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PtdIns(4,5)P2 and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance

A coincidence detection mechanism regulates phagosomal sealing and couples it with phosphoinositide conversion from PtdIns(4,5)P2 enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes.


Introduction
Phosphoinositides (PIs) are concentrated on different organelles to direct signaling and membrane trafficking events. For example, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5) P 2 ) is enriched in the inner leaflet of plasma membranes and is involved in almost all cellular events occurring at the cell surface, whereas phosphatidylinositol-3-phosphate (PtdIns3P) is concentrated on early endosomes and is a key determinant of endosome maturation (Di Paolo and De Camilli, 2006;Bohdanowicz and Grinstein, 2013). The differential distribution of PIs is determined by PI kinases and phosphatases that catalyze local PI synthesis and turnover. PIs mediate signaling or trafficking events by affecting activities of integral membrane proteins or recruiting cytosolic effectors to specific membrane compartments (Di Paolo and De Camilli, 2006;Balla, 2013). PI-protein interactions mediated by electrostatic interaction or by protein modules are of low affinity, and stable protein-membrane association requires additional lipid, protein, or geometry determinants within the membrane (Carlton and Cullen, 2005;Di Paolo and De Camilli, 2006). For instance, EEA1 binds both PtdIns3P and phosphatidylserine through distinct sites in the FYVE domain, suggesting that it can be enriched on membranes containing both lipids (Kutateladze et al., 2004;Carlton and Cullen, 2005). EEA1 also binds RAB-5 and is recruited to early endosomes through both lipid and RAB-5 binding (Lawe et al., 2000). Moreover, SNX1 contains both a PtdIns3P-binding PX domain and a curvature-sensing BAR (Bin-Amphiphysin-Rvs) domain and localizes to high-curvature membrane tubules emanating from endosomes, indicating that membrane geometry can also be a localization cue (Carlton et al., 2004;Carlton and Cullen, 2005). Thus, multiple components of a given membrane serve as the coincidence detection code to define organelle identity and direct membrane trafficking by controlling the spatial and temporal recruitment and release of effectors. Nascent transport vesicles must change identity through interconversion of PIs before fusing with the next membrane. How PI conversion is controlled and coordinated with fusion and/or fission events remains poorly understood.
Eukaryotic cells engulf and eliminate large particles like microorganisms and apoptotic cells through phagocytosis, which involves many typical features of vesicle biogenesis and maturation (Flannagan et al., 2012). In Caenorhabditis elegans hermaphrodites, 131 somatic cells and about half of the germ cells die through apoptosis, and the resulting cell corpses are phagocytosed and cleared by neighboring cells (Pinto and Hengartner, 2012). Apoptotic cells are recognized and engulfed through evolutionarily conserved pathways, leading to cytoskeleton reorganization and formation of a Phagocytosis requires phosphoinositides (PIs) as both signaling molecules and localization cues. How PIs coordinate to control phagosomal sealing and the accompanying switch of organelle identity is unclear. In this study, we followed dynamic changes in PIs during apoptotic cell clearance in Caenorhabditis elegans. We found that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) and phosphatidylinositol-3-phosphate (PtdIns3P), which accumulate transiently on unsealed and fully sealed phagosomes, respectively, are both involved in phagosome closure. We identified PtdIns3P phosphatase MTM-1 as an effector of PtdIns(4,5)P 2 to promote phagosomal sealing. MTM-1 coordinates with the class II PI3 kinase PIKI-1 to control PtdIns3P levels on unsealed phagosomes. The SNX9 family protein LST-4 is required for sealing, and its association with unsealed phagosomes is regulated by PtdIns(4,5)P 2 , PIKI-1, and MTM-1. Loss of LST-4 or its retention on phagosomes disrupts sealing and suppresses PtdIns3P accumulation, indicating close coupling of the two events. Our findings support a coincidence detection mechanism by which phagosomal sealing is regulated and coupled with conversion from PtdIns(4,5)P 2 enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes.
