upon perturbation of ESCRTs, previous studies with Ptpn23^−/+ animals have observed tumorigenesis (Manteghi et al., 2016). Because Ptpn23^H/H mice have a shortened life span, it is possible their reduced HD-PTP expression compared to Ptpn23^+/− mice simply does not allow them to live long enough to develop tumors. In addition to differences in lifespan, it is possible that the decreased gene dosage of the Ptpn23^H/H mice relative to Ptpn23^+/− mice confers manifestation of different defects associated with loss of HD-PTP function and its role in receptor signaling.

Based on decreased adipocyte and lipid droplet size in Ptpn23^H/H WAT, and defective signaling stemming from defective receptor phosphorylation downstream of ligand binding, we interrogated cellular cholesterol homeostasis in HD-PTP-deficient cells to assess whether there were alterations in this process that contributed to defective signaling. Although HD-PTP has not previously been shown to regulate cellular cholesterol, other ESCRT-associated proteins, namely, Hrs and Vps4, have been proposed to impact cholesterol homeostasis through disruption of Lys to ER transfer (Du et al., 2012, 2013). Our data support the idea that there is a disruption in Lys–ER contact site formation upon loss of HD-PTP and suggest that HD-PTP might play an indirect role in this process through affecting endosomal maturation. However, a direct role for HD-PTP in contact site formation cannot be eliminated. Knockdown of other ESCRT-I, -II, or -III subunits does not lead to the same cholesterol phenotype (Du et al., 2013), suggesting some level of specificity in which subunits are necessary for cholesterol homeostasis and highlighting the need to further understand what cellular processes HD-PTP is involved in. Fitting with the assertion of subunit specificity, the mammalian ESCRT subunits necessary for cholesterol homeostasis [Hrs (Du et al., 2012), Vps4 (Du et al., 2013) and HD-PTP] are homologs of the Drosophila subunits necessary for promoting receptor activation [Hrs (Huang et al., 2010), Vps4 (Legent et al., 2015), and Mop (Miura et al., 2008)], further drawing links between these two functions. Additionally, this role in cholesterol homeostasis appears to be evolutionarily conserved, as yeast lacking the HD-PTP homolog Bro1 also exhibit a reduction in plasma membrane ergosterol and show a corresponding increase in punctate intracellular structures. Thus, at the levels of animal (mouse), primary cell lines and yeast, we observe comparable defects in cholesterol or ergosterol homeostasis.

MVB sorting starts at PI(3)P endosomes (Katzmann et al., 2003), prior to the Rab5–Rab7 switch during endosomal maturation. As documented previously in the context of yeast (Russell et al., 2012) and mammalian cells (Parkinson et al., 2021), and supported in the present work, perturbation of ESCRTs is associated with accumulation of Rab5 and ubiquitylated cargo, thus suggesting a block in endosomal maturation prior to Rab5 to Rab7 conversion. A block at this stage of endosomal maturation would thereby impact trafficking of both proteins and lipids to the late endosome and hinder subsequent delivery to the lysosome. Although the mechanism by which depletion of ESCRTs or HD-PTP leads to Rab5 accumulation is unknown, two similar hypotheses involving the Rab5 GEF Rabaptin have been proposed. In the first proposed mechanism, accumulation of ubiquitylated cargoes leads to recruitment of Rabex-5–Rabaptin-5, and therefore Rab5 hyperactivation that cannot be terminated by the usual removal of ubiquitin from these cargoes (Mattera and Bonifacino, 2008; Doyotte et al., 2008; Lauer et al., 2019). In the second model, a more direct role for HD-PTP has been proposed wherein its binding to Rabaptin-5 leads to Rab5 inactivation to promote the Rab5–Rab7 switch (Parkinson et al., 2021). Although our data are consistent with an accumulation of both ubiquitylated cargo and Rab5, they do not shed further light on how HD-PTP and MVB sorting are mechanistically related to Rab conversion. Utilization of a Rab5 inhibitor NAP restored EGFR signaling and cholesterol transporter localization, likely owing to decreasing the amount of active Rab5, and promoting the switch to Rab7, thereby restoring lipid movement. However, aside from its known role as a Rab5 inhibitor (Zhang et al., 2020), we cannot eliminate the possibility of unknown off-target functions of NAP. Consistent with the observed suppression by NAP being related to Rab5 inhibition, similar studies have been done in yeast where depletion of the Rab5 homolog Vps21 reverted typical Class E phenotypes observed upon deletion of ESCRT subunits (Russell et al., 2012). Therefore, further studies are necessary to unravel the regulation of endosomal maturation by HD-PTP and the relationship to cholesterol transport. Cumulatively, these studies span from yeast to mice and show an evolutionarily conserved role for HD-PTP in cholesterol homeostasis, thereby enabling a positive role in receptor signaling.

MATERIALS AND METHODS

Mouse strains

Mouse protocols were reviewed and approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). Ptpn23 hypomorphic allele (H) containing a neomycin selectable cassette in the reverse orientation between exons 6 and 7 was generated by standard gene targeting techniques and embryonic stem cell technology. Ptpn23^H/+ mutant mice were maintained on a mixed 129Sv/E×C57BL/6 genetic background or backcrossed for six generations to the C57BL/6 strain, and Ptpn23^H/+ intercrosses were used to generate Ptpn23^H/H mice. Ptpn23^H/H mice in the mixed background or C57BL/6 background behaved equivalently. The neomycin cassette was removed by crossing Ptpn23^H/+ mice with FLP-expressing mice to generate Ptpn23^flox. Intercrosses with R26-M2rtTA and TetOp-CRE mice were used to generate Ptpn23^flox/flox R26-M2rtTA/R26-M2rtTA+/+ and Ptpn23^flox/flox R26-M2rtTA/R26-M2rtTA+/TetOp-CRE mice for intercrossing to generate Ptpn23^flox/flox R26-M2rtTA/R26-M2rtTA+/TetOp-CRE mice used to isolate preadipocytes and MEFs for doxycycline-induced knockout studies. Exons 5 and 6 were removed by crossing Ptpn23^H/+ mice with Hprt-Cre transgenic mice to generate the null allele (−). Ptpn23^−/+ intercrosses were performed to examine the viability of Ptpn23^−/− mice. Ptpn23^H/−, Ptpn23^−/+, Ptpn23^H/+ and Ptpn23^+/+ pups were generated by crossing Ptpn23^H/+ and Ptpn23^−/+ mice; Ptpn23^H/H and Ptpn23^+/+ pups were generated by intercrossing Ptpn23^H/+ mice. Male and female mice were used for experimentation. To genotype for hypomorphic allele, tails were clipped and heated in 25 mM NaOH and 0.2 mM EDTA for 1 h at 95°C, then neutralized with 40 mM Tris-HCl pH 5.5. PCRs were run with Choice Taq Blue MasterMix [Denville CB4065-7] with primer sequences 5′-GCTGGGCTGACTGTCACAAGCG-3′ and 5′-CAGCCAACAGCACAGCTGAC-3′.

Body composition, physiological and behavioral analyses

Body composition and total body fat were determined using an EchoMRI-100 QNMR instrument (Echo Medical Systems) as previously described (Sieben et al., 2020). Food intake, activity and metabolic rate were measured using a Comprehensive Lab Animal Monitoring System (CLAMS) as described previously (Xu et al., 2015). Individual experiments analyzed ∼6-week-old Ptpn23^H/H and Ptpn23^+/+ mice (n=4 female and n=4 male for each) and were repeated for total of three sets of animals. Energy expenditure was normalized to either total body mass or lean body mass, as determined using the EchoMRI-100. Individual fat depot mass and additional organs (liver, heart, lung and gastrocnemius) were determined through dissection and normalized to total body mass. Skin and fat (IAT) were fixed in 10% normal buffered formalin, embedded in paraffin, and sections were stained with hematoxylin and eosin (H&E) as previously described (Sieben et al., 2020).

RNA isolation, library preparation, sequencing and bioinformatics analyses

RNA was extracted from cryo-pulverized inguinal adipose tissue from ad libitum-fed 14-day-old mice (n=3 male and n=3 female for Ptpn23^H/H and Ptpn23^+/+) using the RNeasy Mini Kit (Qiagen 74104) according to the manufacturer's instructions. The on-column DNase digestion step was included. RNA library preparation, sequencing and downstream analysis were performed as previously described (Aziz et al., 2019; Limzerwala et al., 2020); ∼200 ng RNA was used for RNA library preparation. Sequencing was performed by the Mayo Clinic Medical Genome Facility Genome Analysis Core. Genes with significantly altered expression between Ptpn23^H/H and Ptpn23^+/+ IAT were analyzed with Ingenuity Pathway Analysis (Qiagen) to identify impacted pathways and candidate upstream drivers. Expression variation between male and female animals was also examined to confirm results. Heat maps were created with the Next-Generation Clustered Heat Map (NG-CHM) viewer web interface (Ryan et al., 2019).

Cell lines and culture

iMEFs were generated from Ptpn23^flox/flox R26-M2rtTA/R26-M2rtTA+/TetOp-CRE mice and immortalized using SV40LT virus as previously described (Hamada et al., 2011). iMEFs were cultured in 5% CO₂ and normal oxygen in complete DMEM [DMEM (Gibco 12100-038), 10% fetal bovine serum (Gibco 10437-028), 20 mM L-glutamine (Corning 25-005-CI), 1× MEM nonessential amino acids (Corning 25-025-CI), 10 mM sodium pyruvate (Corning 25-000-CI), 1× antibiotic-antimycotic (Gibco 15240-062)]. To induce HD-PTP knock-out, iMEFs were incubated with 1 μg/ml doxycycline (BD Biosciences 8634-1) for 4 days prior to experiments.

VapA KO iMEFS were generated by CRISPR-Cas9 and utilized at day 7 for experiments, with guide sequences 5′-CAGACCTCAAATTCAAAGGT-3′, 5′-TGAAGACTACAGCACCTCGC-3′ and 5′-ACAGTGGAATTATTGACCCA-3′. Briefly, guide RNA complexes were prepared by heating equimolar amounts of crRNA and tracrRNA at 95°C for 5 min as previously described (Wen et al., 2022). The resulting complexes were allowed to cool to room temperature and incubated with NLS-Cas9 (QB3 Macrolab, Berkeley, CA, USA) for 10 min. For each target, three crRNAs were pooled so that 450 pmols of crRNA-tracrRNA complex were delivered with 180 pmol Cas9. iMEFs were nucleofected by Lonza 4D nucleofector program CG-104 in a nucleofection buffer consisting of 5 mM KCl, 15 mM MgCl₂, 15 mM HEPES and 105 mM Na₂HPO₄ and 50 mM mannitol, all supplied by Sigma (St Louis, MO, USA). crRNAs were designed using CRISPick tool from Broad (Doench et al., 2016; Sanson et al., 2018). crRNA and tracrRNA were ordered from Integrated DNA Technologies (Coralville, IA, USA).

Preadipocyte isolation was performed by mincing IAT in Hanks’ balanced salt solution (HBSS) and digesting with Type 2 collagenase for ∼1 h at 37°. Preadipocytes were pelleted with a 10 min 335 g spin at room temperature. Preadipocytes were plated in αMEM (Life Technologies) with 10% heat inactivated calf serum (CFS; Sigma) and incubated overnight. The following day, preadipocytes were recovered by trypsinization, replated at 50,000 cells/cm² in αMEM with 10% CFS, and cultured at 10% CO₂ and 3% O₂, changing the medium every other day. Knockout of Ptpn23 was induced through addition of 1 μg/ml doxycycline to the culture medium of Ptpn23^flox/flox R26-M2rtTA/R26-M2rtTA+/TetOp-CRE preadipocytes. Cells were cultured a minimum of 3 days before initiating adipogenesis. Preadipocyte differentiation was initiated at 85–90% confluence by feeding with differentiation medium [DMEM:F12 (Life Technologies 11330-032) with 10% FBS (Sigma F4135), 1 μg/ml insulin (Sigma), 250 nM dexamethasone (Sigma), 0.5 mM IBMX (Sigma) and 2.5 μM Rosiglitazone (Sigma)] for 48 h. Preadipocytes were then refed with DMEM:F12 10% FBS, 1 μg/ml insulin (Sigma) for 7 days harvesting samples each day. All cell lines were tested regularly for mycoplasma contamination.

Immunofluorescence

iMEFs were plated at a density of 30,000 cells per coverslip and incubated overnight. Coverslips were then washed with PBS and incubated in 4% PFA for 15 min at room temperature. Permeabilization was undertaken with either 3 min in 0.15% Triton X-100 or 5 min in 0.01% digitonin. Coverslips were blocked for at least 1 h in blocking buffer [PBS, 5% goat serum (Vector Laboratories), 1% glycerol (Baxter Scientific Products), 0.1% BSA (Sigma), 0.1% fish skin gelatin (Sigma), 0.04% sodium azide (Fisher)], inverted onto 90 µl of primary antibody (see Table S1 for details) in blocking buffer, and incubated overnight at 4°C in a humidity chamber. The next day, coverslips were washed three times in 250 µl of PBS for 5 min, then incubated on 90 µl of secondary antibody, 5 mg/ml Hoechst 33342 (1:500, Thermo Fisher Scientific), and phalloidin (1:100; Invitrogen) for 1 h at room temperature in a humidity chamber. Coverslips were then washed three times with PBS and three times with water, dried, mounted onto slides with slowfade [50 mM Tris-HCl, 50% glycerol and 1% n-propylgallate] for 60 min, and sealed with nail polish. Z-stacks were collected in 25 μm increments on Zeiss LSM 800 with Airyscan utilizing the 60× oil or 60× water objectives. Images were analyzed in FIJI software, utilizing phalloidin to identify single cells, for puncta size, puncta number, and percent overlap. Cells were selected for imaging based upon DAPI and phalloidin staining, independent of experimental channels.

