Sequential conversion of PtdIns3P to PtdIns(3,5)P₂ via endosome maturation couples nutrient signaling to lysosome reformation and basal autophagy

ABSTRACT

Macroautophagy/autophagy occurs basally under nutrient-rich conditions in most mammalian cells, contributing to protein and organelle quality control, and protection against aging and neurodegeneration. During autophagy, lysosomes are heavily utilized via their fusion with autophagosomes and must be repopulated to maintain autophagic degradative capacity. During starvation-induced autophagy, lysosomes are generated via de novo biogenesis under the control of TFEB (transcription factor EB), or by the recycling of autolysosome membranes via autophagic lysosome reformation (ALR). However, these lysosome repopulation processes do not operate under nutrient-rich conditions. In our recent study, we identify a sequential phosphoinositide conversion pathway that enables lysosome repopulation under nutrient-rich conditions to facilitate basal autophagy. Phosphatidylinositol-3,4-bisphosphate (PtdIns[3,4]P₂) signals generated downstream of phosphoinositide 3-kinase alpha (PI3Kα) during growth factor stimulation are converted to phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes by INPP4B (inositol polyphosphate-4-phosphatase type II B). We show that PtdIns3P is retained as endosomes mature into endolysosomes, and serves as a substrate for PIKFYVE (phosphoinositide kinase, FYVE-type zinc finger containing) to generate phosphatidylinositol-3,5-bisphosphate (PtdIns[3,5]P₂) to promote SNX2-dependent lysosome reformation, basal autophagic flux and protein aggregate degradation. Therefore, endosome maturation couples nutrient signaling to lysosome repopulation during basal autophagy by delivering PI3Kα-derived PtdIns3P to endolysosomes for PtdIns(3,5)P₂-dependent lysosome reformation.

Abbreviations: ALR: autophagic lysosome reformation; INPP4B: inositol polyphosphate-4-phosphatase type II B; PI3Kα: phosphoinositide 3-kinase alpha; PIKFYVE: phosphoinositide kinase FYVE-type zinc finger containing; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,4)P₂: phosphatidylinositol-3,4-bisphosphate; PtdIns(3,5)P₂ phosphatidylinositol-3,5-bisphosphate; SNX2 sorting nexin 2; PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3.

Phosphoinositides are a family of seven distinct membrane-bound signaling lipids transiently generated on the inner leaflet of the plasma membrane and intracellular membranes via the coordinated functions of phosphoinositide kinases and phosphatases. Phosphoinositides elicit localized changes to membrane ultrastructure and signaling, and are essential for autophagy regulation. PtdIns3P is generated during starvation-induced autophagy by PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) to promote autophagosome biogenesis including phagophore expansion and closure, as well as autophagosome-lysosome fusion and ALR. An alternate pathway for PtdIns3P generation also occurs in response to growth factor stimulation, which activates PI3Kα at the plasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate (PtdIns[3,4,5]P₃). INPP5 (inositol polyphosphate-5-phosphatase) enzymes hydrolyze PtdIns(3,4,5)P₃ to PtdIns(3,4)P₂, which is subsequently hydrolyzed to PtdIns3P by INPP4B on endosomes, however, the function of INPP4B-generated PtdIns3P during autophagy is unclear.

We undertook a systematic evaluation of autophagy in mammalian cells with INPP4B depletion or overexpression to determine whether PtdIns3P synthesis by INPP4B, downstream of PI3Kα, contributes to autophagy regulation [1]. Our analysis reveals that INPP4B depletion reduces the number of lysosomes and suppresses basal autophagic flux under nutrient-rich conditions in multiple cell lines but has little effect during starvation-induced autophagy. In contrast, INPP4B overexpression increases lysosome numbers and basal autophagic flux, events that are blocked using PI3Kα pharmacological inhibition or depletion. These data suggest that PI3Kα-derived PtdIns3P is required for lysosome homeostasis and basal autophagy during nutrient-rich conditions. Critically, INPP4B regulation of basal autophagy is independent of the canonical PIK3C3-dependent PtdIns3P autophagosome biogenesis pathway.

Our previous study revealed that INPP4B localizes to endosomes where it dephosphorylates PtdIns(3,4)P₂ to PtdIns3P and recruits the PtdIns3P-binding effector HGS/HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), a member of the endosomal sorting complex required for transport (ESCRT) that is required for intralumenal vesicle formation, endosome maturation and the subsequent fusion of endosomes with lysosomes to form endolysosomes. In the current study, we show that although INPP4B does not localize to lysosomes, PtdIns3P signals generated by INPP4B on endosomes are retained as endosomes mature into endolysosomes. This is functionally significant as inhibition of endosome maturation via HGS depletion reduces lysosomal membrane-associated PtdIns3P, lysosome numbers and basal autophagic flux, suggesting that delivery of endosomal PtdIns3P to endolysosomes is required for lysosome homeostasis and basal autophagy.