PtdIns(4,5)P 2 plays a key role in forming phagosomes that enclose invading microorganisms or opsonized particles (Flannagan et al., 2012;Bohdanowicz and Grinstein, 2013). PtdIns(4,5)P 2 is present constitutively in the inner leaflet of plasma membranes, enriches transiently in advancing pseudopods, and decreases rapidly when phagosomes seal (Botelho et al., 2000;Scott et al., 2005). The dynamic change in PtdIns(4,5)P 2 levels during phagocytosis is thought to contribute to the localized increase in actin polymerization required for pseudopod extension and the subsequent actin disassembly needed for completion of phagocytosis (Scott et al., 2005;Sarantis et al., 2012). PtdIns(4,5)P 2 and PtdIns3P are key markers of unsealed and fully sealed phagosomes, respectively, but how their conversion is controlled to couple with phagosome sealing is unknown. In fact, very little is known about phagosome scission. In clathrin-mediated endocytosis, the large GTPase dynamin and its partner protein sorting nexin 9 (SNX9) are important for vesicle scission (Ramachandran, 2011). SNX9 contains an SH3 domain that interacts with dynamin, a PI-binding PX domain, and a BAR domain that modulates and stabilizes membrane shape (Lundmark and Carlsson, 2009). It contributes to vesicle scission by recruiting and stimulating the GTPase activity of dynamin, as well as coordinating actin polymerization Carlsson, 2004, 2009;Soulet et al., 2005). In C. elegans, apoptotic cell removal requires both DYN-1, the C. elegans dynamin, and LST-4, an SNX9 family protein, which are thought to function early in phagosome maturation (Yu et al., 2006;Chen et al., 2013). LST-4 interacts with DYN-1 and promotes phagosomal association of the latter (Lu et al., 2010;Chen et al., 2013). However, it is unclear whether LST-4 and DYN-1 regulate early maturation events or are involved in the sealing process.
Here, we identify a coincidence detection mechanism that regulates phagosomal sealing through LST-4 and couples sealing with the switch of membrane identity from PtdIns(4,5)P 2 -enriched unsealed phagosomes to PtdIns3P-enriched fully sealed phagosomes.

1(lf) mutants
We found that MTM-1 was enriched on PtdIns(4,5)P 2 -positive extending pseudopods and phagosomes and was released before the appearance of 2xFYVE, RAB-5, and RAB-7, indicating that MTM-1 acts very early during phagosome formation and/or maturation ( Fig. 1 D and Fig. S2, H and I). We isolated a recessive mutation of mtm-1, qx322, which contained a C to T transition that resulted in an early stop codon after Lys99. qx322 worms contained significantly more germ cell corpses than the wild type ( Fig. S2 J). This phenotype is similar to inactivation of mtm-1 by RNAi as previously reported (Neukomm et al., 2011) or gk890934, which contains two missense mutations in mtm-1 (Fig. S2 J). Expression of wild-type, but not catalytically inactive, MTM-1 significantly reduced germ cell corpses in qx322 and gk890934, indicating that phosphatase activity is important for MTM-1 function in apoptotic cell clearance ( Fig. S2 K). Expression of human myotubularin MTM1, which localized to plasma membranes and phagosomal surfaces like worm MTM-1, rescued the cell corpse phenotype of mtm-1(qx322) (Fig. S2, L-N'), suggesting that human MTM1 can substitute for worm MTM-1 in removing germ cell corpses. The persistent cell corpses in mtm-1RNAi animals were all surrounded by Myri-GFP, a plasma membrane reporter, as in gla-3RNAi worms, suggesting that cell corpse recognition and initiation of engulfment are not disrupted (Fig. S2, O-Q). However, mtm-1(lf) phagosomes were not stained by LysoSensor green, indicating defects in phagosomal acidification (Fig. 2, C-D' and O). Moreover, phagosomal association of LST-4, DYN-1, RAB-5, and RAB-7, which are recruited at early or late maturation stages, were all disrupted, and the lysosome reporter NUC-1 was also absent from phagosomes in mtm-1RNAi animals (Fig. 2, E-O; Guo et al., 2010). In contrast, phagosomal labeling by PLCδ1-PH was significantly increased in mtm-1(lf) worms (Fig. 2, A -B' and O). These data indicate that apoptotic cell-containing phagosomes are arrested very early at the PtdIns(4,5)P 2 -positive stage in mtm-1(lf) worms. This is consistent with association of MTM-1 with PtdIns(4,5)P 2 -positive phagosomes and strongly supports the hypothesis that MTM-1 acts at the PtdIns(4,5)P 2 -positive stage to regulate phagocytosis of apoptotic cells.
As sustained MTM-1 abrogates 2xFYVE appearance on phagosomes, we next examined whether loss of mtm-1 increases PtdIn3P accumulation. In mtm-1(qx322) or mtm-1RNAi worms, almost all germ cell corpses were labeled by YFP::2x-FYVE, whereas <50% of them were YFP positive in wild type (Fig. 5, A-C' and G). Moreover, extending pseudopods, which are positive for PLCδ1-PH and MTM-1(C378S) but not 2xFYVE in wild type, were labeled by YFP::2xFYVE in mtm-1(lf) (Fig. 5, H and J; Fig. S3, J-K''; and Video 6). C. elegans germ cell corpses are removed by gonadal sheath cells, which form a single layer covering germline components (Fig. 5 K). In wild-type sheath cells, 2xFYVE formed small puncta in the cytosol but was absent from the cell surface (Fig. 5 L). In mtm-1(qx322), however, YFP::2xFYVE appeared on the surface of sheath cells, similar to PLCδ1-PH, which labels plasma membranes (Fig. 5,M,and N). This suggests that PtdIns3P accumulates on the cell surface in mtm-1(lf) sheath cells. Endosomal and phagosomal labeling by YFP::2xFYVE were also seen in mtm-1(qx322) when images were taken at the top focal plane because of the extremely thin cytosol of the sheath cell layer (Fig. 5,M and N,arrowheads). The cell surface and increased phagosomal labeling of 2xFYVE in mtm-1(qx322) can be suppressed by expressing wild-type but not catalytically inactive MTM-1, indicating that increased PtdIns3P accumulation is caused by loss of MTM-1 function (Fig. S3, L-P). Together, these data indicate that loss of MTM-1 leads to increased Pt-dIns3P accumulation, causing the appearance of 2xFYVE on cell surfaces, extending pseudopods, and phagosomes.
