Lois S. Weisman

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 ETn2β (early transposon 2β) 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.

PI(3,5)P 2 controls membrane trafficking by direct activation of mucolipin Ca 2+ release channels in the endolysosome

Membrane fusion and fission events in intracellular trafficking are controlled by both intraluminal Ca(2+) release and phosphoinositide (PIP) signalling. However, the molecular identities of the Ca(2+) release channels and the target proteins of PIPs are elusive. In this paper, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P(2), an endolysosome-specific PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specificity and potency. Both PI(3,5)P(2)-deficient cells and cells that lack TRPML1 exhibited enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathway. We find that the enlarged vacuole phenotype observed in PI(3,5)P(2)-deficient mouse fibroblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P(2)-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca(2+) release from the vacuole. In this study, we show that this release requires both PI(3,5)P(2) production and a yeast functional TRPML homologue. We propose that TRPMLs regulate membrane trafficking by transducing information regarding PI(3,5)P(2) levels into changes in juxtaorganellar Ca(2+), thereby triggering membrane fusion/fission events.

Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p.

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P2) was first identified as a nonabundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P2 via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P2 synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P2 levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P2 are 18–28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P2. After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P2 rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P2 plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P2 continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P2 and provide insight into why PtdIns(3,5)P2 levels rise in response to osmotic stress.

Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice

The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P2 synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4, presents the unexpected finding that the housekeeping lipid, PI(3,5)P2, is critical for the survival of neural cells.

VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P2 in yeast and mouse

The signalling lipid PI(3,5)P2 is generated on endosomes and regulates retrograde traffic to the trans‐Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P2 levels. Mutations that lower PI(3,5)P2 cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P2 was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P2 regulatory complex by direct contact with the known regulators of PI(3,5)P2: Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P2 regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P2. Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse.

Structural basis for myosin V discrimination between distinct cargoes

Myosin V molecular motors move cargoes on actin filaments. A myosin V may move multiple cargoes to distinct places at different times. The cargoes attach to the globular tail of myosin V via cargo‐specific receptors. Here we report the crystal structure at 2.2 Å of the myosin V globular tail. The overall tertiary structure has not been previously observed. There are several patches of highly conserved regions distributed on the surface of the tail. These are candidate attachment sites for cargo‐specific receptors. Indeed, we identified a region of five conserved surface residues that are solely required for vacuole inheritance. Likewise, we identified a region of five conserved surface residues that are required for secretory vesicle movement, but not vacuole movement. These two regions are at opposite ends of the oblong‐shaped cargo‐binding domain, and moreover are offset by 180°. The fact that the cargo‐binding areas are distant from each other and simultaneously exposed on the surface of the globular tail suggests that major targets for the regulation of cargo attachment are organelle‐specific myosin V receptors.

The Vac14p–Fig4p complex acts independently of Vac7p and couples PI3,5P2 synthesis and turnover

Phosphoinositide-signaling lipids function in diverse cellular pathways. Dynamic changes in the levels of these signaling lipids regulate multiple processes. In particular, when Saccharomyces cerevisiae cells are exposed to hyperosmotic shock, PI3,5P2 (phosphatidylinositol [PI] 3,5-bisphosphate) levels transiently increase 20-fold. This causes the vacuole to undergo multiple acute changes. Control of PI3,5P2 levels occurs through regulation of both its synthesis and turnover. Synthesis is catalyzed by the PI3P 5-kinase Fab1p, and turnover is catalyzed by the PI3,5P2 5-phosphatase Fig4p. In this study, we show that two putative Fab1p activators, Vac7p and Vac14p, independently regulate Fab1p activity. Although Vac7p only regulates Fab1p, surprisingly, we find that Vac14 regulates both Fab1p and Fig4p. Moreover, Fig4p itself functions in both PI3,5P2 synthesis and turnover. In both the absence and presence of Vac7p, the Vac14p–Fig4p complex controls the hyperosmotic shock–induced increase in PI3,5P2 levels. These findings suggest that the dynamic changes in PI3,5P2 are controlled through a tight coupling of synthesis and turnover.

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)P2] lead to profound neurodegeneration in mice. Moreover, mutations in human FIG4 predicted to lower PI(3,5)P2 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)P2 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)P2, suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we characterize a Pikfyve gene-trap mouse (Pikfyveβ-geo/β-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)P2 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)P2, likely via 3′-phosphatase activity. Analysis of tissues from the Pikfyveβ-geo/β-geo mouse mutants reveals that Pikfyve is critical in neural tissues, heart, lung, kidney, thymus, and spleen. Thus, PI(3,5)P2 and PI5P have major roles in multiple organs. Understanding the regulation of these lipids may provide insights into therapies for multiple diseases.

Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology.

During cell division, the vacuole of Saccharomyces cerevisiae partitions between mother and daughter cells. A portion of the parental vacuole membrane moves into the bud, and ultimately membrane scission divides the vacuole into two separate structures. Here we characterize two yeast mutations causing defects in vacuole membrane scission, vac7-1 and vac14-1. A third mutant, afab1-2 strain, isolated in a nonrelated screen (A. Yamamoto et al., Mol. Biol. Cell 6:525-539, 1995) shares the vacuolar phenotypes of the vac7-1 and vac14-1 strains. Unlike the wild type, mutant vacuoles are not multilobed structures; in many cases, a single vacuole spans both the mother and bud, with a distinct gap in the mother-bud neck. Thus, even where the membranes are closely opposed, vacuole fission is arrested. Simply enlarging the vacuole does not produce this mutant phenotype. An additional common phenotype of these mutants is a defect in vacuole acidification; however, vacuole scission in most other vacuole acidification mutants is normal. An alteration in vacuole membrane lipids could account for both the vacuole membrane scission and acidification defects. Because a directed screen has not identified additional class III complementation groups, it is likely that all three genes are involved in a similar process. Interestingly, FAB1, was previously shown to encode a putative phosphatidylinositol-4-phosphate 5-kinase. Moreover, overexpression of FAB1 suppresses the vac14-1 mutation, which suggests that VAC14 and FAB1 act at a common step. VAC7 encodes a novel 128-kDa protein that is localized at the vacuole membrane. This location of Vac7p is consistent with its involvement in vacuole morphology and inheritance.

Regulated degradation of a class V myosin receptor directs movement of the yeast vacuole

Normal cellular function requires that organelles be positioned in specific locations. The direction in which molecular motors move organelles is based in part on the polarity of microtubules and actin filaments. However, this alone does not determine the intracellular destination of organelles. For example, the yeast class V myosin, Myo2p, moves several organelles to distinct locations during the cell cycle. Thus the movement of each type of Myo2p cargo must be regulated uniquely. Here we report a regulatory mechanism that specifically provides directionality to vacuole movement. The vacuole-specific Myo2p receptor, Vac17p, has a key function in this process. Vac17p binds simultaneously to Myo2p and to Vac8p, a vacuolar membrane protein. The transport complex, Myo2p–Vac17p–Vac8p, moves the vacuole to the bud, and is then disrupted through the degradation of Vac17p. The vacuole is ultimately deposited near the centre of the bud. Removal of a PEST sequence (a potential signal for rapid protein degradation) within Vac17p causes its stabilization and the subsequent ‘backward’ movement of vacuoles, which mis-targets them to the neck between the mother cell and the bud. Thus the regulated disruption of this transport complex places the vacuole in its proper location. This may be a general mechanism whereby organelles are deposited at their terminal destination.