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Published in final edited form as: Biochem Biophys Res Commun. 2024 Apr 16;712-713:149923. doi: 10.1016/j.bbrc.2024.149923

Stasimon/Tmem41b is required for cell proliferation and adult mouse survival

Maria J Carlini a,b, Meaghan Van Alstyne a,b,1, Hua Yang a,b, Shubhi Yadav a,b, Neil A Shneider a,b, Livio Pellizzoni a,b,c,*
PMCID: PMC11066899  NIHMSID: NIHMS1988859  PMID: 38640735

Abstract

Stasimon/Tmem41b is a transmembrane protein with phospholipid scrambling activity that resides in the endoplasmic reticulum and has been implicated in autophagy, lipid metabolism, and viral replication. Stasimon/Tmem41b has also been linked to the function of sensory-motor circuits and the pathogenesis of spinal muscular atrophy. However, the early embryonic lethality of constitutive knockout in mice has hindered the analysis of spatial and temporal requirements of Stasimon/Tmem41b in vivo. To address this, we developed a novel mouse lines harboring a conditional knockout allele of the Stasimon/Tmem41b gene in which exon 4 has been flanked by loxP sites (Stas/Tmem41bCKO). Cre-mediated recombination of Stas/Tmem41bCKO generates a functionally null allele (Stas/Tmem41bΔ4) resulting in loss of protein expression and embryonic lethality in the homozygous mouse mutant. Here, using a ubiquitously expressed, tamoxifen inducible Cre recombinase in the homozygous Stas/Tmem41bCKO mice, we demonstrate that postnatal depletion of Stasimon/Tmem41b rapidly arrests weight gain in adult mice and causes motor dysfunction and death approximately three weeks after tamoxifen treatment. Moreover, we show that depletion of Stasimon/Tmem41b severely affects cell proliferation in mouse embryonic fibroblasts. This study provides new insights into the essential requirement of Stasimon/Tmem41b for cellular and organismal fitness and expands the experimental toolkit to investigate its functions in the mammalian system.

Keywords: Stasimon/Tmem41b, phospholipid scramblase, lipid homeostasis, endoplasmic reticulum (ER), cell proliferation, spinal muscular atrophy (SMA)

1. Introduction

Stasimon (also known as Transmembrane protein 41b, and hereafter referred to as Stas/Tmem41b) is an ER-resident transmembrane protein that localizes to contact sites with mitochondria [1–3] and has been shown to possess phospholipid scrambling activity in vitro [4–6]. Expanding earlier discoveries of a key role for Stas/Tmem41b in autophagosome biogenesis and lipid homeostasis [1,2,7], recent studies showed that the scramblase activity of Stas/Tmem41b is necessary to support the transport of lipids that fuel the growth of autophagic membranes at ER contact sites [4] as well as for the biogenesis of lipoproteins [5]. Stas/Tmem41b is also required for the replication of several families of RNA viruses that occurs at specific ER factories [8,9]. However, since most of these studies employed in vitro model systems, the biological relevance of Stas/Tmem41b in vivo is less well understood.

Stas/Tmem41b was originally discovered as a target of splicing dysfunction induced by SMN deficiency that contributes to sensory-motor circuit pathology in animal models of spinal muscular atrophy (SMA) spanning from flies to mice [10–12]. Stas/Tmem41b restoration corrects neurotransmission deficits in the motor circuit of Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish embryos [10]. Furthermore, Stas/Tmem41b gene delivery restores proprioceptive afferent synapses and motor neuron survival in SMA mice [11]. Lastly, Stas/Tmem41b loss of function disrupts the development and function of neural circuits that control movement in Drosophila and zebrafish models [10]. However, characterization of the normal requirement of Stas/Tmem41b in the mammalian nervous system has been hindered by the embryonic lethality of its ubiquitous knockout in mice [3].

Here we describe the generation and functional characterization of novel cellular and mouse models harboring a conditional allele for spatially and temporally regulated knockout of Stas/Tmem41b as novel tools to study its general and tissue-specific requirement in the mammalian system.

