PIKfyve Regulates Vacuole Maturation and Nutrient Recovery following Engulfment

Abstract

Summary The scavenging of extracellular macromolecules by engulfment can sustain cell growth in a nutrient-depleted environment. Engulfed macromolecules are contained within vacuoles that are targeted for lysosome fusion to initiate degradation and nutrient export. We have shown that vacuoles containing engulfed material undergo mTORC1-dependent fission that redistributes degraded cargo back into the endosomal network. Here we identify the lipid kinase PIKfyve as a regulator of an alternative pathway that distributes engulfed contents in support of intracellular macromolecular synthesis during macropinocytosis, entosis, and phagocytosis. We find that PIKfyve regulates vacuole size in part through its downstream effector the cationic transporter TRPML1. Furthermore, PIKfyve promotes recovery of nutrients from vacuoles, suggesting a potential link between PIKfyve activity and lysosomal nutrient export. During nutrient depletion, PIKfyve activity protects Ras-mutant cells from starvation-induced cell death and supports their proliferation. These data identify PIKfyve as a critical regulator of vacuole maturation and nutrient recovery during engulfment.

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

“…However, S6K remained phosphorylated in apilimod-treated cells, suggesting that mTORC1 was active. These observations are consistent with Wang et al and Krishna et al who also showed that mTORC1 retains its activity in PIKfyve-abrogated cells (Wang et al, 2015;Krishna et al, 2016). We considered the possibility that mTORC1 and starvation controls PIKfyve activity instead, which may then modulate TFEB, perhaps by a PtdIns(3,5)P 2 -dependent localization of TFEB to lysosomes, as has been shown for Tup1, a transcription factor in yeast that controls galactose metabolism (Han and Emr, 2011).…”

“…We do not understand how PIKfyve modulates lysosome fission dynamics. However, we speculate that mechanisms that offset lysosome coalescence are distinct from those that drive vesiculation and recycling (Krishna et al, 2016;Bryant et al, 1998;Rutherford et al, 2006). One interesting possibility may relate to contact sites between endo/lysosome and the endoplasmic reticulum since these have been shown to demarcate and assemble molecular machines that catalyse endolysosome fission (Friedman et al, 2013;Rowland et al, 2014).…”

“…In addition, PIKfyve has been suggested to control mTORC1 in adipocytes , though others have noted that mTOR activity is sustained in PIKfyveinhibited cells (Wang et al, 2015;Krishna et al, 2016). To better assess the possibility that PIKfyve controls TFEB by coordinating with mTOR in our cells, we examined the phosphorylation state of S6K, a major mTORC1 target, in cells treated with apilimod.…”

A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles

“…Because of this specialized function, lysosomes receive cargo via multiple pathways, including macroautophagy (autophagy hereafter) and macropinocytosis, which allows for scavenging of intracellular and extracellular nutrients, respectively (1). Autophagy and macropinocytosis are both regulated by the mammalian target of rapamycin complex 1 (mTORC1) (2). In turn, the lysosome membrane directly regulates mTORC1 by housing key mTOR regulators.…”

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“…Macropinocytosis has been shown to act similarly to allow cancer cells to scavenge extracellular proteins [5]. Entosis instead supplies bulk nutrients in the form of whole cells, where, on average, winner cells ingest two losers, providing a large nutrient supply that is well suited to support the outgrowth of selected cells in the population.…”