Close encounters of the lysosome-peroxisome kind.

Lysosomes provide a major source for cellular cholesterol; however, most of this cholesterol is trafficked to the plasma membrane via unknown mechanisms. Chu et al. identify an unexpected role for peroxisomes in the transport of cholesterol from the lysosome to the plasma membrane via a lysosome-peroxisome membrane contact site.

Insights into Lysosomal PI(3,5)P2 Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex.

The phosphoinositide PI(3,5)P2, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P2 levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P2 from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P2 production by PIKfyve in vivo.

Bur1 functions with TORC1 for vacuole-mediated cell cycle progression.

The vacuole/lysosome plays essential roles in the growth and proliferation of many eukaryotic cells via the activation of target of rapamycin complex 1 (TORC1). Moreover, the yeast vacuole/lysosome is necessary for progression of the cell division cycle, in part via signaling through the TORC1 pathway. Here, we show that an essential cyclin-dependent kinase, Bur1, plays a critical role in cell cycle progression in cooperation with TORC1. A mutation in BUR1 combined with a defect in vacuole inheritance shows a synthetic growth defect. Importantly, the double mutant, as well as a bur1-267 mutant on its own, has a severe defect in cell cycle progression from G1 phase. In further support that BUR1 functions with TORC1, mutation of bur1 alone results in high sensitivity to rapamycin, a TORC1 inhibitor. Mechanistic insight for Bur1 function comes from the findings that Bur1 directly phosphorylates Sch9, a target of TORC1, and that both Bur1 and TORC1 are required for the activation of Sch9. Together, these discoveries suggest that multiple signals converge on Sch9 to promote cell cycle progression.

The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.

Let it go: mechanisms that detach myosin V from the yeast vacuole.

A major question in cell biology is, how are organelles and macromolecular machines moved within a cell? The delivery of cargoes to the right place at the right time within a cell is critical to cellular health. Failure to do so is often catastrophic for animal physiology and results in diseases of the gut, brain, and skin. In budding yeast, a myosin V motor, Myo2, moves cellular materials from the mother cell into the growing daughter bud. Myo2-based transport ensures that cellular contents are shared during cell division. During transport, Myo2 is often linked to its cargo via cargo-specific adaptor proteins. This simple organism thus serves as a powerful tool to study how myosin V moves cargo, such as organelles. Some critical questions include how myosin V moves along the actin cytoskeleton, or how myosin V attaches to cargo in the mother. Other critical questions include how the cargo is released from myosin V when it reaches its final destination in the bud. Here, we review the mechanisms that regulate the vacuole-specific adaptor protein, Vac17, to ensure that Myo2 delivers the vacuole to the bud and releases it at the right place and the right time. Recent studies have revealed that Vac17 is regulated by ubiquitylation and phosphorylation events that coordinate its degradation and the detachment of the vacuole from Myo2. Thus, multiple post-translational modifications tightly coordinate cargo delivery with cellular events. It is tempting to speculate that similar mechanisms regulate other cargoes and molecular motors.

PI5P and PI(3,5)P2: Minor, but Essential Phosphoinositides.

In most eukaryotes, phosphoinositides (PIs) have crucial roles in multiple cellular functions. Although the cellular levels of phosphatidylinositol 5-phosphate (PI5P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) are extremely low relative to some other PIs, emerging evidence demonstrates that both lipids are crucial for the endocytic pathway, intracellular signaling, and adaptation to stress. Mutations that causes defects in the biosynthesis of PI5P and PI(3,5)P2 are linked to human diseases including neurodegenerative disorders. Here, we review recent findings on cellular roles of PI5P and PI(3,5)P2, as well as the pathophysiological importance of these lipids.Key words: Phosphoinositides, Membrane trafficking, Endocytosis, Vacuoles/Lysosomes, Fab1/PIKfyve.

Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression.

Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Normal steady-state levels of the signalling lipids PI(3,5)P(2) and PI(5)P require the lipid kinase FAB1/PIKfyve and its regulators, VAC14 and FIG4. Mutations in the PIKfyve/VAC14/FIG4 pathway are associated with Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis in humans, and profound neurodegeneration in mice. Hence, tight regulation of this pathway is critical for neural function. Here, we examine the localization and physiological role of VAC14 in neurons. We report that endogenous VAC14 localizes to endocytic organelles in fibroblasts and neurons. Unexpectedly, VAC14 exhibits a pronounced synaptic localization in hippocampal neurons, suggesting a role in regulating synaptic function. Indeed, the amplitude of miniature excitatory postsynaptic currents is enhanced in both Vac14(-/-) and Fig4(-/-) neurons. Re-introduction of VAC14 in postsynaptic Vac14(-/-) cells reverses this effect. These changes in synaptic strength in Vac14(-/-) neurons are associated with enhanced surface levels of the AMPA-type glutamate receptor subunit GluA2, an effect that is due to diminished regulated endocytosis of AMPA receptors. Thus, VAC14, PI(3,5)P(2) and/or PI(5)P play a role in controlling postsynaptic function via regulation of endocytic cycling of AMPA receptors.