The SH3-PX-BAR protein LST-4 acts at the PtdIns(4,5)P 2 -positive stage to regulate phagosomal sealing Scission of endocytic vesicles requires the large GTPase dynamin and its partner SNX9. In C. elegans, the SNX9 family protein LST-4 acts through DYN-1, the worm dynamin, to regulate the early stage of phagosome maturation Chen et al., 2013). In lst-4 mutants, phagosomes are arrested at the early stage like in mtm-1(lf) (Chen et al., 2013). We found that GFP::LST-4 appeared on extending pseudopods and nascent phagosomes simultaneously with MTM-1(C378S), and its release coincided with, or preceded the enrichment of, 2xFYVE on phagosomes (  Table S1). These data indicate that LST-4 acts at the PtdIns(4,5)P 2 -positive stage in the same pathway with MTM-1 and PIKI-1 to regulate phagosomal sealing.

Sealing of apoptotic cell-containing phagosomes involves both PtdIns(4,5)P 2 and PtdIns3P
PtdIns(4,5)P 2 and PtdIns3P are key determinants of phagosome formation and maturation, respectively (Bohdanowicz and Grinstein, 2013). Here, we found that both are involved in closure of apoptotic cell-containing phagosomes. PtdIns(4,5)P 2 associates with extending pseudopods and unsealed but not sealed phagosomes. Inhibition of PtdIns(4,5)P 2 hydrolysis by ocrl-1 RNAi or its increased generation by PPK-1 overexpression causes defective clearance of apoptotic cells and accumulation of unsealed phagosomes. Thus, like mammalian phagocytosis, sealing of apoptotic cell-containing phagosomes coincides with, and requires timely depletion of, PtdIns(4,5)P 2 . The inositol 5-phosphatase OCRL-1, which contributes to PtdIns(4,5) P 2 elimination in mammalian phagocytosis (Bohdanowicz et al., 2012), is important for this process. Loss of ocrl-1 leads to prolonged association of MTM-1, LST-4, and DYN-1 with unsealed phagosomes, suggesting that defective sealing may be caused by sequestration of sealing factors by PtdIns(4,5)P 2 -enriched membranes. Our finding is consistent with a recent study that shows OCRL is required for dissociation of SNX9 from endocytic vesicles during uncoating (Nández et al., 2014).
Our data indicate that PtdIns3P is produced on plasma membranes and forming phagosomes where PtdIns(4,5)P 2 is enriched, but its accumulation is suppressed by the PtdIns(4,5) P 2 effector MTM-1. Loss of MTM-1 causes PtdIns3P accumulation on plasma membranes, extending pseudopods, and nascent phagosomes and numerous unsealed PtdIns(4,5)P 2 -positive phagosomes. Inactivation of the PI3 kinases PIKI-1 and VPS-34 completely suppresses PtdIns3P accumulation caused by mtm-1 mutation but also leads to defects in phagosomal sealing, indicating that PtdIns3P must be tightly controlled in this process. Consistent with this, the 2xFYVE reporter is only visible on plasma or unsealed phagosomal membranes in mtm-1(lf) but not wild-type worms. The timely depletion of PtdIns(4,5) P 2 by OCRL-1 may release MTM-1, LST-4, and DYN-1, which completes the scission process and allows subsequent accumulation of PtdIns3P on fully sealed phagosomes (Fig. 7 F). In this way, phagosomal sealing and the switch of membrane identity from unsealed (PtdIns(4,5)P 2 enriched) to sealed (PtdIns3P enriched) phagosomes are coregulated and precisely coupled. More work is needed to determine how OCRL-1 is regulated when apoptotic cell-containing phagosomes are being sealed.