2. Materials and methods

2.1. Mouse lines and behavioral analysis

All mouse work was performed in accordance with the National Institutes of Health (NIH) Guidelines on the Care and Use of Animals, complied with all ethical regulations and was approved by the IACUC committee of Columbia University. Equal number of male and female mice were used in all experiments and aggregated data are presented because gender-specific differences were not found. C57BL/6J (JAX stock # 000664), Protamine::Cre (JAX stock # 003328), Pgk1::FLPo (JAX stock # 011065), and CAGG::Cre-ER (JAX stock # 004682) mouse lines were obtained from the Jackson Laboratory. The mouse embryonic stem (ES) cell clone EPD0122_4_H06 (C57BL/6N background) harboring a knockout-first allele (reporter-tagged insertion with conditional potential) of Stas/Tmem41b (Tmem41btm1a(KOMP)Wtsi) was obtained by the UC Davis KOMP Repository [13] and injected into mouse blastocysts to generate chimeras, which were then backcrossed to the C57BL/6J strain to establish the Stas/Tmem41bKOMP mouse line. The conditional Stas/Tmem41bCKO mouse line in which exon 4 is flanked by LoxP sites was generated by crossing Stas/Tmem41bKOMP and Pgk1::FLPo mice. Stas/Tmem41bΔ4 mice with constitutive deletion of exon 4 were generated by crossing Stas/Tmem41bCKO and Protamine::Cre mice. Stas/Tmem41bCKO and CAGG::Cre-ER mice were crossed to generate double heterozygous mice with potential for tamoxifen inducible, ubiquitous Cre recombination. All newly generated mouse lines were backcrossed to the C57BL/6J strain for a minimum of 10 generations. To induce Cre recombination, Tamoxifen (SIGMA, #T5648) was dissolved in corn oil at a concentration of 20mg/ml and a dose of 100mg per kg of body weight was administered to 4 weeks old adult mice via oral gavage once every 24 hours for three to five consecutive days. Body weight, inverted grid and rotarod analyses were performed weekly from 3 to 20 weeks of age as previously described [14]. Survival was monitored daily until study termination at 5 months of age. Genotyping was carried out with genomic DNA isolated from mouse tails or dissected tissues by PCR amplification using the primers listed in Supplementary Table 1. β-galactosidase activity in whole E12.5 mouse embryos was analyzed as previously described [15].

2.2. Cell lines and treatments

Primary mouse embryonic fibroblasts (MEFs) were derived from E17.5 embryos generated by crossing Stas/Tmem41bCKO/CKO and Stas/Tmem41bCKO/WT; CAGG::Cre-ER+/− mice following standard procedures [16]. The resulting Stas/Tmem41bCKO/CKO MEFs with or without the CAGG::Cre-ER allele were expanded and immortalized by lentiviral transduction followed by puromycin selection (3μg/ml) using the SV40 T Antigen Immortalization Kit according to the manufacturer’s instructions (ALSTEM, #CILV01). MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (Gibco, #004682) supplemented with 10% fetal bovine serum (Genclone, #25–550), 2 mM L-glutamine (Gibco, #25-030-164), and penicillin/streptomycin (Gibco, #15140122). Mycoplasma contamination was routinely assessed using the Universal Mycoplasma Detection Kit (ATCC, #30–1012K) kit. MEFs were treated with 2μM 4Hydroxytamoxifen (4-OHT; Sigma-Aldrich, #508225) for 24 hours to induce Cre recombination. Following a change of medium, control (Stas/Tmem41bCKO/CKO) and Stas/Tmem41b knockout (Stas/Tmem41bCKO/CKO; CAGG::Cre-ER+/−) MEFs were cultured for additional 7 days prior to RNA and protein analysis.