Western blotting and reverse phase protein array analyses

Tissues were flash frozen, cryo-pulverized, and resuspended in Reverse Phase Protein Array (RPPA) lysis buffer [1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na₃VO₄, 10% glycerol, 100 μm AEBSF (Thermo Fisher Scientific BP2644-100), with Complete EDTA-free Protease Inhibitor Cocktail Tablet (Roche 04693132001) and PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche 04906837001)]. Cells were lysed in RPPA lysis buffer. Cleared lysate concentrations were measured with a Bio-Rad Bradford protein assay and normalization was performed. Cleared lysates were combined with 5× Laemmli sample buffer, denatured, resolved by SDS-PAGE, and transferred to nitrocellulose. Primary antibodies and concentrations used for western blotting are listed in Table S1. Blots were developed with fluorescent secondary antibodies [goat anti-rabbit-IgG IRDye 680LT (1:5000, 926-68021, Li-Cor), goat anti-mouse-IgG IRDye 800CW (1:5000, 926-32210, Li-Cor)] (uncropped images for blots shown in this paper can be viewed in Fig. S7). Signal was detected using the Odyssey Infrared Imager with ImageStudio software (LiCor) and analyzed using ImageJ (Schneider et al., 2012), ImageQuantTL (GE Life Sciences) and Prism 7 (GraphPad) to calculate the ratio of protein to loading control (actin or tubulin) or phosphorylated to total protein. RPPA was performed by the MD Anderson Functional Proteomics RPPA Core Facility with 377 antibodies as described previously (Davies et al., 2009). Heat maps were created with the Next-Generation Clustered Heat Map (NG-CHM) Viewer web interface (Ryan et al., 2019).

Signaling assays

In vitro stimulation of IAT was performed with minced IAT in HBSS with protease and phosphatase inhibitors. Stimulation with 100 ng/ml insulin was performed after 1 h equilibration, and samples were lysed in RPPA lysis buffer with a dounce homogenizer at 5, 15, 30, 60 and 120 min after stimulation. Western blotting was used to assess Akt activation. In vitro stimulation of preadipocytes was performed following 1 h serum starvation in DMEM:F12. Stimulation with 100 ng/ml insulin was performed, and samples were lysed in RPPA lysis buffer at 5, 10, 15, 30 and 60 min after stimulation. Western blotting was used to assess Akt activation. iMEFs were plated at 75,000 cells/well in 12-well tissue culture plate for 2 days, then incubated in serum-free DMEM for 2 h prior to stimulation. Wells were stimulated with 100 ng/ml EGF (Gibco, PHG0311) and samples lysed at 0, 5, 10, 15, 20, 30, 45 and 60 min in RPPA lysis buffer. Western blotting was used to assess EGFR (Y1068) and ERK1/2 activation. For flow cytometry analysis, iMEFs were plated at 3×10⁶ cells/dish in 10 cm tissue culture plates and incubated overnight in complete DMEM. 0.5 M Water-soluble cholesterol (Sigma C4951) or Neoandrographolide (Sigma 49879) were added for ∼24 h before harvest. Cells were removed from plates by 5 min TrypLE (Gibco 12604-013) incubation and then replated in 96-well round bottom plate at 10⁶ cells/well in serum-free DMEM 2 h prior to stimulation. 5 mM methyl-β-cyclodextran (Sigma C4555) was added for cholesterol depletion 30 min prior to stimulation. 100 ng/ml EGF was used to stimulate cells, and fixable viability dye (Thermo Fisher Scientific L23105) was added prior to fixation in 4% PFA at 0 and 10 min for analysis of ERK1/2 phosphorylation by flow cytometry on BD FACS Canto II, with analysis in FlowJo.

Ligand binding and plasma membrane receptor content

iMEFs were plated at 10⁶ cells/well in a 96-well round bottom plate in triplicate and incubated 2 h in either complete DMEM (basal condition) or serum-free DMEM (time 0 of stimulations). Cells were rinsed once in cold PBS and moved to ice for a 20-min incubation with 488–EGF (100 ng/ml; Thermo Fisher Scientific E13345) or extracellular EGFR antibody (1:200, Cell Signaling 54359) and fixable viability dye (Thermo Fisher Scientific L23105). Control samples for ligand binding were preincubated with 1000 ng/ml unlabeled EGF on ice 15 min prior to 488–EGF to assess background. Cells were washed once with cold PBS and fixed in 4% PFA for 15 min. 488–EGF binding was assessed by flow cytometry immediately and after a 45-min incubation with Alexa Fluor 647-conjugated anti-rabbit-IgG (1:800; Invitrogen A31573) in 0.5% BSA was done to visualize the extracellular EGFR antibody for flow cytometry detection. For EGFR downregulation from the plasma membrane, cells were processed as above but collected at 0, 5, 10, 15, 20 and 30 min of 100 ng/ml EGF incubation before being moved to ice for anti-EGFR and fixable viability incubation.

Yeast distribution of ergosterol

SEY6210 WT and bro1Δ strains were previously described (Pashkova et al., 2013). Plasma membrane ergosterol was assessed in yeast strains by monitoring the localization of the ergosterol sensor D4H–GFP to the plasma membrane. Yeast were transformed with a plasmid encoding the sensor pRS316-CUP1-D4H-GFP (Koselny et al., 2018). In this plasmid, D4H, which encodes for the fourth domain of Clostridium perfringens O theta-toxin (residues 391–500) containing an S434D mutation (Maekawa and Fairn, 2015), is C-terminally tagged with GFP, and placed under the control of the CUP1 promoter, which is activated by Cu²⁺ ions. D4H localization was assessed in yeast after growth overnight at 29°C in liquid synthetic SD-Ura-Met medium (RPI YNB Y20040-1000.0, CSM dropout-MET-URA ref #DCS0659) to OD₆₀₀=0.4–0.6 with shaking, followed by induction of the reporter construct with 1 mM CuCl₂ for 1 h. Yeast were imaged using an Olympus fluorescent microscope BX60 controlled by iVision software (BioVision Technologies) and equipped with UPlanSApo100×/1.40 oil objective and Hamamatsu Orca-R2 digital camera (Hamamatsu, JP). Images were processed using FIJI software. ROIs were drawn around each cell, and D4H–GFP localization was assessed to be either at the plasma membrane (PM), intracellular puncta (Dots), or at both plasma membrane and puncta (PM+Dots).

Di-4-ANEPPDHQ staining

iMEFs were plated at 3×10⁶ cells/dish in 10 cm tissue culture plates and incubated overnight. Water-soluble cholesterol was added (as described above for stimulations) during this incubation. Methyl-β-cyclodextran was added 30 min prior to harvest. For flow cytometry, cells were incubated for 5 min in TrypLE and moved to a 15 ml conical flask for resuspension in PBS with 2.5 μg/ml Di-4-ANEPPDHQ (Invitrogen D36802) at 10⁶ cells/ml; ∼10⁶ cells were then moved to each uncapped flow tube and incubated for 45 min at 37°C in 5% CO₂ and normal oxygen. Samples were immediately run on a flow cytometer and analyzed as described previously (Waddington et al., 2019). For microscopy analysis, iMEFs were plated at 15,000 cells/well in an 8-well chamber coverslip (Thermo Fisher Scientific 155411) and incubated overnight. Wells were washed once with PBS and then incubated with 2.5 μg/ml DI-4-ANEPPDHQ in FluoroBrite DMEM (Gibco A1896701) with 10% heat-inactivated FBS for 45 min. Wells were imaged live at 37°C in 5% CO₂ chamber on a Zeiss LSM 800 microscope with a 60× water objective, and images were analyzed with a Fiji macro as previously described (Owen et al., 2012).

Cholesterol detection

Total cellular cholesterol was measured with an Amplex Red cholesterol assay (Invitrogen). Briefly, confluent iMEFs in 12-well tissue culture plate were lysed in 500 µl RPPA lysis buffer and 50 μl of lysate was plated per well in an ELISA plate and read out on a Molecular Devices SpectraMax M3 plate reader. Each sample was assessed in triplicate. For measurement of esterified cholesterol, the same protocol was followed without addition of esterase, and then subtracted from the total value. For cellular cholesterol distribution, iMEFs were plated at 50,000 cells/coverslip and incubated overnight. Coverslips were rinsed once with PBS, fixed for 15 min in 4% PFA, and permeabilized for 2 min in 0.15% Triton X-100. Coverslips were then incubated overnight with anti-CD107 antibody (see Table S1 for details) at 4°C. The following day, coverslips were rinsed three times with PBS and then incubated 45 min in Filipin (Maxfield and Wüstner, 2012) (Cayman 70440) and Alexa Fluor 647-conjugated anti-rat-IgG antibody. Coverslips were then rinsed three times with PBS and three times with water before mounting in slowfade solution for visualization on Zeiss Axio Observer with Colibri 7 and Definite Focus.2. Manders analysis was done in FIJI software with the JaCOP plugin (Bolte and Cordelières, 2006), and intensity of Filipin within lysosomes also assessed in FIJI software. For visualization of exogenous cholesterol, cells were plated as above and incubated with 2 μg/ml TopFluor-cholesterol (Avanti 810255) for 18 h. Coverslips then fixed and processed for immunofluorescence as above, with visualization Zeiss LSM 800 utilizing the 60× oil objective. Analysis was done in FIJI software. For plasma membrane CTxB staining, iMEFs in suspension were incubated with 1 μg/ml CTxB (Invitrogen C34775) and viability dye on ice for 30 min. Cells then rinsed with cold PBS and fixed in 4% PFA for 15 min prior to running on a BD FACS Canto II cytometer. Analysis was done in FlowJo software.

Membrane fractionation

Membrane fractionation was undertaken as described previously (MacDonald and Pike, 2005). Briefly, iMEFs were grown to confluency in a T75 tissue culture flask. Cells were then washed three times with cold PBS, incubated in 1 ml isotonic buffer (20 mM Tris-HCl pH 7.8, 250 mM sucrose, 1 mM CaCl₂, 1 mM MgCl₂, 1 μg/ml aprotinin, 10 μg/ml leupeptin) on ice for ∼10 min and scraped into 1.5 ml Eppendorf tube. Lysate was passed through a 22 G needle 15 times, followed by a 27 G needle five times. Lysates were cleared in a 10-min spin at 1000 g at 4°C and supernatant transferred to a new tube. The pellet was then resuspended in 1 ml of isotonic lysis buffer and passed through a 27 G needle 10 times followed by another clearing spin. Supernatant from second spin was combined with that from the first. Then, 2 ml of 50% OptiPrep (Sigma D1556) was added to cleared lysate to give 25% OptiPrep. Samples were loaded into a 20 ml OptiPrep gradient [4 ml 100/50/25(sample)%, 1.6 ml 20/15/10/5/0%] and spun at 17,000 rpm in a Beckman SW28 rotor for 90 min at 4°C. 29 fractions were then collected in 670 μl aliquots and used for western blotting, Amplex Red cholesterol assay and determining total protein concentration by use of Bradford reagent. Lipid microdomain fractions were identified as those with no transferrin receptor content and prior to fractions containing most intense CAV1 by western blotting, as done previously for HeLa cells (MacDonald and Pike, 2005).

Lipid droplet visualization

Lipid droplet visualization was undertaken as described previously (Li et al., 2016). Briefly, iMEFs were plated at 15,000 cells/coverslip and incubated overnight. Cells were then loaded with 500 μM oleate in complete DMEM for ∼24 h. Coverslips were washed 1× in PBS, fixed for 15 min in 4% PFA, and washed twice with PBS prior to Oil Red O staining. Coverslips were then incubated 25 s in 60% isopropanol, followed by 2 min in 60% Oil Red O solution, and then incubated 25 s in 60% isopropanol. Coverslips were then washed 2× with PBS and incubated in Hoechst 33342 (1:500, Thermo Fisher Scientific) and AlexaFluor488–Phalloidin (1:100; see Table S1) for 30 min at room temperature. Rinses and mounting of coverslips were done as described above in the Immunofluorescence section.

Quantitative PCR for cholesterol synthesis and transporter proteins

Quantitative (q)PCR was undertaken as described previously (Ding et al., 2017). Briefly, RNA was isolated from 5×10⁶ iMEFs utilizing an RNeasy mini kit (Qiagen 74104) for RNA extraction and reverse transcription performed with Superscript III RT-PCR kit (Invitrogen 18080400). For qPCR, a master mix was made with iTaq Universal SYBR Green Supermix (Bio-Rad L006363) and analyzed utilizing the ABI StepOnePlus Sequence Detection System (Applied Biosystems). Actin was used as housekeeping gene for normalization of gene expression and the double ΔCt method used to analyze gene expression. Experiments were performed in triplicate utilizing three independent cDNAs. TaqMan probes utilized were Idi1 (Thermo Fisher Scientific #Mm01337454_m1), Abca1 (Thermo Fisher Scientific #Mm00442646_m1), Sqle (Thermo Fisher Scientific #Mm00436772_m1), Hmgcr (Thermo Fisher Scientific #Mm01282499_m1) and Abcg1 (Thermo Fisher Scientific #Mm00437390_m1).