Phosphatidylinositol 3,5-bisphosphate: regulation of cellular events in space and time.

Phosphorylated phosphatidylinositol lipids are crucial for most eukaryotes and have diverse cellular functions. The low-abundance signalling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is critical for cellular homoeostasis and adaptation to stimuli. A large complex of proteins that includes the lipid kinase Fab1-PIKfyve, dynamically regulates the levels of PI(3,5)P2. Deficiencies in PI(3,5)P2 are linked to some human diseases, especially those of the nervous system. Future studies will probably determine new, undiscovered regulatory roles of PI(3,5)P2, as well as uncover mechanistic insights into how PI(3,5)P2 contributes to normal human physiology.

Phosphatidylinositol 3,5-bisphosphate: low abundance, high significance.

Recent studies of the low abundant signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2 ), reveal an intriguingly diverse list of downstream pathways, the intertwined relationship between PI(3,5)P2 and PI5P, as well as links to neurodegenerative diseases. Derived from the structural lipid phosphatidylinositol, PI(3,5)P2 is dynamically generated on multiple cellular compartments where interactions with an increasing list of effectors regulate many cellular pathways. A complex of proteins that includes Fab1/PIKfyve, Vac14, and Fig4/Sac3 mediates the biosynthesis of PI(3,5)P2 , and mutations that disrupt complex function and/or formation cause profound consequences in cells. Surprisingly, mutations in this pathway are linked with neurological diseases, including Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis. Future studies of PI(3,5)P2 and PI5P are likely to expand the roles of these lipids in regulation of cellular functions, as well as provide new approaches for treatment of some neurological diseases.

Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics.

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P2 exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P2-metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI(3,5)P2, however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI(3,5)P2 binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P2-specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P2. Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P2 probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P2-specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.

In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the major precursor for PI5P.

Mutations that cause defects in levels of the signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P(2)] lead to profound neurodegeneration in mice. Moreover, mutations in human FIG4 predicted to lower PI(3,5)P(2) levels underlie Charcot-Marie-Tooth type 4J neuropathy and are present in selected cases of amyotrophic lateral sclerosis. In yeast and mammals, PI(3,5)P(2) is generated by a protein complex that includes the lipid kinase Fab1/Pikfyve, the scaffolding protein Vac14, and the lipid phosphatase Fig4. Fibroblasts cultured from Vac14(-/-) and Fig4(-/-) mouse mutants have a 50% reduction in the levels of PI(3,5)P(2), suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we characterize a Pikfyve gene-trap mouse (Pikfyve(ß-geo/ß-geo)), a hypomorph with ~10% of the normal level of Pikfyve protein. shRNA silencing of the residual Pikfyve transcript in fibroblasts demonstrated that Pikfyve is required to generate all of the PI(3,5)P(2) pool. Surprisingly, Pikfyve also is responsible for nearly all of the phosphatidylinositol-5-phosphate (PI5P) pool. We show that PI5P is generated directly from PI(3,5)P(2), likely via 3'-phosphatase activity. Analysis of tissues from the Pikfyve(ß-geo/ß-geo) mouse mutants reveals that Pikfyve is critical in neural tissues, heart, lung, kidney, thymus, and spleen. Thus, PI(3,5)P(2) and PI5P have major roles in multiple organs. Understanding the regulation of these lipids may provide insights into therapies for multiple diseases.

Pathogenic mechanism of the FIG4 mutation responsible for Charcot-Marie-Tooth disease CMT4J.

CMT4J is a severe form of Charcot-Marie-Tooth neuropathy caused by mutation of the phosphoinositide phosphatase FIG4/SAC3. Affected individuals are compound heterozygotes carrying the missense allele FIG4-I41T in combination with a null allele. Analysis using the yeast two-hybrid system demonstrated that the I41T mutation impairs interaction of FIG4 with the scaffold protein VAC14. The critical role of this interaction was confirmed by the demonstration of loss of FIG4 protein in VAC14 null mice. We developed a mouse model of CMT4J by expressing a Fig4-I41T cDNA transgene on the Fig4 null background. Expression of the mutant transcript at a level 5 × higher than endogenous Fig4 completely rescued lethality, whereas 2 × expression gave only partial rescue, providing a model of the human disease. The level of FIG4-I41T protein in transgenic tissues is only 2% of that predicted by the transcript level, as a consequence of the protein instability caused by impaired interaction of the mutant protein with VAC14. Analysis of patient fibroblasts demonstrated a comparably low level of mutant I41T protein. The abundance of FIG4-I41T protein in cultured cells is increased by treatment with the proteasome inhibitor MG-132. The data demonstrate that FIG4-I41T is a hypomorphic allele encoding a protein that is unstable in vivo. Expression of FIG4-I41T protein at 10% of normal level is sufficient for long-term survival, suggesting that patients with CMT4J could be treated by increased production or stabilization of the mutant protein. The transgenic model will be useful for testing in vivo interventions to increase the abundance of the mutant protein.

Genetic interaction between MTMR2 and FIG4 phospholipid phosphatases involved in Charcot-Marie-Tooth neuropathies.