2.3. RNA analysis

Total RNA was isolated from control and Stas/Tmem41b knockout MEFs using TRIzol reagent (Invitrogen, # 15596026), followed by treatment with RNAse-free DNaseI (Ambion, # AM2222). Reverse transcription was performed using RevertAid RT Reverse Transcription Kit (ThermoFisher, # K1691) with random hexamer and oligo dT primers. RT-qPCR analysis was performed using SYBR Green (Applied Biosystems, #4368702) in technical triplicates for each biological replicate using primers specific for mouse Stas/Tmem41b mRNA sequences spanning either exons 1–3 (E1-E3) or exons 3–4 (E3-E4) and Gapdh mRNA as a normalizer. The primers are listed in Supplementary Table 1.

2.4. Protein analysis

Total protein extracts were obtained by homogenization of control and Stas/Tmem41b knockout MEFs or isolated mouse tissue in ice-cold lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton) with protease and phosphatase inhibitors, followed by brief sonication and centrifugation at 16,000 g for 10 minutes at 4°C. Protein extracts were quantified using RC DC™ protein assay (BioRad, # 5000121) and run on 12% polyacrylamide gels and transferred to nitrocellulose membranes for probing. Blocking was done in 5% milk in PBS/0.1% Tween. Primary and secondary antibodies were diluted in PBS/0.1% Tween. Chemiluminescence was carried out using a SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, #PI34580). Signal was detected using an iBrigth CL1500 Imaging System (Thermo Fisher Scientific) and images processed with the iBright Analysis Software (version 5.1.0). The antibodies are listed in Supplementary Table 2.

2.5. Cell proliferation assay

Stas/Tmem41bCKO/CKO and Stas/Tmem41bCKO/CKO; CAGG::Cre-ER+/− MEFs were cultured in the presence of vehicle (ethanol) or 2μM 4-OHT for 24 hours and, after change of media, for additional 48 hours prior to seeding in 96-well optical plates (Greiner Bio-One, #655090) at 250 cells per well and six replicates per experimental group for each biological replicate and time point. Cell number was determined by whole-well imaging acquisition using a plate imaging system (TROPHOS Plate RUNNER H) followed by nuclear counting with the TINA software (TROPHOS) as previously described [17].

2.6. Statistical analysis

All data are presented as mean and SEM. Statistics were performed with two-tailed unpaired Student’s t-test or two-way ANOVA followed by the Sidak’s multiple comparisons test as indicated. Comparison of survival curves was performed with the Mantel-Cox log-rank test. Data were analyzed using GraphPad Prism 10 and P values are indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P< 0.0001.

3. Results

3.1. Development of mouse lines for ubiquitous and conditional knockout of Stas/Tmem41b

We established novel mouse lines with potential for either ubiquitous or conditional knockout of Stas/Tmem41b using a targeted ES cell line harboring an allele (Stas/Tmem41bKOMP) containing a lacZ reporter/stop cassette and a neomycin resistance cassette surrounded by FRT sites inserted within intron 3 of the mouse Stas/Tmem41b gene as well as the exon 4 flanked by loxP sites (Fig. 1A), which is expected to be a functionally null allele. We generated heterozygous Stas/Tmem41bKOMP/WT mice that are viable, fertile and display no obvious phenotypes (Fig. 1B). However, Stas/Tmem41bKOMP/WT crosses did not yield any homozygous Stas/Tmem41bKOMP/KOMP offspring (Fig. 1E). Given the embryonic lethality of ubiquitous Stas/Tmem41b knockout [3], these results confirm that Stas/Tmem41bKOMP is a null allele. Histochemical detection of β-galactosidase expression in whole E12.5 embryos revealed ubiquitous expression of Stas/Tmem41b (Fig. 1H). Next, we crossed Stas/Tmem41bKOMP/WT mice with a Flp deleter line to generate Stas/Tmem41bCKO mice harboring a conditional knockout allele with floxed exon 4 (Fig.1A and C). Crosses of Stas/Tmem41bCKO/WT mice yielded viable and fertile mice with the expected Mendelian ratio of genotypes (Fig. 1F), indicating that Stas/Tmem41bCKO is a functional allele. Lastly, we crossed Stas/Tmem41bCKO/WT mice with a Cre deleter line to generate a Stas/Tmem41bΔ4 allele lacking exon 4 (Fig. 1A and D). Cre-mediated deletion of exon 4 introduces a frame shift mutation starting at codon 124 and a premature stop after codon 137 in the resulting Stas/Tmem41b mRNA, which is predicted to undergo degradation by nonsense mediated decay. Additionally, if any stable protein product is generated, it would contain only the initial 123 of 291 amino acids of mouse Stas and is expected to be non-functional. To confirm this, we analyzed crosses of Stas/Tmem41bΔ4/WT mice, which yielded no homozygous Stas/Tmem41bΔ4/Δ4 mutant mice (Fig. 1G), demonstrating that deletion of exon 4 creates a null Stas/Tmem41b allele. In summary, we generated and validated novel mouse lines harboring alleles for either ubiquitous or conditional knockout of Stas/Tmem41b that will be useful for the study of this gene’s function in vivo.