Proximity ligation assay

Protocol based on PLA kit instructions (Sigma) and primary antibody combination described previously (Atakpa et al., 2018; Yun et al., 2023). Following fixation, permeabilization and blocking described above in the Immunofluorescence section, iMEFs were incubated in primary antibody (see Table S1 for details) overnight at 4°C. Coverslips were then washed twice with PBS and inverted onto 50 μl of PLA secondary antibodies (1:5; Table S1) for 1 h at 37°C in a humidity chamber. Following another two 5 min PBS washes, coverslips were incubated in 50 μl ligation reaction for 30 min at 37°C in a humidity chamber. After two 5 min PBS washes, coverslips were incubated in 50 μl amplification reaction (Sigma DUO92013) in a humidity chamber for 1 h 40 min at 37°C. Finally, coverslips were washed 2× in PBS and incubated in Hoechst 33342 (1:500) and AlexaFluor488-phalloidin (1:100) for 20 min at room temperature, then mounted as above. Coverslips were imaged on Zeiss LSM 800 utilizing the 60× water objective. Quantitation of PLA puncta/cell was done in FIJI.

Lysosome fractionation

Lysosome fractionation was performed as described previously (Phatarpekar et al., 2020; Schenkman and Cinti, 1978). Briefly, 50×10⁶ WT and KO iMEFs were resuspended in 1.5 ml of 0.25 M sucrose in 10 mM Tris-HCl (pH 7.4) and homogenized with a dounce tissue homogenizer. Lysates were then spun for 5 min at 603 g at 4°C and the supernatant (post-nuclear lysate) spun again at 12,000 g for 10 min at 4°C. Supernatant was collected (post-mitochondrial fraction) and CaCl₂ added to a final concentration of 8 mM, then vortexed well. Lysate was then spun at 18,000 g for 15 min at 4°C and all supernatant collected as the non-lysosomal fraction. The pellet was washed once in 1 ml of 150 mM KCl in 10 mM Tris-HCl pH 7.4 at 18,000 g for 15 min at 4°C and supernatant collected (wash fraction). The pellet was then resuspended in 50 μl of 150 mM KCl in 10 mM Tris-HCl pH 7.4 and saved as crude lysosome fraction. Samples from each fraction were assessed by western blot for markers indicated. Analysis of the NPC1 to LAMP1 ratio in crude lysosome fractions was completed in FIJI software.

Statistical analyses

All analyses completed in Prism and error bars represented as mean±s.e.m. unless otherwise noted. *P<0.05, **P<0.01, ***P<0.001. Data with more than two experimental groups were assessed by two-way ANOVA followed by Tukey test or Kruskal–Wallis followed by Mann–Whitney. Data with two experimental groups were assessed for normality by a Shapiro–Wilk normality test, then assessed by Mann–Whitney if normality was not met or a two-tailed unpaired t-test if normally distributed. If n=3 for both experimental groups, a two-tailed unpaired t-test was used due to Mann–Whitney being underpowered for this sample size. In the cases where samples were normalized to a control group across multiple experiments, significance was assessed by a one sample t-test. Flow cytometry MFI and immunofluorescence quantitation were assessed by a two-tailed paired t-test on the experimental means, rather than individual cells, thus highlighting data reproducibility and accounting for variation across experiments (Lord et al., 2020). Time course experiments were assessed by area under curve, which was then used to generate a P-value.

Supplementary Material

joces-137-262032_review_history.pdf^{(297.5KB, pdf)}

Supplementary information

joces-137-262032-s1.pdf^{(65.7MB, pdf)}

DOI: 10.1242/joces.262032_sup1

Table S2. Raw RPPA data.

joces-137-262032-TableS2.xlsx^{(203.1KB, xlsx)}

Acknowledgements

HD-PTP mice were generated by the Transgenic and Knockout Core of Mayo Clinic. We thank members of the Shapiro lab at Mayo Clinic for discussion of cholesterol reagents, members of the McNiven lab at Mayo Clinic for microscope assistance, and Zhigang He for experimental assistance. The Functional Proteomics Reverse Phase Protein Array Core was supported in part by The University of Texas MD Anderson Cancer Center, P30CA016672 (National Institutes of Health), and R50CA221675 (National Institutes of Health).

Footnotes

Author contributions

Conceptualization: D.F.S., B.A.D., D.J.K.; Methodology: D.F.S., B.A.D., D.J.B., J.v.; Validation: D.F.S., B.A.D.; Formal analysis: D.F.S., C.Z., H.C.; Investigation: D.F.S., B.A.D., J.A.P., C.P.M., A.Y.M., B.G.C., T.W., A.d., L.H., S.J.; Resources: K.B.J., I.S., M.A.D., N.K.L., R.C.P., H.L., D.J.B., J.v., D.D.B.; Writing - original draft: D.F.S.; Writing - review & editing: D.F.S., B.A.D., R.C.P., D.D.B., D.J.K.; Visualization: D.F.S., B.A.D.; Supervision: D.J.K.; Funding acquisition: D.J.K.

Funding

This work was funded by National Institutes of Health (NIH) (R01GM116826 awarded to D.J.K.). D.F.S. is supported by the NIH (R25GM55252 and T32DK124190). M.A.D. is supported by the Dr Miriam and Sheldon G. Adelson Medical Research Foundation, the AIM at Melanoma Foundation, the NIH (NCI; P50CA221703), the American Cancer Society and the Melanoma Research Alliance, Cancer Fighters of Houston, the Anne and John Mendelsohn Chair for Cancer Research, and philanthropic contributions to the Melanoma Moon Shots Program of MD Anderson. Deposited in PMC for release after 12 months.

Data availability

All relevant data can be found within the article and its supplementary information.

Peer review history

The peer review history is available online at https://journals.biologists.com/jcs/lookup/doi/10.1242/jcs.262032.reviewer-comments.pdf