We previously reported that autosomal recessive demyelinating Charcot-Marie-Tooth (CMT) type 4B1 neuropathy with myelin outfoldings is caused by loss of MTMR2 (Myotubularin-related 2) in humans, and we created a faithful mouse model of the disease. MTMR2 dephosphorylates both PtdIns3P and PtdIns(3,5)P(2), thereby regulating membrane trafficking. However, the function of MTMR2 and the role of the MTMR2 phospholipid phosphatase activity in vivo in the nerve still remain to be assessed. Mutations in FIG4 are associated with CMT4J neuropathy characterized by both axonal and myelin damage in peripheral nerve. Loss of Fig4 function in the plt (pale tremor) mouse produces spongiform degeneration of the brain and peripheral neuropathy. Since FIG4 has a role in generation of PtdIns(3,5)P(2) and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P(2) homeostasis and their mutations might have compensatory effects in vivo. To explore the role of the MTMR2 phospholipid phosphatase activity in vivo, we generated and characterized the Mtmr2/Fig4 double null mutant mice. Here we provide strong evidence that Mtmr2 and Fig4 functionally interact in both Schwann cells and neurons, and we reveal for the first time a role of Mtmr2 in neurons in vivo. Our results also suggest that imbalance of PtdIns(3,5)P(2) is at the basis of altered longitudinal myelin growth and of myelin outfolding formation. Reduction of Fig4 by null heterozygosity and downregulation of PIKfyve both rescue Mtmr2-null myelin outfoldings in vivo and in vitro.

Rab5 proteins regulate activation and localization of target of rapamycin complex 1.

The mechanistic target of rapamycin (mTOR) complex 1 is regulated by small GTPase activators and localization signals. We examine here the role of the small GTPase Rab5 in the localization and activation of TORC1 in yeast and mammalian cells. Rab5 mutants disrupt mTORC1 activation and localization in mammalian cells, whereas disruption of the Rab5 homolog in yeast, Vps21, leads to decreased TORC1 function. Additionally, regulation of PI(3)P synthesis by Rab5 and Vps21 is essential for TORC1 function in both contexts.

Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J.

Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells. Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a phospholipid present in small quantities that regulates membrane trafficking in the endosome-lysosome axis in yeast. Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2beta (early transposon 2beta) into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC (suppressor of actin) domain PtdIns(3,5)P2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns(3,5)P2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosome-lysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated patients with hereditary motor and sensory neuropathy. This novel form of autosomal recessive Charcot-Marie-Tooth disorder is designated CMT4J.

A signaling lipid drives synapse formation.

Phosphatidylinositol 3,5-bisphosphate enables transport of proteins to synaptic sites.

Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

Trafficking of cell-surface proteins from endosomes to the plasma membrane is a key mechanism to regulate synaptic function. In non-neuronal cells, proteins recycle to the plasma membrane either via the SNX27-Retromer-WASH pathway or via the recently discovered SNX17-Retriever-CCC-WASH pathway. While SNX27 is responsible for the recycling of key neuronal receptors, the roles of SNX17 in neurons are less understood. Here, using cultured hippocampal neurons, we demonstrate that the SNX17 pathway regulates synaptic function and plasticity. Disruption of this pathway results in a loss of excitatory synapses and prevents structural plasticity during chemical long-term potentiation (cLTP). cLTP drives SNX17 recruitment to synapses, where its roles are in part mediated by regulating the surface expression of ß1-integrin. SNX17 recruitment relies on NMDAR activation, CaMKII signaling, and requires binding to the Retriever and PI(3)P. Together, these findings provide molecular insights into the regulation of SNX17 at synapses and define key roles for SNX17 in synaptic maintenance and in regulating enduring forms of synaptic plasticity.

Upregulation of the ESCRT pathway and multivesicular bodies accelerates degradation of proteins associated with neurodegeneration.

Many neurodegenerative diseases, including Huntington's disease (HD) and Alzheimer's disease (AD), occur due to an accumulation of aggregation-prone proteins, which results in neuronal death. Studies in animal and cell models show that reducing the levels of these proteins mitigates disease phenotypes. We previously reported a small molecule, NCT-504, which reduces cellular levels of mutant huntingtin (mHTT) in patient fibroblasts as well as mouse striatal and cortical neurons from an HdhQ111 mutant mouse. Here, we show that NCT-504 has a broader potential, and in addition reduces levels of Tau, a protein associated with Alzheimer's disease, as well as other tauopathies. We find that in untreated cells, Tau and mHTT are degraded via autophagy. Notably, treatment with NCT-504 diverts these proteins to multivesicular bodies (MVB) and the ESCRT pathway. Specifically, NCT-504 causes a proliferation of endolysosomal organelles including MVB, and an enhanced association of mHTT and Tau with endosomes and MVB. Importantly, depletion of proteins that act late in the ESCRT pathway blocked NCT-504 dependent degradation of Tau. Moreover, NCT-504-mediated degradation of Tau occurred in cells where Atg7 is depleted, which indicates that this pathway is independent of canonical autophagy. Together, these studies reveal that upregulation of traffic through an ESCRT-dependent MVB pathway may provide a therapeutic approach for neurodegenerative diseases.