Fig. 1.

Fig. 1.

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Development and characterization of novel mouse lines for ubiquitous and conditional knockout of Stas/Tmem41b. (A) Schematic of the mouse Stas/Tmem41b (Stas/Tmem41bWT) gene, the targeting vector used to generate the corresponding knockout-first allele (Stas/Tmem41bKOMP) and its derivatives obtained through sequential recombination with Flp (Stas/Tmem41bCKO) and Cre (Stas/Tmem41bΔ4) recombinases. The location of genotyping primers is indicated by arrows. (B) Genomic PCR analysis of Stas/Tmem41bWT (primers (a) Stas/Tmem41b-F and (c) Stas/Tmem41b-ttR) and Stas/Tmem41bKOMP (primers (b) neo-F and (c) Stas/Tmem41b-ttR) alleles from P1 mice of the indicated genotypes. (C) Genomic PCR analysis of Stas/Tmem41bWT and Stas/Tmem41bCKO (primers (a) Stas/Tmem41b-F and (c) Stas/Tmem41b-ttR) alleles, and Stas/Tmem41bKOMP (primers (b) neo-F and (c) Stas/Tmem41b-ttR) allele from P1 mice of the indicated genotypes before and after Flp recombination. (D) Genomic PCR analysis of Stas/Tmem41bWT and Stas/Tmem41bCKO (primers (a) Stas/Tmem41b-F and (c) Stas/Tmem41b-ttR) alleles, and Stas/Tmem41bΔ4 (primers (a) Stas/Tmem41b-F and (d) Stas/Tmem41b-R) allele from P1 mice of the indicated genotypes before and after Cre recombination. (E) Percentage of expected and observed newborn mice with the indicated genotypes from crosses of Stas/Tmem41bKOMP/WT mice. (F) Percentage of expected and observed newborn mice with the indicated genotypes from crosses of Stas/Tmem41bCKO/WT mice. (G) Percentage of expected and observed newborn mice with the indicated genotypes from crosses of Stas/Tmem41bΔ4/WT mice. (H) β-galactosidase staining of E12.5 whole mouse embryos with or without the Stas/Tmem41bKOMP allele.

3.2. Postnatal depletion of Stas/Tmem41b affects growth, motor function, and survival in adult mice

To determine the postnatal requirement of Stas/Tmem41b, we generated homozygous Stas/Tmem41bCKO/CKO mice with or without a single CAGG::Cre-ER allele and studied the effects of ubiquitous, tamoxifen-dependent knockout of Stas/Tmem41b in adult mice. For these experiments, four weeks old Stas/Tmem41bCKO/CKO; CAGG::Cre-ER+/− mice and Stas/Tmem41bCKO/CKO control littermates without Cre-ER were treated with tamoxifen and tissues were isolated one week later for analysis of Cre-mediated recombination. Genomic PCR showed strong recombination of the floxed Stas/Tmem41bCKO allele in all tissues from mice harboring Cre-ER but not in those from control mice lacking the recombinase as expected (Fig. 2A). Furthermore, Western blot analysis highlighted a robust reduction in the levels of the Stas/Tmem41b protein expressed in tissues from Stas/Tmem41bCKO/CKO mice following Cre dependent recombination (Fig. 2B). These experiments validated the tamoxifen-dependent, Cre-mediated depletion of Stas/Tmem41b in adult mice.