References

Ahmed, S. N., Brown, D. A. and London, E. (1997). On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36, 10944-10953. 10.1021/bi971167g [DOI] [PubMed] [Google Scholar]
Ali, N., Zhang, L., Taylor, S., Mironov, A., Urbé, S. and Woodman, P. (2013). Recruitment of UBPY and ESCRT exchange drive HD-PTP-dependent sorting of EGFR to the MVB. Curr. Biol. 23, 453-461. 10.1016/j.cub.2013.02.033 [DOI] [PubMed] [Google Scholar]
Atakpa, P., Thillaiappan, N. B., Mataragka, S., Prole, D. L. and Taylor, C. W. (2018). IP(3) receptors preferentially associate with ER-lysosome contact sites and selectively deliver Ca(2+) to lysosomes. Cell Rep. 25, 3180-3193.e7. 10.1016/j.celrep.2018.11.064 [DOI] [PMC free article] [PubMed] [Google Scholar]
Aziz, K., Limzerwala, J. F., Sturmlechner, I., Hurley, E., Zhang, C., Jeganathan, K. B., Nelson, G., Bronk, S., Fierro Velasco, R. O., Van Deursen, E.-J.et al. (2019). Ccne1 overexpression causes chromosome instability in liver cells and liver tumor development in mice. Gastroenterology 157, 210-226.e12. 10.1053/j.gastro.2019.03.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
Babst, M., Odorizzi, G., Estepa, E. J. and Emr, S. D. (2000). Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic 1, 248-258. 10.1034/j.1600-0854.2000.010307.x [DOI] [PubMed] [Google Scholar]
Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T. and Emr, S. D. (2002a). Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3, 271-282. 10.1016/S1534-5807(02)00220-4 [DOI] [PubMed] [Google Scholar]
Babst, M., Katzmann, D. J., Snyder, W. B., Wendland, B. and Emr, S. D. (2002b). Endosome-associated complex, ESCRT-Ii, recruits transport machinery for protein sorting at the multivesicular body. Dev. Cell 3, 283-289. 10.1016/S1534-5807(02)00219-8 [DOI] [PubMed] [Google Scholar]
Baietti, M. F., Zhang, Z., Mortier, E., Melchior, A., Degeest, G., Geeraerts, A., Ivarsson, Y., Depoortere, F., Coomans, C., Vermeiren, E.et al. (2012). Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat. Cell Biol. 14, 677-685. 10.1038/ncb2502 [DOI] [PubMed] [Google Scholar]
Baker, D. J., Jeganathan, K. B., Cameron, J. D., Thompson, M., Juneja, S., Kopecka, A., Kumar, R., Jenkins, R. B., De Groen, P. C., Roche, P.et al. (2004). BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat. Genet. 36, 744-749. 10.1038/ng1382 [DOI] [PubMed] [Google Scholar]
Bend, R., Cohen, L., Carter, M. T., Lyons, M. J., Niyazov, D., Mikati, M. A., Rojas, S. K., Person, R. E., Si, Y., Wentzensen, I. M.et al. (2020). Phenotype and mutation expansion of the PTPN23 associated disorder characterized by neurodevelopmental delay and structural brain abnormalities. Eur. J. Hum. Genet. 28, 76-87. 10.1038/s41431-019-0487-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
Bishop, N. and Woodman, P. (2000). ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking. Mol. Biol. Cell 11, 227-239. 10.1091/mbc.11.1.227 [DOI] [PMC free article] [PubMed] [Google Scholar]
Blüher, M., Michael, M. D., Peroni, O. D., Ueki, K., Carter, N., Kahn, B. B. and Kahn, C. R. (2002). Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev. Cell 3, 25-38. 10.1016/S1534-5807(02)00199-5 [DOI] [PubMed] [Google Scholar]
Bolte, S. and Cordelières, F. P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213-232. 10.1111/j.1365-2818.2006.01706.x [DOI] [PubMed] [Google Scholar]
Brown, D. A. and Rose, J. K. (1992). Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533-544. 10.1016/0092-8674(92)90189-J [DOI] [PubMed] [Google Scholar]
Byers, D. M., Rastogi, S. R., Cook, H. W., Palmer, F. B. S. C. and Spence, M. W. (1989). Defective activity of acyl-CoA:cholesterol O-acyltransferase in Niemann-Pick type C and type D fibroblasts. Biochem. J. 262, 713-719. 10.1042/bj2620713 [DOI] [PMC free article] [PubMed] [Google Scholar]
Carlton, J. G., Agromayor, M. and Martin-Serrano, J. (2008). Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release. Proc. Natl Acad. Sci. USA 105, 10541. 10.1073/pnas.0802008105 [DOI] [PMC free article] [PubMed] [Google Scholar]
Chang, T. Y., Chang, C. C. Y. and Cheng, D. (1997). Acyl-coenzyme a: cholesterol acyltransferase. Annu. Rev. Biochem. 66, 613-638. 10.1146/annurev.biochem.66.1.613 [DOI] [PubMed] [Google Scholar]
Christ, L., Wenzel, E. M., Liestøl, K., Raiborg, C., Campsteijn, C. and Stenmark, H. (2016). ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters in cytokinetic abscission. J. Cell Biol. 212, 499-513. 10.1083/jcb.201507009 [DOI] [PMC free article] [PubMed] [Google Scholar]
Christ, L., Raiborg, C., Wenzel, E. M., Campsteijn, C. and Stenmark, H. (2017). Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem. Sci. 42, 42-56. 10.1016/j.tibs.2016.08.016 [DOI] [PubMed] [Google Scholar]
Cianciola, N. L., Greene, D. J., Morton, R. E. and Carlin, C. R. (2013). Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1. Mol. Biol. Cell 24, 3309-3325. 10.1091/mbc.e12-10-0760 [DOI] [PMC free article] [PubMed] [Google Scholar]
Cox, L. E., Ferraiuolo, L., Goodall, E. F., Heath, P. R., Higginbottom, A., Mortiboys, H., Hollinger, H. C., Hartley, J. A., Brockington, A., Burness, C. E.et al. (2010). Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS). PLoS ONE 5, e9872. 10.1371/journal.pone.0009872 [DOI] [PMC free article] [PubMed] [Google Scholar]
Davies, M. A., Stemke-Hale, K., Lin, E., Tellez, C., Deng, W., Gopal, Y. N., Woodman, S. E., Calderone, T. C., Ju, Z., Lazar, A. J.et al. (2009). Integrated molecular and clinical analysis of AKT activation in metastatic melanoma. Clin. Cancer Res. 15, 7538-7546. 10.1158/1078-0432.CCR-09-1985 [DOI] [PMC free article] [PubMed] [Google Scholar]
Dinchuk, J. E., Cao, C., Huang, F., Reeves, K. A., Wang, J., Myers, F., Cantor, G. H., Zhou, X., Attar, R. M., Gottardis, M.et al. (2010). Insulin receptor (IR) pathway hyperactivity in IGF-IR null cells and suppression of downstream growth signaling using the dual IGF-IR/IR inhibitor, BMS-754807. Endocrinology 151, 4123-4132. 10.1210/en.2010-0032 [DOI] [PubMed] [Google Scholar]
Ding, L., Liou, G. Y., Schmitt, D. M., Storz, P., Zhang, J. S. and Billadeau, D. D. (2017). Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia. J. Pathol. 243, 65-77. 10.1002/path.4928 [DOI] [PMC free article] [PubMed] [Google Scholar]
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R.et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184-191. 10.1038/nbt.3437 [DOI] [PMC free article] [PubMed] [Google Scholar]
Dowlatshahi, D. P., Sandrin, V., Vivona, S., Shaler, T. A., Kaiser, S. E., Melandri, F., Sundquist, W. I. and Kopito, R. R. (2012). ALIX is a Lys63-specific polyubiquitin binding protein that functions in retrovirus budding. Dev. Cell 23, 1247-1254. 10.1016/j.devcel.2012.10.023 [DOI] [PMC free article] [PubMed] [Google Scholar]
Doyotte, A., Mironov, A., Mckenzie, E. and Woodman, P. (2008). The Bro1-related protein HD-PTP/PTPN23 is required for endosomal cargo sorting and multivesicular body morphogenesis. Proc. Natl. Acad. Sci. USA 105, 6308-6313. 10.1073/pnas.0707601105 [DOI] [PMC free article] [PubMed] [Google Scholar]
Du, X., Kumar, J., Ferguson, C., Schulz, T. A., Ong, Y. S., Hong, W., Prinz, W. A., Parton, R. G., Brown, A. J. and Yang, H. (2011). A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking. J. Cell Biol. 192, 121-135. 10.1083/jcb.201004142 [DOI] [PMC free article] [PubMed] [Google Scholar]
Du, X., Kazim, A. S., Brown, A. J. and Yang, H. (2012). An essential role of Hrs/Vps27 in endosomal cholesterol trafficking. Cell Rep. 1, 29-35. 10.1016/j.celrep.2011.10.004 [DOI] [PubMed] [Google Scholar]
Du, X., Kazim, A. S., Dawes, I. W., Brown, A. J. and Yang, H. (2013). The AAA ATPase VPS4/SKD1 regulates endosomal cholesterol trafficking independently of ESCRT-III. Traffic 14, 107-119. 10.1111/tra.12015 [DOI] [PubMed] [Google Scholar]
Farazi Fard, M. A., Rebelo, A. P., Buglo, E., Nemati, H., Dastsooz, H., Gehweiler, I., Reich, S., Reichbauer, J., Quintáns, B., Ordóñez-Ugalde, A.et al. (2019). Truncating mutations in UBAP1 cause hereditary spastic paraplegia. Am. J. Hum. Genet. 104, 767-773. 10.1016/j.ajhg.2019.03.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
Futter, C. E., Pearse, A., Hewlett, L. J. and Hopkins, C. R. (1996). Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. 132, 1011-1023. 10.1083/jcb.132.6.1011 [DOI] [PMC free article] [PubMed] [Google Scholar]
Gingras, M.-C., Kharitidi, D., Chénard, V., Uetani, N., Bouchard, M., Tremblay, M. L. and Pause, A. (2009a). Expression analysis and essential role of the putative tyrosine phosphatase His-domain-containing protein tyrosine phosphatase (HD-PTP). Int. J. Dev. Biol. 53, 1069-1074. 10.1387/ijdb.082820mg [DOI] [PubMed] [Google Scholar]
Gingras, M.-C., Zhang, Y. L., Kharitidi, D., Barr, A. J., Knapp, S., Tremblay, M. L. and Pause, A. (2009b). HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain. PLoS ONE 4, e5105. 10.1371/journal.pone.0005105 [DOI] [PMC free article] [PubMed] [Google Scholar]
Gingras, M.-C., Kazan, J. M. and Pause, A. (2017). Role of ESCRT component HD-PTP/PTPN23 in cancer. Biochem. Soc. Trans. 45, 845-854. 10.1042/BST20160332 [DOI] [PubMed] [Google Scholar]
Hamada, M., Haeger, A., Jeganathan, K. B., Van Ree, J. H., Malureanu, L., Wälde, S., Joseph, J., Kehlenbach, R. H. and Van Deursen, J. M. (2011). Ran-dependent docking of importin-beta to RanBP2/Nup358 filaments is essential for protein import and cell viability. J. Cell Biol. 194, 597-612. 10.1083/jcb.201102018 [DOI] [PMC free article] [PubMed] [Google Scholar]
Hofman, E. G., Bader, A. N., Gerritsen, H. C. and Van Bergen En Henegouwen, P. M. P. (2009). EGF induces rapid reorganization of plasma membrane microdomains. Commun. Integr. Biol. 2, 213-214. 10.4161/cib.2.3.7877 [DOI] [PMC free article] [PubMed] [Google Scholar]
Höglinger, D., Burgoyne, T., Sanchez-Heras, E., Hartwig, P., Colaco, A., Newton, J., Futter, C. E., Spiegel, S., Platt, F. M. and Eden, E. R. (2019). NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress. Nat. Commun. 10, 4276. 10.1038/s41467-019-12152-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
Huang, H.-R., Chen, Z. J., Kunes, S., Chang, G.-D. and Maniatis, T. (2010). Endocytic pathway is required for Drosophila Toll innate immune signaling. Proc. Natl. Acad. Sci. USA 107, 8322-8327. 10.1073/pnas.1004031107 [DOI] [PMC free article] [PubMed] [Google Scholar]
Infante, R. E., Wang, M. L., Radhakrishnan, A., Kwon, H. J., Brown, M. S. and Goldstein, J. L. (2008). NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc. Natl. Acad. Sci. USA 105, 15287-15292. 10.1073/pnas.0807328105 [DOI] [PMC free article] [PubMed] [Google Scholar]
Irwin, M. E., Mueller, K. L., Bohin, N., Ge, Y. and Boerner, J. L. (2011). Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. J. Cell. Physiol. 226, 2316-2328. 10.1002/jcp.22570 [DOI] [PMC free article] [PubMed] [Google Scholar]
Katoh, K., Shibata, H., Suzuki, H., Nara, A., Ishidoh, K., Kominami, E., Yoshimori, T. and Maki, M. (2003). The ALG-2-interacting protein alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. J. Biol. Chem. 278, 39104-39113. 10.1074/jbc.M301604200 [DOI] [PubMed] [Google Scholar]
Katzmann, D. J., Babst, M. and Emr, S. D. (2001). Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145-155. 10.1016/S0092-8674(01)00434-2 [DOI] [PubMed] [Google Scholar]
Katzmann, D. J., Stefan, C. J., Babst, M. and Emr, S. D. (2003). Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 162, 413-423. 10.1083/jcb.200302136 [DOI] [PMC free article] [PubMed] [Google Scholar]
Khalaf-Nazzal, R., Fasham, J., Ubeyratna, N., Evans, D. J., Leslie, J. S., Warner, T. T., Al-Hijawi, F., Alshaer, S., Baker, W., Turnpenny, P. D.et al. (2021). Final Exon Frameshift Biallelic PTPN23 variants are associated with microcephalic complex hereditary spastic paraplegia. Brain Sci. 11, 614. 10.3390/brainsci11050614 [DOI] [PMC free article] [PubMed] [Google Scholar]
Komada, M. and Soriano, P. (1999). Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis. Genes Dev. 13, 1475-1485. 10.1101/gad.13.11.1475 [DOI] [PMC free article] [PubMed] [Google Scholar]
Koselny, K., Mutlu, N., Minard, A. Y., Kumar, A., Krysan, D. J. and Wellington, M. (2018). A genome-wide screen of deletion mutants in the filamentous Saccharomyces cerevisiae background identifies ergosterol as a direct trigger of macrophage pyroptosis. mBio 9, e01204-18. 10.1128/mBio.01204-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
Kwon, H. J., Abi-Mosleh, L., Wang, M. L., Deisenhofer, J., Goldstein, J. L., Brown, M. S. and Infante, R. E. (2009). Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 137, 1213-1224. 10.1016/j.cell.2009.03.049 [DOI] [PMC free article] [PubMed] [Google Scholar]
Larios, J., Mercier, V., Roux, A. and Gruenberg, J. (2020). ALIX- and ESCRT-III–dependent sorting of tetraspanins to exosomes. J. Cell Biol. 219, e201904113. 10.1083/jcb.201904113 [DOI] [PMC free article] [PubMed] [Google Scholar]
Lauer, J., Segeletz, S., Cezanne, A., Guaitoli, G., Raimondi, F., Gentzel, M., Alva, V., Habeck, M., Kalaidzidis, Y., Ueffing, M.et al. (2019). Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding. eLife 8, e46302. 10.7554/eLife.46302 [DOI] [PMC free article] [PubMed] [Google Scholar]
Legent, K., Liu, H. H. and Treisman, J. E. (2015). Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation. Development 142, 1480-1491. 10.1242/dev.117960 [DOI] [PMC free article] [PubMed] [Google Scholar]
Li, Z., Schulze, R. J., Weller, S. G., Krueger, E. W., Schott, M. B., Zhang, X., Casey, C. A., Liu, J., Stöckli, J., James, D. E.et al. (2016). A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets. Sci. Adv. 2, e1601470. 10.1126/sciadv.1601470 [DOI] [PMC free article] [PubMed] [Google Scholar]
Lim, C.-Y., Davis, O. B., Shin, H. R., Zhang, J., Berdan, C. A., Jiang, X., Counihan, J. L., Ory, D. S., Nomura, D. K. and Zoncu, R. (2019). ER–lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann–Pick type C. Nat. Cell Biol. 21, 1206-1218. 10.1038/s41556-019-0391-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
Limzerwala, J. F., Jeganathan, K. B., Kloeber, J. A., Davies, B. A., Zhang, C., Sturmlechner, I., Zhong, J., Fierro Velasco, R., Fields, A. P., Yuan, Y.et al. (2020). FoxM1 insufficiency hyperactivates Ect2-RhoA-mDia1 signaling to drive cancer. Nat. Cancer 1, 1010-1024. 10.1038/s43018-020-00116-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
Lin, X., Su, H.-Z., Dong, E.-L., Lin, X.-H., Zhao, M., Yang, C., Wang, C., Wang, J., Chen, Y.-J., Yu, H.et al. (2019). Stop-gain mutations in UBAP1 cause pure autosomal-dominant spastic paraplegia. Brain 142, 2238-2252. 10.1093/brain/awz158 [DOI] [PubMed] [Google Scholar]
Lloyd, T. E., Atkinson, R., Wu, M. N., Zhou, Y., Pennetta, G. and Bellen, H. J. (2002). Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila. Cell 108, 261-269. 10.1016/S0092-8674(02)00611-6 [DOI] [PubMed] [Google Scholar]
Lord, S. J., Velle, K. B., Mullins, R. D. and Fritz-Laylin, L. K. (2020). SuperPlots: communicating reproducibility and variability in cell biology. J. Cell Biol. 219, e202001064. 10.1083/jcb.202001064 [DOI] [PMC free article] [PubMed] [Google Scholar]
Lunati, A., Petit, A., Lapillonne, H., Gameiro, C., Saillour, V., Garel, C., Doummar, D., Qebibo, L., Aissat, A., Fanen, P.et al. (2021). VPS4A mutation in syndromic congenital hemolytic anemia without obvious signs of dyserythropoiesis. Am. J. Hematol. 96, E121-e123. 10.1002/ajh.26099 [DOI] [PubMed] [Google Scholar]