PIKFYVE phosphorylates endolysosomal PtdIns3P to PtdIns(3,5)P₂ to promote lysosome reformation from endolysosomes, a process whereby endolysosome membranes undergo tubulation and scission to regenerate new lysosomes. Our investigation reveals that PIKFYVE depletion or pharmacological inhibition prevents the increased lysosome numbers and enhanced basal autophagic flux in INPP4B-overexpressing cells. PIKFYVE inactivation in INPP4B-overexpressing cells also additively increases lysosomal PtdIns3P, suggesting that INPP4B-generated PtdIns3P functions as a substrate for PIKFYVE phosphorylation to PtdIns(3,5)P₂. Using rapid live cell imaging via spinning disk microscopy, our report demonstrates that INPP4B enhances the reformation of lysosomes from endolysosomes. Furthermore, the PtdIns(3,5)P₂-binding protein SNX2, which detects and promotes membrane curvature, is recruited to endolysosomes to facilitate the formation of lysosome reformation tubules. Finally, inactivation of INPP4B or PIKFYVE results in protein aggregate accumulation and cell death during proteotoxic stress, suggesting INPP4B-PIKFYVE-dependent lysosome reformation is required for the autophagic clearance of protein aggregates and cytoprotection.

Collectively, our findings uncover a molecular pathway that regulates lysosome repopulation during basal autophagy, whereby PI3Kα signaling initiates a phosphoinositide conversion pathway under nutrient-rich conditions resulting in PtdIns3P generation on late endosomes that contributes to PtdIns(3,5)P₂-dependent lysosome reformation (Figure 1). However, this pathway is dispensable during starvation-induced autophagy, consistent with evidence that low growth factor availability suppresses PI3Kα-dependent function. Whether cells utilize distinct membrane sources for lysosome reformation during autophagy under different nutrient conditions, whereby lysosomes are derived from endolysosomes under nutrient-rich conditions versus autolysosomes under starvation conditions during ALR, remains an unanswered question for future investigation.

Figure 1.

Under nutrient-rich conditions, sequential phosphoinositide conversion via endosomes delivers PtdIns3P to endolysosomes for conversion to PtdIns(3,5)P₂ to regulate lysosome reformation, basal autophagy and proteostasis. Basal autophagy enables protein quality control by degrading protein aggregates, thereby protecting against cellular stress. PtdIns(4,5)P₂ is converted to PtdIns(3,4,5)P₃ by PI3Kα at the plasma membrane in response to growth factor stimulation, which is subsequently dephosphorylated by INPP5 (inositol polyphosphate-5-phosphatase) enzymes to form PtdIns(3,4)P₂. A pool of PtdIns(3,4)P₂ is internalized during endocytosis and is maintained on endosomes where it is dephosphorylated by INPP4B to form PtdIns3P. Following endosome maturation to endolysosomes, PtdIns3P is phosphorylated by PIKFYVE to generate PtdIns(3,5)P₂, which recruits SNX2. SNX2 promotes lysosome reformation to facilitate basal autophagic degradation and protein aggregate clearance. When INPP4B is inactivated, PtdIns(3,4)P₂ is not converted to PtdIns3P at endosomes resulting in inhibition of endosome maturation and lysosome reformation. PIKFYVE inactivation prevents SNX2 recruitment to endolysosomes, suppressing lysosome reformation. As a consequence, inactivation of INPP4B or PIKFYVE decreases lysosomes thereby reducing autophagy function and leading to the accumulation of protein aggregates and increased proteotoxic stress.

Critically, we demonstrate that endosome maturation is essential for coupling nutrient availability to lysosome homeostasis during basal autophagy. There is significant crosstalk between the endosomal and autophagy pathways, including the derivation of autophagic membranes from endocytic structures. Our study reports that the maturation of endosomes into endolysosomes is required to deliver PI3Kα-derived PtdIns3P to endolysosomes as a substrate for PtdIns(3,5)P₂ conversion by PIKFYVE to facilitate lysosome reformation.

Interestingly, loss of function of PIKFYVE complex proteins (PIKFYVE, FIG4 [FIG4 phosphoinositide 5-phosphatase] and VAC14 [VAC14 component of PIKFYVE complex]) are associated with neurological disorders in humans and mice via unknown mechanisms, but the presence of enlarged lysosomes is a common pathological feature observed in these diseases. Given that a defective proteotoxic stress response is associated with neurological disease, our findings raise the intriguing question as to whether dysregulated lysosome reformation and impaired protein aggregate clearance may contribute to the pathogenesis of PIKFYVE complex disorders.

Funding Statement

The work was supported by the Australian Research Council [DP220103810]; Australian Research Council [DP190102499]; Australian Government Research Training Program [N/A]

Disclosure statement

No potential conflict of interest was reported by the author(s).