Fig. 2.

Fig. 2.

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Postnatal depletion of Stas/Tmem41b affects growth, motor behavior, and survival in adult mice. (A) Genomic PCR analysis of Stas/Tmem41bCKO recombination (primers Stas/Tmem41b-F and Stas/Tmem41b-R) in the indicated tissues from tamoxifen-treated Stas/Tmem41bCKO/CKO and Stas/Tmem41bCKO/CKO; CAGG::Cre-ER+/− mice. Tamoxifen treatment in mice was initiated at four weeks of age and tissues were isolated one week later. (B) Western blot analysis of Stas/Tmem41b expression in the indicated tissues from the same experimental groups as in (A). The asterisk marks a non-specific protein. (C-F) Behavioral analysis of postnatal depletion of Stas/Tmem41b in adult mice (n=15 per group). Inverted grid (C), rotarod (D), weight (E), and survival (F) were monitored in tamoxifen-treated Stas/Tmem41bCKO/CKO and Stas/Tmem41bCKO/CKO; CAGG::Cre-ER+/− mice from 3 to 20 weeks of age. Tamoxifen treatment was initiated at four weeks of age. Statistics in (C-E) were performed with two-way ANOVA. ** P<0.01; **** P<0.0001. Comparison of survival curves in (F) was performed with the Mantel-Cox log-rank test. **** P<0.0001.

Next, we used this experimental system to investigate the behavioral effects of postnatal loss of Stas/Tmem41b. To do so, we monitored weight gain, motor function, and survival from 3 to 20 weeks of age in Stas/Tmem41bCKO/CKO mice with and without Cre-ER that were subjected to tamoxifen treatment starting at 4 weeks of age. Analysis of muscle strength with the inverted grid assay showed a steep decline caused by Stas/Tmem41b depletion between 6 and 7 weeks of age (Fig. 2C). A similar temporal onset of phenotypic impairment induced by the loss of Stas/Tmem41b was observed when testing motor coordination and endurance with the rotarod assay (Fig. 2D). Furthermore, the weight gain of Stas/Tmem41bCKO/CKO mice was severely compromised shortly after induction of Stasimon/Tmem41b depletion (Fig. 2E) and their median survival was 48 days (Fig. 2E). None of these phenotypic effects were observed in tamoxifen treated Stas/Tmem41bCKO/CKO mice that were used as controls. Together, these results demonstrate an essential postnatal requirement of Stas/Tmem41b for growth, motor function and survival in adult mice.

3.3. Stas/Tmem41b is required for cell proliferation in mouse embryonic fibroblasts

To develop a cellular model with inducible knockout of Stas/Tmem41b, we generated homozygous Stas/Tmem41bCKO/CKO mouse embryonic fibroblasts (MEFs) with and without a single copy of the CAGG::Cre-ER allele. In this system, in which Cre activity can be induced by addition of 4-OHT, analysis by genomic PCR showed robust, Cre-mediated recombination of the floxed allele in Stas/Tmem41bCKO/CKO MEFs harboring Cre-ER but not in control cells lacking the recombinase (Fig. 3A). We then analyzed Stas/Tmem41b mRNA expression by RT-qPCR using two sets of primers: one measuring total mRNA levels (E1-E3) and another monitoring specifically exon 4 containing mRNAs (E3-E5). Interestingly, while Cre recombination nearly abolished exon 4 inclusion in Stas/Tmem41bCKO/CKO MEFs containing CRE-ER, similar levels of total Stas/Tmem41b mRNA transcripts were found in both cell lines overall (Fig. 3B). These results validate the efficiency of exon 4 recombination and indicate that Stas/Tmem41b Δ4 mRNAs are not degraded by nonsense mediated decay. Next, we investigated the levels of Stas/Tmem41b protein by Western blot and found it is specifically depleted from Stas/Tmem41bCKO/CKO MEFs following Cre-mediated recombination (Fig. 3C). Thus, exon 4 deletion results in the loss of Stas/Tmem41b protein expression.