MacDonald, J. L. and Pike, L. J. (2005). A simplified method for the preparation of detergent-free lipid rafts. J. Lipid Res. 46, 1061-1067. 10.1194/jlr.D400041-JLR200 [DOI] [PubMed] [Google Scholar]
Maekawa, M. and Fairn, G. D. (2015). Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol. J. Cell Sci. 128, 1422-1433. 10.1242/jcs.164715 [DOI] [PubMed] [Google Scholar]
Manteghi, S., Gingras, M.-C., Kharitidi, D., Galarneau, L., Marques, M., Yan, M., Cencic, R., Robert, F., Paquet, M., Witcher, M.et al. (2016). Haploinsufficiency of the ESCRT component HD-PTP predisposes to cancer. Cell Rep. 15, 1893-1900. 10.1016/j.celrep.2016.04.076 [DOI] [PubMed] [Google Scholar]
Mattera, R. and Bonifacino, J. S. (2008). Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes. EMBO J. 27, 2484-2494. 10.1038/emboj.2008.177 [DOI] [PMC free article] [PubMed] [Google Scholar]
Maxfield, F. R. and Wüstner, D. (2012). Analysis of cholesterol trafficking with fluorescent probes. Methods Cell Biol. 108, 367-393. 10.1016/B978-0-12-386487-1.00017-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
Miura, G. I., Roignant, J.-Y., Wassef, M. and Treisman, J. E. (2008). Myopic acts in the endocytic pathway to enhance signling by the Drosophila EGF receptor. Development 135, 1913-1922. 10.1242/dev.017202 [DOI] [PMC free article] [PubMed] [Google Scholar]
Moberg, K. H., Schelble, S., Burdick, S. K. and Hariharan, I. K. (2005). Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev. Cell 9, 699-710. 10.1016/j.devcel.2005.09.018 [DOI] [PubMed] [Google Scholar]
Mochida, G. H., Ganesh, V. S., De Michelena, M. I., Dias, H., Atabay, K. D., Kathrein, K. L., Huang, H. T., Hill, R. S., Felie, J. M., Rakiec, D.et al. (2012). CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development. Nat. Genet. 44, 1260-1264. 10.1038/ng.2425 [DOI] [PMC free article] [PubMed] [Google Scholar]
Morita, E., Sandrin, V., Chung, H.-Y., Morham, S. G., Gygi, S. P., Rodesch, C. K. and Sundquist, W. I. (2007). Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. EMBO J. 26, 4215-4227. 10.1038/sj.emboj.7601850 [DOI] [PMC free article] [PubMed] [Google Scholar]
Mullock, B. M., Bright, N. A., Fearon, C. W., Gray, S. R. and Luzio, J. P. (1998). Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent. J. Cell Biol. 140, 591-601. 10.1083/jcb.140.3.591 [DOI] [PMC free article] [PubMed] [Google Scholar]
Orr, G., Hu, D., Özçelik, S., Opresko, L. K., Steven Wiley, H. and Colson, S. D. (2005). Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane. Biophys. J. 89, 1362-1373. 10.1529/biophysj.104.056192 [DOI] [PMC free article] [PubMed] [Google Scholar]
Owen, D. M., Rentero, C., Magenau, A., Abu-Siniyeh, A. and Gaus, K. (2012). Quantitative imaging of membrane lipid order in cells and organisms. Nat. Protoc. 7, 24-35. 10.1038/nprot.2011.419 [DOI] [PubMed] [Google Scholar]
Parkinson, N., Ince, P. G., Smith, M. O., Highley, R., Skibinski, G., Andersen, P. M., Morrison, K. E., Pall, H. S., Hardiman, O., Collinge, J.et al. (2006). ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B). Neurology 67, 1074-1077. 10.1212/01.wnl.0000231510.89311.8b [DOI] [PubMed] [Google Scholar]
Parkinson, G., Roboti, P., Zhang, L., Taylor, S. and Woodman, P. (2021). His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation. J. Cell Sci. 134, jcs259192. 10.1242/jcs.259192 [DOI] [PMC free article] [PubMed] [Google Scholar]
Parpal, S., Karlsson, M., Thorn, H. and Strålfors, P. (2001). Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control*. J. Biol. Chem. 276, 9670-9678. 10.1074/jbc.M007454200 [DOI] [PubMed] [Google Scholar]
Pashkova, N., Gakhar, L., Winistorfer, S. C., Sunshine, A. B., Rich, M., Dunham, M. J., Yu, L. and Piper, R. C. (2013). The Yeast Alix Homolog Bro1 functions as a ubiquitin receptor for protein sorting into multivesicular endosomes. Dev. Cell 25, 520-533. 10.1016/j.devcel.2013.04.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
Pentchev, P. G., Brady, R. O., Blanchette-Mackie, E. J., Vanier, M. T., Carstea, E. D., Parker, C. C., Goldin, E. and Roff, C. F. (1994). The Niemann-Pick C lesion and its relationship to the intracellular distribution and utilization of LDL cholesterol. Biochim. Biophys. Acta 1225, 235-243. 10.1016/0925-4439(94)90001-9 [DOI] [PubMed] [Google Scholar]
Phatarpekar, P. V., Overlee, B. L., Leehan, A., Wilton, K. M., Ham, H. and Billadeau, D. D. (2020). The septin cytoskeleton regulates natural killer cell lytic granule release. J. Cell Biol. 219, e202002145. 10.1083/jcb.202002145 [DOI] [PMC free article] [PubMed] [Google Scholar]
Poteryaev, D., Datta, S., Ackema, K., Zerial, M. and Spang, A. (2010). Identification of the switch in early-to-late endosome transition. Cell 141, 497-508. 10.1016/j.cell.2010.03.011 [DOI] [PubMed] [Google Scholar]
Puri, C., Tosoni, D., Comai, R., Rabellino, A., Segat, D., Caneva, F., Luzzi, P., Di Fiore, P. P. and Tacchetti, C. (2005). Relationships between EGFR signaling-competent and endocytosis-competent membrane microdomains. Mol. Biol. Cell 16, 2704-2718. 10.1091/mbc.e04-07-0596 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rink, J., Ghigo, E., Kalaidzidis, Y. and Zerial, M. (2005). Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735-749. 10.1016/j.cell.2005.06.043 [DOI] [PubMed] [Google Scholar]
Rodger, C., Flex, E., Allison, R. J., Sanchis-Juan, A., Hasenahuer, M. A., Cecchetti, S., French, C. E., Edgar, J. R., Carpentieri, G., Ciolfi, A.et al. (2020). De novo VPS4A mutations cause multisystem disease with abnormal neurodevelopment. Am. J. Hum. Genet. 107, 1129-1148. 10.1016/j.ajhg.2020.10.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
Russell, M. R. G., Shideler, T., Nickerson, D. P., West, M. and Odorizzi, G. (2012). Class E compartments form in response to ESCRT dysfunction in yeast due to hyperactivity of the Vps21 Rab GTPase. J. Cell Sci. 125, 5208-5220. 10.1242/jcs.111310 [DOI] [PMC free article] [PubMed] [Google Scholar]
Ryan, M. C., Stucky, M., Wakefield, C., Melott, J. M., Akbani, R., Weinstein, J. N. and Broom, B. M. (2019). Interactive clustered heat map builder: an easy web-based tool for creating sophisticated clustered heat maps. F1000Res 8, 1750. 10.12688/f1000research.20590.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
Sanson, K. R., Hanna, R. E., Hegde, M., Donovan, K. F., Strand, C., Sullender, M. E., Vaimberg, E. W., Goodale, A., Root, D. E., Piccioni, F.et al. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416. 10.1038/s41467-018-07901-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
Schenkman, J. B. and Cinti, D. L. (1978). Preparation of microsomes with calcium. Methods Enzymol. 52, 83-89. 10.1016/S0076-6879(78)52008-9 [DOI] [PubMed] [Google Scholar]
Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671-675. 10.1038/nmeth.2089 [DOI] [PMC free article] [PubMed] [Google Scholar]
Schulze, H. and Sandhoff, K. (2011). Lysosomal lipid storage diseases. Cold Spring Harb. Perspect. Biol. 3, a004804. 10.1101/cshperspect.a004804 [DOI] [PMC free article] [PubMed] [Google Scholar]
Seo, Y., Kim, T. Y., Won, D., Choi, J. R., Seo, G. H., Lee, S. T. and Han, J. (2022). PTPN23 neurodevelopmental disorder presenting with optic atrophy and spasmus nutans-like nystagmus. J. Neuroophthalmol. 43, e316-e318. 10.1097/WNO.0000000000001582 [DOI] [PubMed] [Google Scholar]
Seu, K. G., Trump, L. R., Emberesh, S., Lorsbach, R. B., Johnson, C., Meznarich, J., Underhill, H. R., Chou, S. T., Sakthivel, H., Nassar, N. N.et al. (2020). VPS4A mutations in humans cause syndromic congenital dyserythropoietic anemia due to cytokinesis and trafficking defects. Am. J. Hum. Genet. 107, 1149-1156. 10.1016/j.ajhg.2020.10.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
Shiels, A., Bennett, T. M., Knopf, H. L. S., Yamada, K., Yoshiura, K., Niikawa, N., Shim, S. and Hanson, P. I. (2007). CHMP4b, a novel gene for autosomal dominant cataracts linked to chromosome 20q. Am. J. Hum. Genet. 81, 596-606. 10.1086/519980 [DOI] [PMC free article] [PubMed] [Google Scholar]
Shih, S. C., Katzmann, D. J., Schnell, J. D., Sutanto, M., Emr, S. D. and Hicke, L. (2002). Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat. Cell Biol. 4, 389-393. 10.1038/ncb790 [DOI] [PubMed] [Google Scholar]
Shim, J.-H., Xiao, C., Hayden, M. S., Lee, K.-Y., Trombetta, E. S., Pypaert, M., Nara, A., Yoshimori, T., Wilm, B., Erdjument-Bromage, H.et al. (2006). CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis. J. Cell Biol. 172, 1045-1056. 10.1083/jcb.200509041 [DOI] [PMC free article] [PubMed] [Google Scholar]
Sieben, C. J., Jeganathan, K. B., Nelson, G. G., Sturmlechner, I., Zhang, C., Van Deursen, W. H., Bakker, B., Foijer, F., Li, H., Baker, D. J.et al. (2020). BubR1 allelic effects drive phenotypic heterogeneity in mosaic-variegated aneuploidy progeria syndrome. J. Clin. Invest. 130, 171-188. 10.1172/JCI126863 [DOI] [PMC free article] [PubMed] [Google Scholar]
Simons, K. and Gruenberg, J. (2000). Jamming the endosomal system: lipid rafts and lysosomal storage diseases. Trends Cell Biol. 10, 459-462. 10.1016/S0962-8924(00)01847-X [DOI] [PubMed] [Google Scholar]
Skibinski, G., Parkinson, N. J., Brown, J. M., Chakrabarti, L., Lloyd, S. L., Hummerich, H., Nielsen, J. E., Hodges, J. R., Spillantini, M. G., Thusgaard, T.et al. (2005). Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat. Genet. 37, 806-808. 10.1038/ng1609 [DOI] [PubMed] [Google Scholar]
Smigiel, R., Landsberg, G., Schilling, M., Rydzanicz, M., Pollak, A., Walczak, A., Stodolak, A., Stawinski, P., Mierzewska, H., Sasiadek, M. M.et al. (2018). Developmental epileptic encephalopathy with hypomyelination and brain atrophy associated with PTPN23 variants affecting the assembly of UsnRNPs. Eur. J. Hum. Genet. 26, 1502-1511. 10.1038/s41431-018-0179-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
Sowada, N., Hashem, M. O., Yilmaz, R., Hamad, M., Kakar, N., Thiele, H., Arold, S. T., Bode, H., Alkuraya, F. S. and Borck, G. (2017). Mutations of PTPN23 in developmental and epileptic encephalopathy. Hum. Genet. 136, 1455-1461. 10.1007/s00439-017-1850-3 [DOI] [PubMed] [Google Scholar]
Tabernero, L. and Woodman, P. (2018). Dissecting the role of His domain protein tyrosine phosphatase/PTPN23 and ESCRTs in sorting activated epidermal growth factor receptor to the multivesicular body. Biochem. Soc. Trans. 46, 1037-1046. 10.1042/BST20170443 [DOI] [PMC free article] [PubMed] [Google Scholar]
Thompson, B. J., Mathieu, J., Sung, H.-H., Loeser, E., Rørth, P. and Cohen, S. M. (2005). Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Dev. Cell 9, 711-720. 10.1016/j.devcel.2005.09.020 [DOI] [PubMed] [Google Scholar]
Toyooka, S.-I., Ouchida, M., Jitsumori, Y., Tsukuda, K., Sakai, A., Nakamura, A., Shimizu, N. and Shimizu, K. (2000). HD-PTP: A Novel Protein Tyrosine Phosphatase Gene on Human Chromosome 3p21.3. Biochem. Biophys. Res. Commun. 278, 671-678. 10.1006/bbrc.2000.3870 [DOI] [PubMed] [Google Scholar]
Vaccari, T. and Bilder, D. (2005). The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking. Dev. Cell 9, 687-698. 10.1016/j.devcel.2005.09.019 [DOI] [PubMed] [Google Scholar]
Van Den Boomen, D. J. H., Sienkiewicz, A., Berlin, I., Jongsma, M. L. M., Van Elsland, D. M., Luzio, J. P., Neefjes, J. J. C. and Lehner, P. J. (2020). A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export. Nat. Commun. 11, 5559. Available: http://europepmc.org/abstract/MED/33144569, https://www.nature.com/articles/s41467-020-19032-0.pdf, 10.1038/s41467-020-19032-0, https://europepmc.org/articles/PMC7642327, https://europepmc.org/articles/PMC7642327?pdf=render [Accessed 2020/11//]. 10.1038/s41467-020-19032-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
Van Der Zee, J., Urwin, H., Engelborghs, S., Bruyland, M., Vandenberghe, R., Dermaut, B., De Pooter, T., Peeters, K., Santens, P., De Deyn, P. P.et al. (2008). CHMP2B C-truncating mutations in frontotemporal lobar degeneration are associated with an aberrant endosomal phenotype in vitro. Hum. Mol. Genet. 17, 313-322. 10.1093/hmg/ddm309 [DOI] [PubMed] [Google Scholar]
Waddington, K. E., Pineda-Torra, I. and Jury, E. C. (2019). Analyzing T-cell plasma membrane lipids by flow cytometry. Methods Mol. Biol. 1951, 209-216. 10.1007/978-1-4939-9130-3_16 [DOI] [PubMed] [Google Scholar]
Wagner, K.-U., Krempler, A., Qi, Y., Park, K., Henry, M. L. D., Triplett, A. A., Riedlinger, G., Rucker, E. B. and Hennighausen, L. (2003). Tsg101 is essential for cell growth, proliferation, and cell survival of embryonic and adult tissues. Mol. Cell. Biol. 23, 150-162. 10.1128/MCB.23.1.150-162.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
Wen, T., Barham, W., Li, Y., Zhang, H., Gicobi, J. K., Hirdler, J. B., Liu, X., Ham, H., Peterson Martinez, K. E., Lucien, F.et al. (2022). NKG7 Is a T-cell-intrinsic therapeutic target for improving antitumor cytotoxicity and cancer immunotherapy. Cancer Immunol. Res. 10, 162-181. 10.1158/2326-6066.CIR-21-0539 [DOI] [PMC free article] [PubMed] [Google Scholar]
Wollert, T. and Hurley, J. H. (2010). Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864-869. 10.1038/nature08849 [DOI] [PMC free article] [PubMed] [Google Scholar]
Wurmser, A. E. and Emr, S. D. (1998). Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities. EMBO J. 17, 4930-4942. 10.1093/emboj/17.17.4930 [DOI] [PMC free article] [PubMed] [Google Scholar]
Xu, M., Tchkonia, T., Ding, H., Ogrodnik, M., Lubbers, E. R., Pirtskhalava, T., White, T. A., Johnson, K. O., Stout, M. B., Mezera, V.et al. (2015). JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age. Proc. Natl. Acad. Sci. USA 112, E6301-E6310. 10.1073/pnas.1515386112 [DOI] [PMC free article] [PubMed] [Google Scholar]
Yamada, K., Tomita, H.-A., Kanazawa, S., Mera, A., Amemiya, T. and Niikawa, N. (2000). Genetically distinct autosomal dominant posterior polar cataract in a four-generation Japanese family. Am. J. Ophthalmol. 129, 159-165. 10.1016/S0002-9394(99)00313-X [DOI] [PubMed] [Google Scholar]
Yun, H., Jung, M., Lee, H., Jung, S., Kim, T., Kim, N., Park, S. Y., Kim, W. J., Mun, J. Y. and Yoo, J. Y. (2023). Homotypic SCOTIN assemblies form ER-endosome membrane contacts and regulate endosome dynamics. EMBO Rep. 24, e56538. 10.15252/embr.202256538 [DOI] [PMC free article] [PubMed] [Google Scholar]
Zhang, S., Fan, G., Hao, Y., Hammell, M., Wilkinson, J. E. and Tonks, N. K. (2017). Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase. Genes Dev. 31, 1939-1957. 10.1101/gad.304261.117 [DOI] [PMC free article] [PubMed] [Google Scholar]
Zhang, J., Sun, Y., Zhong, L.-Y., Yu, N.-N., Ouyang, L., Fang, R.-D., Wang, Y. and He, Q.-Y. (2020). Structure-based discovery of neoandrographolide as a novel inhibitor of Rab5 to suppress cancer growth. Comput. Struct. Biotechnol. J. 18, 3936-3946. 10.1016/j.csbj.2020.11.033 [DOI] [PMC free article] [PubMed] [Google Scholar]
Zivony-Elboum, Y., Westbroek, W., Kfir, N., Savitzki, D., Shoval, Y., Bloom, A., Rod, R., Khayat, M., Gross, B., Samri, W.et al. (2012). A founder mutation in Vps37A causes autosomal recessive complex hereditary spastic paraparesis. J. Med. Genet. 49, 462-472. 10.1136/jmedgenet-2012-100742 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

joces-137-262032_review_history.pdf^{(297.5KB, pdf)}

Supplementary information

joces-137-262032-s1.pdf^{(65.7MB, pdf)}

DOI: 10.1242/joces.262032_sup1

Table S2. Raw RPPA data.

joces-137-262032-TableS2.xlsx^{(203.1KB, xlsx)}

[JCS262032C1] Ahmed, S. N., Brown, D. A. and London, E. (1997). On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36, 10944-10953. 10.1021/bi971167g [DOI] [PubMed] [Google Scholar]

[JCS262032C2] Ali, N., Zhang, L., Taylor, S., Mironov, A., Urbé, S. and Woodman, P. (2013). Recruitment of UBPY and ESCRT exchange drive HD-PTP-dependent sorting of EGFR to the MVB. Curr. Biol. 23, 453-461. 10.1016/j.cub.2013.02.033 [DOI] [PubMed] [Google Scholar]

[JCS262032C3] Atakpa, P., Thillaiappan, N. B., Mataragka, S., Prole, D. L. and Taylor, C. W. (2018). IP(3) receptors preferentially associate with ER-lysosome contact sites and selectively deliver Ca(2+) to lysosomes. Cell Rep. 25, 3180-3193.e7. 10.1016/j.celrep.2018.11.064 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C4] Aziz, K., Limzerwala, J. F., Sturmlechner, I., Hurley, E., Zhang, C., Jeganathan, K. B., Nelson, G., Bronk, S., Fierro Velasco, R. O., Van Deursen, E.-J.et al. (2019). Ccne1 overexpression causes chromosome instability in liver cells and liver tumor development in mice. Gastroenterology 157, 210-226.e12. 10.1053/j.gastro.2019.03.016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C5] Babst, M., Odorizzi, G., Estepa, E. J. and Emr, S. D. (2000). Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic 1, 248-258. 10.1034/j.1600-0854.2000.010307.x [DOI] [PubMed] [Google Scholar]

[JCS262032C6] Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T. and Emr, S. D. (2002a). Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3, 271-282. 10.1016/S1534-5807(02)00220-4 [DOI] [PubMed] [Google Scholar]

[JCS262032C7] Babst, M., Katzmann, D. J., Snyder, W. B., Wendland, B. and Emr, S. D. (2002b). Endosome-associated complex, ESCRT-Ii, recruits transport machinery for protein sorting at the multivesicular body. Dev. Cell 3, 283-289. 10.1016/S1534-5807(02)00219-8 [DOI] [PubMed] [Google Scholar]

[JCS262032C8] Baietti, M. F., Zhang, Z., Mortier, E., Melchior, A., Degeest, G., Geeraerts, A., Ivarsson, Y., Depoortere, F., Coomans, C., Vermeiren, E.et al. (2012). Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat. Cell Biol. 14, 677-685. 10.1038/ncb2502 [DOI] [PubMed] [Google Scholar]

[JCS262032C9] Baker, D. J., Jeganathan, K. B., Cameron, J. D., Thompson, M., Juneja, S., Kopecka, A., Kumar, R., Jenkins, R. B., De Groen, P. C., Roche, P.et al. (2004). BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat. Genet. 36, 744-749. 10.1038/ng1382 [DOI] [PubMed] [Google Scholar]

[JCS262032C10] Bend, R., Cohen, L., Carter, M. T., Lyons, M. J., Niyazov, D., Mikati, M. A., Rojas, S. K., Person, R. E., Si, Y., Wentzensen, I. M.et al. (2020). Phenotype and mutation expansion of the PTPN23 associated disorder characterized by neurodevelopmental delay and structural brain abnormalities. Eur. J. Hum. Genet. 28, 76-87. 10.1038/s41431-019-0487-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C11] Bishop, N. and Woodman, P. (2000). ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking. Mol. Biol. Cell 11, 227-239. 10.1091/mbc.11.1.227 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C12] Blüher, M., Michael, M. D., Peroni, O. D., Ueki, K., Carter, N., Kahn, B. B. and Kahn, C. R. (2002). Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev. Cell 3, 25-38. 10.1016/S1534-5807(02)00199-5 [DOI] [PubMed] [Google Scholar]

[JCS262032C113] Bolte, S. and Cordelières, F. P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213-232. 10.1111/j.1365-2818.2006.01706.x [DOI] [PubMed] [Google Scholar]

[JCS262032C13] Brown, D. A. and Rose, J. K. (1992). Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533-544. 10.1016/0092-8674(92)90189-J [DOI] [PubMed] [Google Scholar]

[JCS262032C14] Byers, D. M., Rastogi, S. R., Cook, H. W., Palmer, F. B. S. C. and Spence, M. W. (1989). Defective activity of acyl-CoA:cholesterol O-acyltransferase in Niemann-Pick type C and type D fibroblasts. Biochem. J. 262, 713-719. 10.1042/bj2620713 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C15] Carlton, J. G., Agromayor, M. and Martin-Serrano, J. (2008). Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release. Proc. Natl Acad. Sci. USA 105, 10541. 10.1073/pnas.0802008105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C16] Chang, T. Y., Chang, C. C. Y. and Cheng, D. (1997). Acyl-coenzyme a: cholesterol acyltransferase. Annu. Rev. Biochem. 66, 613-638. 10.1146/annurev.biochem.66.1.613 [DOI] [PubMed] [Google Scholar]

[JCS262032C17] Christ, L., Wenzel, E. M., Liestøl, K., Raiborg, C., Campsteijn, C. and Stenmark, H. (2016). ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters in cytokinetic abscission. J. Cell Biol. 212, 499-513. 10.1083/jcb.201507009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C18] Christ, L., Raiborg, C., Wenzel, E. M., Campsteijn, C. and Stenmark, H. (2017). Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem. Sci. 42, 42-56. 10.1016/j.tibs.2016.08.016 [DOI] [PubMed] [Google Scholar]

[JCS262032C19] Cianciola, N. L., Greene, D. J., Morton, R. E. and Carlin, C. R. (2013). Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1. Mol. Biol. Cell 24, 3309-3325. 10.1091/mbc.e12-10-0760 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C20] Cox, L. E., Ferraiuolo, L., Goodall, E. F., Heath, P. R., Higginbottom, A., Mortiboys, H., Hollinger, H. C., Hartley, J. A., Brockington, A., Burness, C. E.et al. (2010). Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS). PLoS ONE 5, e9872. 10.1371/journal.pone.0009872 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C21] Davies, M. A., Stemke-Hale, K., Lin, E., Tellez, C., Deng, W., Gopal, Y. N., Woodman, S. E., Calderone, T. C., Ju, Z., Lazar, A. J.et al. (2009). Integrated molecular and clinical analysis of AKT activation in metastatic melanoma. Clin. Cancer Res. 15, 7538-7546. 10.1158/1078-0432.CCR-09-1985 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C22] Dinchuk, J. E., Cao, C., Huang, F., Reeves, K. A., Wang, J., Myers, F., Cantor, G. H., Zhou, X., Attar, R. M., Gottardis, M.et al. (2010). Insulin receptor (IR) pathway hyperactivity in IGF-IR null cells and suppression of downstream growth signaling using the dual IGF-IR/IR inhibitor, BMS-754807. Endocrinology 151, 4123-4132. 10.1210/en.2010-0032 [DOI] [PubMed] [Google Scholar]

[JCS262032C23] Ding, L., Liou, G. Y., Schmitt, D. M., Storz, P., Zhang, J. S. and Billadeau, D. D. (2017). Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia. J. Pathol. 243, 65-77. 10.1002/path.4928 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C24] Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R.et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184-191. 10.1038/nbt.3437 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C25] Dowlatshahi, D. P., Sandrin, V., Vivona, S., Shaler, T. A., Kaiser, S. E., Melandri, F., Sundquist, W. I. and Kopito, R. R. (2012). ALIX is a Lys63-specific polyubiquitin binding protein that functions in retrovirus budding. Dev. Cell 23, 1247-1254. 10.1016/j.devcel.2012.10.023 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C26] Doyotte, A., Mironov, A., Mckenzie, E. and Woodman, P. (2008). The Bro1-related protein HD-PTP/PTPN23 is required for endosomal cargo sorting and multivesicular body morphogenesis. Proc. Natl. Acad. Sci. USA 105, 6308-6313. 10.1073/pnas.0707601105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C27] Du, X., Kumar, J., Ferguson, C., Schulz, T. A., Ong, Y. S., Hong, W., Prinz, W. A., Parton, R. G., Brown, A. J. and Yang, H. (2011). A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking. J. Cell Biol. 192, 121-135. 10.1083/jcb.201004142 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C28] Du, X., Kazim, A. S., Brown, A. J. and Yang, H. (2012). An essential role of Hrs/Vps27 in endosomal cholesterol trafficking. Cell Rep. 1, 29-35. 10.1016/j.celrep.2011.10.004 [DOI] [PubMed] [Google Scholar]

[JCS262032C29] Du, X., Kazim, A. S., Dawes, I. W., Brown, A. J. and Yang, H. (2013). The AAA ATPase VPS4/SKD1 regulates endosomal cholesterol trafficking independently of ESCRT-III. Traffic 14, 107-119. 10.1111/tra.12015 [DOI] [PubMed] [Google Scholar]

[JCS262032C30] Farazi Fard, M. A., Rebelo, A. P., Buglo, E., Nemati, H., Dastsooz, H., Gehweiler, I., Reich, S., Reichbauer, J., Quintáns, B., Ordóñez-Ugalde, A.et al. (2019). Truncating mutations in UBAP1 cause hereditary spastic paraplegia. Am. J. Hum. Genet. 104, 767-773. 10.1016/j.ajhg.2019.03.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C31] Futter, C. E., Pearse, A., Hewlett, L. J. and Hopkins, C. R. (1996). Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. 132, 1011-1023. 10.1083/jcb.132.6.1011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C32] Gingras, M.-C., Kharitidi, D., Chénard, V., Uetani, N., Bouchard, M., Tremblay, M. L. and Pause, A. (2009a). Expression analysis and essential role of the putative tyrosine phosphatase His-domain-containing protein tyrosine phosphatase (HD-PTP). Int. J. Dev. Biol. 53, 1069-1074. 10.1387/ijdb.082820mg [DOI] [PubMed] [Google Scholar]

[JCS262032C33] Gingras, M.-C., Zhang, Y. L., Kharitidi, D., Barr, A. J., Knapp, S., Tremblay, M. L. and Pause, A. (2009b). HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain. PLoS ONE 4, e5105. 10.1371/journal.pone.0005105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C34] Gingras, M.-C., Kazan, J. M. and Pause, A. (2017). Role of ESCRT component HD-PTP/PTPN23 in cancer. Biochem. Soc. Trans. 45, 845-854. 10.1042/BST20160332 [DOI] [PubMed] [Google Scholar]