Fig. 3.

Fig. 3.

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Stas/Tmem41b is required for cell proliferation in primary mouse fibroblasts. (A) Genomic PCR analysis of Stas/Tmem41bCKO recombination (primers Stas/Tmem41b-F and Stas/Tmem41b-R) in immortalized MEF lines derived from Stas/Tmem41bCKO/CKO and Stas/Tmem41bCKO/CKO; CAGG::Cre-ER+/− embryos. MEFs were treated with 2mM 4-OHT for 24 hours and cultured for additional 7 days prior to DNA purification. (B) RT-qPCR analysis of Stasimon mRNA levels using either Stas/Tmem41b E1-E3 or Stas/Tmem41b E3-E4 primers and normalized to Gapdh mRNA expression from the same experimental groups as in (A). Data represent mean and SEM (n=3). Statistics were performed with two-tailed unpaired Student’s t-test. ** P < 0.01; ns, not significant. (C) Western blot analysis of Stas/Tmem41b expression from the same experimental groups as in (A). The asterisk marks a non-specific protein. (D) Representative images of Stas/Tmem41bCKO/CKO and Stas/Tmem41bKO/CKO; CAGG::Cre-ER+/− MEFs with or without 4-OHT treatment at 4 hours (T0) and 120 hours post-plating in 96-well format. (E) Analysis of cell proliferation in the same experimental groups as in (D) using the 96-well format assay. Cell number was determined at the indicated times post-plating. Lines are exponential growth fittings. Data are represented as mean and SEM (n=7). Statistics were performed with two-way ANOVA. ** P<0.01; **** P<0.0001.

Lastly, we studied the effects of Stas/Tmem41b knockout on cell proliferation using a previously established assay in 96-well format [17]. Cell number was determined following nuclear staining and whole well imaging at each time point (Fig. 3D). Remarkably, while the proliferation of vehicle treated Stas/Tmem41bCKO/CKO cells with and without Cre-ER was nearly identical, 4-OHT-induced Stas/Tmem41b knockout strongly affected cell proliferation in Cre-ER expressing MEFs (Fig. 3E). Furthermore, the effects were specific because 4-OHT-treated Stas/Tmem41bCKO/CKO cells without Cre-ER proliferated as efficiently as vehicle-treated controls (Fig. 3E). These results demonstrate the essential requirement of Stas/Tmem41b for the proliferation of primary mouse fibroblasts.

4. Discussion

This study describes the development and characterization of cellular and mouse models for conditional depletion of Stas/Tmem41b, which provide new insights into the biology of this ER-resident phospholipid scramblase and novel experimental tools to investigate its spatial and temporal requirement in the mammalian system.

Our analysis of mice harboring the knockout-first allele with tagged insertion of a LacZ reporter in intron 3 indicates that Stas/Tmem41bKOMP is functionally null and provides direct evidence for ubiquitous expression of Stas/Tmem41b in mouse embryos. We also demonstrate that the conditional Stas/Tmem41bCKO allele is functional—supporting both embryonic development and postnatal mouse viability in the homozygous state— and generates a knockout allele (Stas/Tmem41bΔ4) upon Cre-mediated deletion of the floxed exon 4, resulting in stable mRNA expression but loss of protein production.