[JCS262032C35] Hamada, M., Haeger, A., Jeganathan, K. B., Van Ree, J. H., Malureanu, L., Wälde, S., Joseph, J., Kehlenbach, R. H. and Van Deursen, J. M. (2011). Ran-dependent docking of importin-beta to RanBP2/Nup358 filaments is essential for protein import and cell viability. J. Cell Biol. 194, 597-612. 10.1083/jcb.201102018 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C36] Hofman, E. G., Bader, A. N., Gerritsen, H. C. and Van Bergen En Henegouwen, P. M. P. (2009). EGF induces rapid reorganization of plasma membrane microdomains. Commun. Integr. Biol. 2, 213-214. 10.4161/cib.2.3.7877 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C37] Höglinger, D., Burgoyne, T., Sanchez-Heras, E., Hartwig, P., Colaco, A., Newton, J., Futter, C. E., Spiegel, S., Platt, F. M. and Eden, E. R. (2019). NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress. Nat. Commun. 10, 4276. 10.1038/s41467-019-12152-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C38] Huang, H.-R., Chen, Z. J., Kunes, S., Chang, G.-D. and Maniatis, T. (2010). Endocytic pathway is required for Drosophila Toll innate immune signaling. Proc. Natl. Acad. Sci. USA 107, 8322-8327. 10.1073/pnas.1004031107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C39] Infante, R. E., Wang, M. L., Radhakrishnan, A., Kwon, H. J., Brown, M. S. and Goldstein, J. L. (2008). NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc. Natl. Acad. Sci. USA 105, 15287-15292. 10.1073/pnas.0807328105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C40] Irwin, M. E., Mueller, K. L., Bohin, N., Ge, Y. and Boerner, J. L. (2011). Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. J. Cell. Physiol. 226, 2316-2328. 10.1002/jcp.22570 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C41] Katoh, K., Shibata, H., Suzuki, H., Nara, A., Ishidoh, K., Kominami, E., Yoshimori, T. and Maki, M. (2003). The ALG-2-interacting protein alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. J. Biol. Chem. 278, 39104-39113. 10.1074/jbc.M301604200 [DOI] [PubMed] [Google Scholar]

[JCS262032C42] Katzmann, D. J., Babst, M. and Emr, S. D. (2001). Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145-155. 10.1016/S0092-8674(01)00434-2 [DOI] [PubMed] [Google Scholar]

[JCS262032C43] Katzmann, D. J., Stefan, C. J., Babst, M. and Emr, S. D. (2003). Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 162, 413-423. 10.1083/jcb.200302136 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C44] Khalaf-Nazzal, R., Fasham, J., Ubeyratna, N., Evans, D. J., Leslie, J. S., Warner, T. T., Al-Hijawi, F., Alshaer, S., Baker, W., Turnpenny, P. D.et al. (2021). Final Exon Frameshift Biallelic PTPN23 variants are associated with microcephalic complex hereditary spastic paraplegia. Brain Sci. 11, 614. 10.3390/brainsci11050614 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C45] Komada, M. and Soriano, P. (1999). Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis. Genes Dev. 13, 1475-1485. 10.1101/gad.13.11.1475 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C46] Koselny, K., Mutlu, N., Minard, A. Y., Kumar, A., Krysan, D. J. and Wellington, M. (2018). A genome-wide screen of deletion mutants in the filamentous Saccharomyces cerevisiae background identifies ergosterol as a direct trigger of macrophage pyroptosis. mBio 9, e01204-18. 10.1128/mBio.01204-18 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C47] Kwon, H. J., Abi-Mosleh, L., Wang, M. L., Deisenhofer, J., Goldstein, J. L., Brown, M. S. and Infante, R. E. (2009). Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 137, 1213-1224. 10.1016/j.cell.2009.03.049 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C48] Larios, J., Mercier, V., Roux, A. and Gruenberg, J. (2020). ALIX- and ESCRT-III–dependent sorting of tetraspanins to exosomes. J. Cell Biol. 219, e201904113. 10.1083/jcb.201904113 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C49] Lauer, J., Segeletz, S., Cezanne, A., Guaitoli, G., Raimondi, F., Gentzel, M., Alva, V., Habeck, M., Kalaidzidis, Y., Ueffing, M.et al. (2019). Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding. eLife 8, e46302. 10.7554/eLife.46302 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C50] Legent, K., Liu, H. H. and Treisman, J. E. (2015). Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation. Development 142, 1480-1491. 10.1242/dev.117960 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C51] Li, Z., Schulze, R. J., Weller, S. G., Krueger, E. W., Schott, M. B., Zhang, X., Casey, C. A., Liu, J., Stöckli, J., James, D. E.et al. (2016). A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets. Sci. Adv. 2, e1601470. 10.1126/sciadv.1601470 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C52] Lim, C.-Y., Davis, O. B., Shin, H. R., Zhang, J., Berdan, C. A., Jiang, X., Counihan, J. L., Ory, D. S., Nomura, D. K. and Zoncu, R. (2019). ER–lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann–Pick type C. Nat. Cell Biol. 21, 1206-1218. 10.1038/s41556-019-0391-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C53] Limzerwala, J. F., Jeganathan, K. B., Kloeber, J. A., Davies, B. A., Zhang, C., Sturmlechner, I., Zhong, J., Fierro Velasco, R., Fields, A. P., Yuan, Y.et al. (2020). FoxM1 insufficiency hyperactivates Ect2-RhoA-mDia1 signaling to drive cancer. Nat. Cancer 1, 1010-1024. 10.1038/s43018-020-00116-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C54] Lin, X., Su, H.-Z., Dong, E.-L., Lin, X.-H., Zhao, M., Yang, C., Wang, C., Wang, J., Chen, Y.-J., Yu, H.et al. (2019). Stop-gain mutations in UBAP1 cause pure autosomal-dominant spastic paraplegia. Brain 142, 2238-2252. 10.1093/brain/awz158 [DOI] [PubMed] [Google Scholar]

[JCS262032C55] Lloyd, T. E., Atkinson, R., Wu, M. N., Zhou, Y., Pennetta, G. and Bellen, H. J. (2002). Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila. Cell 108, 261-269. 10.1016/S0092-8674(02)00611-6 [DOI] [PubMed] [Google Scholar]

[JCS262032C56] Lord, S. J., Velle, K. B., Mullins, R. D. and Fritz-Laylin, L. K. (2020). SuperPlots: communicating reproducibility and variability in cell biology. J. Cell Biol. 219, e202001064. 10.1083/jcb.202001064 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C57] Lunati, A., Petit, A., Lapillonne, H., Gameiro, C., Saillour, V., Garel, C., Doummar, D., Qebibo, L., Aissat, A., Fanen, P.et al. (2021). VPS4A mutation in syndromic congenital hemolytic anemia without obvious signs of dyserythropoiesis. Am. J. Hematol. 96, E121-e123. 10.1002/ajh.26099 [DOI] [PubMed] [Google Scholar]

[JCS262032C58] MacDonald, J. L. and Pike, L. J. (2005). A simplified method for the preparation of detergent-free lipid rafts. J. Lipid Res. 46, 1061-1067. 10.1194/jlr.D400041-JLR200 [DOI] [PubMed] [Google Scholar]

[JCS262032C59] Maekawa, M. and Fairn, G. D. (2015). Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol. J. Cell Sci. 128, 1422-1433. 10.1242/jcs.164715 [DOI] [PubMed] [Google Scholar]

[JCS262032C60] Manteghi, S., Gingras, M.-C., Kharitidi, D., Galarneau, L., Marques, M., Yan, M., Cencic, R., Robert, F., Paquet, M., Witcher, M.et al. (2016). Haploinsufficiency of the ESCRT component HD-PTP predisposes to cancer. Cell Rep. 15, 1893-1900. 10.1016/j.celrep.2016.04.076 [DOI] [PubMed] [Google Scholar]

[JCS262032C61] Mattera, R. and Bonifacino, J. S. (2008). Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes. EMBO J. 27, 2484-2494. 10.1038/emboj.2008.177 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C62] Maxfield, F. R. and Wüstner, D. (2012). Analysis of cholesterol trafficking with fluorescent probes. Methods Cell Biol. 108, 367-393. 10.1016/B978-0-12-386487-1.00017-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C63] Miura, G. I., Roignant, J.-Y., Wassef, M. and Treisman, J. E. (2008). Myopic acts in the endocytic pathway to enhance signling by the Drosophila EGF receptor. Development 135, 1913-1922. 10.1242/dev.017202 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C64] Moberg, K. H., Schelble, S., Burdick, S. K. and Hariharan, I. K. (2005). Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev. Cell 9, 699-710. 10.1016/j.devcel.2005.09.018 [DOI] [PubMed] [Google Scholar]

[JCS262032C65] Mochida, G. H., Ganesh, V. S., De Michelena, M. I., Dias, H., Atabay, K. D., Kathrein, K. L., Huang, H. T., Hill, R. S., Felie, J. M., Rakiec, D.et al. (2012). CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development. Nat. Genet. 44, 1260-1264. 10.1038/ng.2425 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C66] Morita, E., Sandrin, V., Chung, H.-Y., Morham, S. G., Gygi, S. P., Rodesch, C. K. and Sundquist, W. I. (2007). Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. EMBO J. 26, 4215-4227. 10.1038/sj.emboj.7601850 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C67] Mullock, B. M., Bright, N. A., Fearon, C. W., Gray, S. R. and Luzio, J. P. (1998). Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent. J. Cell Biol. 140, 591-601. 10.1083/jcb.140.3.591 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C68] Orr, G., Hu, D., Özçelik, S., Opresko, L. K., Steven Wiley, H. and Colson, S. D. (2005). Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane. Biophys. J. 89, 1362-1373. 10.1529/biophysj.104.056192 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C69] Owen, D. M., Rentero, C., Magenau, A., Abu-Siniyeh, A. and Gaus, K. (2012). Quantitative imaging of membrane lipid order in cells and organisms. Nat. Protoc. 7, 24-35. 10.1038/nprot.2011.419 [DOI] [PubMed] [Google Scholar]

[JCS262032C70] Parkinson, N., Ince, P. G., Smith, M. O., Highley, R., Skibinski, G., Andersen, P. M., Morrison, K. E., Pall, H. S., Hardiman, O., Collinge, J.et al. (2006). ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B). Neurology 67, 1074-1077. 10.1212/01.wnl.0000231510.89311.8b [DOI] [PubMed] [Google Scholar]

[JCS262032C71] Parkinson, G., Roboti, P., Zhang, L., Taylor, S. and Woodman, P. (2021). His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation. J. Cell Sci. 134, jcs259192. 10.1242/jcs.259192 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C72] Parpal, S., Karlsson, M., Thorn, H. and Strålfors, P. (2001). Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control*. J. Biol. Chem. 276, 9670-9678. 10.1074/jbc.M007454200 [DOI] [PubMed] [Google Scholar]

[JCS262032C73] Pashkova, N., Gakhar, L., Winistorfer, S. C., Sunshine, A. B., Rich, M., Dunham, M. J., Yu, L. and Piper, R. C. (2013). The Yeast Alix Homolog Bro1 functions as a ubiquitin receptor for protein sorting into multivesicular endosomes. Dev. Cell 25, 520-533. 10.1016/j.devcel.2013.04.007 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C74] Pentchev, P. G., Brady, R. O., Blanchette-Mackie, E. J., Vanier, M. T., Carstea, E. D., Parker, C. C., Goldin, E. and Roff, C. F. (1994). The Niemann-Pick C lesion and its relationship to the intracellular distribution and utilization of LDL cholesterol. Biochim. Biophys. Acta 1225, 235-243. 10.1016/0925-4439(94)90001-9 [DOI] [PubMed] [Google Scholar]

[JCS262032C75] Phatarpekar, P. V., Overlee, B. L., Leehan, A., Wilton, K. M., Ham, H. and Billadeau, D. D. (2020). The septin cytoskeleton regulates natural killer cell lytic granule release. J. Cell Biol. 219, e202002145. 10.1083/jcb.202002145 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C76] Poteryaev, D., Datta, S., Ackema, K., Zerial, M. and Spang, A. (2010). Identification of the switch in early-to-late endosome transition. Cell 141, 497-508. 10.1016/j.cell.2010.03.011 [DOI] [PubMed] [Google Scholar]

[JCS262032C77] Puri, C., Tosoni, D., Comai, R., Rabellino, A., Segat, D., Caneva, F., Luzzi, P., Di Fiore, P. P. and Tacchetti, C. (2005). Relationships between EGFR signaling-competent and endocytosis-competent membrane microdomains. Mol. Biol. Cell 16, 2704-2718. 10.1091/mbc.e04-07-0596 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C78] Rink, J., Ghigo, E., Kalaidzidis, Y. and Zerial, M. (2005). Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735-749. 10.1016/j.cell.2005.06.043 [DOI] [PubMed] [Google Scholar]