To investigate the postnatal requirement of Stas/Tmem41b in vivo, we generated homozygous Stas/Tmem41bCKO mice harboring a tamoxifen inducible Cre recombinase allele driven by a ubiquitous driver. Using these mice, we show that widespread depletion of Stas/Tmem41b in adult mice rapidly impairs weight gain, followed by a sharp decline in motor strength and coordination, and ultimately death within three weeks. Expanding on our earlier study [3], these results demonstrate that Stas/Tmem41b is not only essential for embryonic development but also for motor function and survival in the adult mouse. A detailed investigation of the effects of Stas/Tmem41b loss in specific tissues and cell types was beyond the scope of our present investigation. However, a previous study using transient viral-mediated CRISPR/Cas9 knockout in adult mice demonstrated that loss of Stas/Tmem41b restricted to the liver causes severe deficits in lipoprotein biogenesis and early death [5]. It is therefore likely that dysfunction of the liver together with other organs contributes to the onset and progression of the severe phenotypes elicited by postnatal depletion of Stas/Tmem41b. The essential requirement of Stas/Tmem41b for health and survival of adult mice has implications for the development of potential therapeutic approaches targeting this gene as a strategy against infections by coronavirus and flavivirus among other viruses [8,9]. Viable approaches will need to be specifically tailored towards Stas/Tmem41b’s activities related to the viral life cycle without interfering with those essential for cellular and organism viability.

Our analysis of the effects of Stas/Tmem41b depletion using an inducible model system revealed that loss of Stas/Tmem41b severely affects the proliferation of primary mouse fibroblasts, highlighting the protein’s critical role for maintaining cellular fitness in vitro. Interestingly, proliferation or viability deficits of Stas/Tmem41b knockout cells were not documented in previous studies [1,2,6–8]. It is possible that these deficits were present but not analyzed in detail or that they could be masked by adaptation of non-regulated knockout models following extended growth in culture. Alternatively, there might be differential vulnerability of distinct cell types to the loss of Stas/Tmem41b. Nevertheless, we established an inducible cell system with a robust phenotypic readout that will be useful for structure-function relationship studies as well as for screening of chemical or genetic modifiers of Stas/Tmem41b biology.

This functionally validated, conditional allele of Stas/Tmem41b will make it possible to dissect the tissue-specific requirements for this ER-resident phospholipid scramblase in vivo through depletion in specific cell types in the mouse. Given the links between Stas/Tmem41b and sensory-motor circuit pathology in SMA models [10–12], the characterization of the role of Stas/Tmem41b in the nervous system will be of special interest.

Supplementary Material

1
2

Highlights.

  • Development of a mouse model for conditional knockout of Stas/Tmem41b.

  • Depletion of Stas/Tmem41b in adult mice causes motor dysfunction and death.

  • Stas/Tmem41b is required for cell proliferation in primary mouse fibroblasts.

Acknowledgements

The Tmem41b mouse strain used for this research project was created from ES cell clone EPD0122_4_H06, obtained from the KOMP Repository (www.komp.org) and generated by the Wellcome Trust Sanger Institute (WTSI). Targeting vectors used were generated by the Wellcome Trust Sanger Institute and the Children’s Hospital Oakland Research Institute as part of the Knockout Mouse Project (3U01HG004080).

Funding

This work was supported by the NIH/NINDS grants R01NS114218 and R01NS116400 to LP.

Footnotes

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Ethical approval

All animal care and experimental procedures were approved by the IACUC committee of Columbia University. Experiments comply with the ARRIVE guidelines and were carried out in accordance with the NIH Guidelines on the Care and Use of Animals.

CRediT authorship contribution statement

Maria Carlini: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft preparation, Writing – review & editing. Meaghan Van Alstyne: Investigation, Methodology, Writing – review & editing. Hua Yang: Data curation, Investigation, Methodology. Shubhi Yadav: Investigation. Neil Shneider: Methodology, Resources, Writing – review & editing. Livio Pellizzoni: Conceptualization, Funding acquisition, Supervision, Visualization, Writing – original draft preparation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online.

Data availability

The data presented in this study are available upon reasonable request from the corresponding author.

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Associated Data

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

Supplementary Materials

1
NIHMS1988859-supplement-1.xlsx (9.8KB, xlsx)
2
NIHMS1988859-supplement-2.xlsx (10.1KB, xlsx)

Data Availability Statement

The data presented in this study are available upon reasonable request from the corresponding author.

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