[JCS262032C79] Rodger, C., Flex, E., Allison, R. J., Sanchis-Juan, A., Hasenahuer, M. A., Cecchetti, S., French, C. E., Edgar, J. R., Carpentieri, G., Ciolfi, A.et al. (2020). De novo VPS4A mutations cause multisystem disease with abnormal neurodevelopment. Am. J. Hum. Genet. 107, 1129-1148. 10.1016/j.ajhg.2020.10.012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C80] Russell, M. R. G., Shideler, T., Nickerson, D. P., West, M. and Odorizzi, G. (2012). Class E compartments form in response to ESCRT dysfunction in yeast due to hyperactivity of the Vps21 Rab GTPase. J. Cell Sci. 125, 5208-5220. 10.1242/jcs.111310 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C81] Ryan, M. C., Stucky, M., Wakefield, C., Melott, J. M., Akbani, R., Weinstein, J. N. and Broom, B. M. (2019). Interactive clustered heat map builder: an easy web-based tool for creating sophisticated clustered heat maps. F1000Res 8, 1750. 10.12688/f1000research.20590.1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C82] Sanson, K. R., Hanna, R. E., Hegde, M., Donovan, K. F., Strand, C., Sullender, M. E., Vaimberg, E. W., Goodale, A., Root, D. E., Piccioni, F.et al. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416. 10.1038/s41467-018-07901-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C83] Schenkman, J. B. and Cinti, D. L. (1978). Preparation of microsomes with calcium. Methods Enzymol. 52, 83-89. 10.1016/S0076-6879(78)52008-9 [DOI] [PubMed] [Google Scholar]

[JCS262032C84] Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671-675. 10.1038/nmeth.2089 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C85] Schulze, H. and Sandhoff, K. (2011). Lysosomal lipid storage diseases. Cold Spring Harb. Perspect. Biol. 3, a004804. 10.1101/cshperspect.a004804 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C86] Seo, Y., Kim, T. Y., Won, D., Choi, J. R., Seo, G. H., Lee, S. T. and Han, J. (2022). PTPN23 neurodevelopmental disorder presenting with optic atrophy and spasmus nutans-like nystagmus. J. Neuroophthalmol. 43, e316-e318. 10.1097/WNO.0000000000001582 [DOI] [PubMed] [Google Scholar]

[JCS262032C87] Seu, K. G., Trump, L. R., Emberesh, S., Lorsbach, R. B., Johnson, C., Meznarich, J., Underhill, H. R., Chou, S. T., Sakthivel, H., Nassar, N. N.et al. (2020). VPS4A mutations in humans cause syndromic congenital dyserythropoietic anemia due to cytokinesis and trafficking defects. Am. J. Hum. Genet. 107, 1149-1156. 10.1016/j.ajhg.2020.10.013 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C88] Shiels, A., Bennett, T. M., Knopf, H. L. S., Yamada, K., Yoshiura, K., Niikawa, N., Shim, S. and Hanson, P. I. (2007). CHMP4b, a novel gene for autosomal dominant cataracts linked to chromosome 20q. Am. J. Hum. Genet. 81, 596-606. 10.1086/519980 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C89] Shih, S. C., Katzmann, D. J., Schnell, J. D., Sutanto, M., Emr, S. D. and Hicke, L. (2002). Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat. Cell Biol. 4, 389-393. 10.1038/ncb790 [DOI] [PubMed] [Google Scholar]

[JCS262032C90] Shim, J.-H., Xiao, C., Hayden, M. S., Lee, K.-Y., Trombetta, E. S., Pypaert, M., Nara, A., Yoshimori, T., Wilm, B., Erdjument-Bromage, H.et al. (2006). CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis. J. Cell Biol. 172, 1045-1056. 10.1083/jcb.200509041 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C91] Sieben, C. J., Jeganathan, K. B., Nelson, G. G., Sturmlechner, I., Zhang, C., Van Deursen, W. H., Bakker, B., Foijer, F., Li, H., Baker, D. J.et al. (2020). BubR1 allelic effects drive phenotypic heterogeneity in mosaic-variegated aneuploidy progeria syndrome. J. Clin. Invest. 130, 171-188. 10.1172/JCI126863 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C92] Simons, K. and Gruenberg, J. (2000). Jamming the endosomal system: lipid rafts and lysosomal storage diseases. Trends Cell Biol. 10, 459-462. 10.1016/S0962-8924(00)01847-X [DOI] [PubMed] [Google Scholar]

[JCS262032C93] Skibinski, G., Parkinson, N. J., Brown, J. M., Chakrabarti, L., Lloyd, S. L., Hummerich, H., Nielsen, J. E., Hodges, J. R., Spillantini, M. G., Thusgaard, T.et al. (2005). Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat. Genet. 37, 806-808. 10.1038/ng1609 [DOI] [PubMed] [Google Scholar]

[JCS262032C94] Smigiel, R., Landsberg, G., Schilling, M., Rydzanicz, M., Pollak, A., Walczak, A., Stodolak, A., Stawinski, P., Mierzewska, H., Sasiadek, M. M.et al. (2018). Developmental epileptic encephalopathy with hypomyelination and brain atrophy associated with PTPN23 variants affecting the assembly of UsnRNPs. Eur. J. Hum. Genet. 26, 1502-1511. 10.1038/s41431-018-0179-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C95] Sowada, N., Hashem, M. O., Yilmaz, R., Hamad, M., Kakar, N., Thiele, H., Arold, S. T., Bode, H., Alkuraya, F. S. and Borck, G. (2017). Mutations of PTPN23 in developmental and epileptic encephalopathy. Hum. Genet. 136, 1455-1461. 10.1007/s00439-017-1850-3 [DOI] [PubMed] [Google Scholar]

[JCS262032C96] Tabernero, L. and Woodman, P. (2018). Dissecting the role of His domain protein tyrosine phosphatase/PTPN23 and ESCRTs in sorting activated epidermal growth factor receptor to the multivesicular body. Biochem. Soc. Trans. 46, 1037-1046. 10.1042/BST20170443 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C97] Thompson, B. J., Mathieu, J., Sung, H.-H., Loeser, E., Rørth, P. and Cohen, S. M. (2005). Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Dev. Cell 9, 711-720. 10.1016/j.devcel.2005.09.020 [DOI] [PubMed] [Google Scholar]

[JCS262032C98] Toyooka, S.-I., Ouchida, M., Jitsumori, Y., Tsukuda, K., Sakai, A., Nakamura, A., Shimizu, N. and Shimizu, K. (2000). HD-PTP: A Novel Protein Tyrosine Phosphatase Gene on Human Chromosome 3p21.3. Biochem. Biophys. Res. Commun. 278, 671-678. 10.1006/bbrc.2000.3870 [DOI] [PubMed] [Google Scholar]

[JCS262032C99] Vaccari, T. and Bilder, D. (2005). The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking. Dev. Cell 9, 687-698. 10.1016/j.devcel.2005.09.019 [DOI] [PubMed] [Google Scholar]

[JCS262032C100] Van Den Boomen, D. J. H., Sienkiewicz, A., Berlin, I., Jongsma, M. L. M., Van Elsland, D. M., Luzio, J. P., Neefjes, J. J. C. and Lehner, P. J. (2020). A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export. Nat. Commun. 11, 5559. Available: http://europepmc.org/abstract/MED/33144569, https://www.nature.com/articles/s41467-020-19032-0.pdf, 10.1038/s41467-020-19032-0, https://europepmc.org/articles/PMC7642327, https://europepmc.org/articles/PMC7642327?pdf=render [Accessed 2020/11//]. 10.1038/s41467-020-19032-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C101] Van Der Zee, J., Urwin, H., Engelborghs, S., Bruyland, M., Vandenberghe, R., Dermaut, B., De Pooter, T., Peeters, K., Santens, P., De Deyn, P. P.et al. (2008). CHMP2B C-truncating mutations in frontotemporal lobar degeneration are associated with an aberrant endosomal phenotype in vitro. Hum. Mol. Genet. 17, 313-322. 10.1093/hmg/ddm309 [DOI] [PubMed] [Google Scholar]

[JCS262032C102] Waddington, K. E., Pineda-Torra, I. and Jury, E. C. (2019). Analyzing T-cell plasma membrane lipids by flow cytometry. Methods Mol. Biol. 1951, 209-216. 10.1007/978-1-4939-9130-3_16 [DOI] [PubMed] [Google Scholar]

[JCS262032C103] Wagner, K.-U., Krempler, A., Qi, Y., Park, K., Henry, M. L. D., Triplett, A. A., Riedlinger, G., Rucker, E. B. and Hennighausen, L. (2003). Tsg101 is essential for cell growth, proliferation, and cell survival of embryonic and adult tissues. Mol. Cell. Biol. 23, 150-162. 10.1128/MCB.23.1.150-162.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C104] Wen, T., Barham, W., Li, Y., Zhang, H., Gicobi, J. K., Hirdler, J. B., Liu, X., Ham, H., Peterson Martinez, K. E., Lucien, F.et al. (2022). NKG7 Is a T-cell-intrinsic therapeutic target for improving antitumor cytotoxicity and cancer immunotherapy. Cancer Immunol. Res. 10, 162-181. 10.1158/2326-6066.CIR-21-0539 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C105] Wollert, T. and Hurley, J. H. (2010). Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864-869. 10.1038/nature08849 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C106] Wurmser, A. E. and Emr, S. D. (1998). Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities. EMBO J. 17, 4930-4942. 10.1093/emboj/17.17.4930 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C107] Xu, M., Tchkonia, T., Ding, H., Ogrodnik, M., Lubbers, E. R., Pirtskhalava, T., White, T. A., Johnson, K. O., Stout, M. B., Mezera, V.et al. (2015). JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age. Proc. Natl. Acad. Sci. USA 112, E6301-E6310. 10.1073/pnas.1515386112 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C108] Yamada, K., Tomita, H.-A., Kanazawa, S., Mera, A., Amemiya, T. and Niikawa, N. (2000). Genetically distinct autosomal dominant posterior polar cataract in a four-generation Japanese family. Am. J. Ophthalmol. 129, 159-165. 10.1016/S0002-9394(99)00313-X [DOI] [PubMed] [Google Scholar]

[JCS262032C109] Yun, H., Jung, M., Lee, H., Jung, S., Kim, T., Kim, N., Park, S. Y., Kim, W. J., Mun, J. Y. and Yoo, J. Y. (2023). Homotypic SCOTIN assemblies form ER-endosome membrane contacts and regulate endosome dynamics. EMBO Rep. 24, e56538. 10.15252/embr.202256538 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C110] Zhang, S., Fan, G., Hao, Y., Hammell, M., Wilkinson, J. E. and Tonks, N. K. (2017). Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase. Genes Dev. 31, 1939-1957. 10.1101/gad.304261.117 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C111] Zhang, J., Sun, Y., Zhong, L.-Y., Yu, N.-N., Ouyang, L., Fang, R.-D., Wang, Y. and He, Q.-Y. (2020). Structure-based discovery of neoandrographolide as a novel inhibitor of Rab5 to suppress cancer growth. Comput. Struct. Biotechnol. J. 18, 3936-3946. 10.1016/j.csbj.2020.11.033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[JCS262032C112] Zivony-Elboum, Y., Westbroek, W., Kfir, N., Savitzki, D., Shoval, Y., Bloom, A., Rod, R., Khayat, M., Gross, B., Samri, W.et al. (2012). A founder mutation in Vps37A causes autosomal recessive complex hereditary spastic paraparesis. J. Med. Genet. 49, 462-472. 10.1136/jmedgenet-2012-100742 [DOI] [PubMed] [Google Scholar]

PERMALINK

Loss of HD-PTP function results in lipodystrophy, defective cellular signaling and altered lipid homeostasis

Destiny F Schultz

Brian A Davies

Johanna A Payne

Cole P Martin

Annabel Y Minard

Bennett G Childs

Cheng Zhang

Karthik B Jeganathan

Ines Sturmlechner

Thomas A White

Alain de Bruin

Liesbeth Harkema

Huiqin Chen

Michael A Davies

Sarah Jachim

Nathan K LeBrasseur

Robert C Piper

Hu Li

Darren J Baker

Jan van Deursen

Daniel D Billadeau

David J Katzmann

ABSTRACT

INTRODUCTION

RESULTS

HD-PTP hypomorphic mice display lipodystrophy

Fig. 1.

Insulin and RTK signaling is reduced in adipose tissue of HD-PTP hypomorphic mice

Fig. 2.

HD-PTP impacts cellular cholesterol distribution to promote EGFR phosphorylation

Fig. 3.

Fig. 4.

Loss of HD-PTP negatively impacts lysosome–ER contact sites and cholesterol transport

Fig. 5.

Inhibition of Rab5 suppresses EGFR signaling defect in Ptpn23 KO

Fig. 6.

Fig. 7.

DISCUSSION

MATERIALS AND METHODS

Mouse strains

Body composition, physiological and behavioral analyses

RNA isolation, library preparation, sequencing and bioinformatics analyses

Cell lines and culture

Immunofluorescence

Western blotting and reverse phase protein array analyses

Signaling assays

Ligand binding and plasma membrane receptor content

Yeast distribution of ergosterol

Di-4-ANEPPDHQ staining

Cholesterol detection

Membrane fractionation

Lipid droplet visualization

Quantitative PCR for cholesterol synthesis and transporter proteins

Proximity ligation assay

Lysosome fractionation

Statistical analyses

Supplementary Material

Acknowledgements

Footnotes

Peer review history

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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