The PtdIns(4,5)P 2 effector MTM-1 coordinates with PIKI-1 to control PtdIns3P levels on unsealed phagosomes
Previously, we and others reported that MTM-1 localizes to plasma membranes and negatively regulates cell corpse engulfment through the CED-5/12/10 module (Zou et al., 2009;Neukomm et al., 2011). Here, we found that MTM-1 associates with forming phagosomes to promote sealing. MTM-1 binds specifically to PtdIns(4,5)P 2 , and its membrane association is regulated by the latter. Thus, MTM-1 acts as an effector of Pt-dIns(4,5)P 2 to regulate both initiation and completion of engulfment. The acceleration in the initiation step but impairment in the sealing of phagosomes caused by loss of mtm-1 suggest that PtdIns3P turnover may have distinct effects on apoptotic cell clearance at different stages, a phenomenon that is also observed in autophagy regulation (Vergne et al., 2009;Taguchi-Atarashi et al., 2010;Cebollero et al., 2012;Wu et al., 2014).
Our data indicate that PIKI-1, the class II PI3 kinase, acts with MTM-1 to control a subpool of PtdIns3P on plasma membranes and unsealed phagosomes for cell corpse removal. The class III PI3 kinase VPS-34 may play a major role in producing PtdIns3P on sealed phagosomes, as both MTM-1 and PIKI-1 are released at this stage. Human class II PI3 kinase C2α is reported to control clathrin-mediated endocytosis by generating Pt-dIns(3,4)P 2 (Posor et al., 2013). In our study, however, 2xFYVE but not TAPP1, a PtdIns(3,4)P 2 reporter, is readily seen on phagosomes and abrogated by loss of PIKI-1. Loss or overexpression of R01H10.7, the C. elegans homologue of INPP4A, which converts PtdIns(3,4)P 2 to PtdIns3P, has no effect on cell corpse removal. Moreover, TAPP1 was not observed on phagosomes in r01h10.7(lf) or r01h10.7(lf);mtm-1RNAi worms (Fig. S1, Y and Z). Thus, PtdIns(3,4)P 2 is probably not involved in apoptotic cell clearance either directly or as a precursor of PtdIns3P.
The PI3 kinases PIKI-1 and VPS-34 were reported to regulate phagosome maturation by acting sequentially to control PtdIns3P oscillation on phagosomes, a process antagonized by MTM-1 (Lu et al., 2012). By examining >100 embryonic cell corpses, however, we found that PtdIns3P oscillation is not  readily observed on cell corpses other than the three designated corpses (C1, C2, and C3) on which two waves of 2xFYVE were previously observed and characterized. Thus, PtdIns3P oscillation may associate with clearance of specific corpses like C1, C2, and C3. MTM-1(C378S) and 2xFYVE appear sequentially on the C3 phagosome, and MTM-1 release coincides with 2xFYVE enrichment (Fig. S5 H and Video 9), confirming that MTM-1 acts before PtdIns3P accumulation on phagosomes. MTM-1 remains absent when 2xFYVE disappears and reappears on the C3 phagosome, indicating that MTM-1 is probably not involved in regulating PtdIns3P oscillation on the C3 phagosome (Fig. S5 H and Video 9).
The copresence of PtdIns(4,5)P 2 , PtdIns3P, and MTM-1 recruits LST-4 to unsealed phagosomes LST-4 was reported as a PtdIns3P effector that promotes phagosome maturation (Lu et al., 2010). However, we found that LST-4 associates with phagosomes at the PtdIns(4,5)P 2 -positive stage and is released before PtdIns3P accumulates. This is consistent with the lipid-binding property of LST-4 and two studies showing that LST-4 acts earlier than RAB-5 in cell corpse removal Chen et al., 2013). We observed a large number of unsealed phagosomes in worms lacking LST-4 or DYN-1, indicating essential roles of these proteins in the sealing process. Thus, LST-4 may act with DYN-1 to control scission of apoptotic cell-containing phagosomes as in endocytosis.
Our data strongly support the hypothesis that PtdIns(4,5) P 2 , PtdIns3P, and MTM-1 serve as a coincidence detection code on unsealed phagosomes to recruit LST-4 for the sealing of apoptotic cell-containing phagosomes (Fig. 7 F). LST-4 binds efficiently to PtdIns(4,5)P 2 liposomes in vitro and associates with PtdIns(4,5)P 2 -but not PtdIns3P-enriched phagosomes in vivo, indicating an essential role of PtdIns(4,5)P 2 in membrane recruitment of LST-4. Loss of the PtdIns3P-producing kinase PIKI-1 or the LST-4-interacting PtdIns3P phosphatase MTM-1 disrupts phagosomal association of LST-4, causing accumulation of unsealed phagosomes. This indicates that PtdIns3P is important for LST-4 recruitment and its levels may be tightly controlled during sealing. Expression of MTM-1(C378S), which interacts with LST-4 but lacks catalytic activity, failed to rescue the defective phagosomal association of LST-4 in mtm-1(lf);piki-1(lf) double mutants (Fig. S5, F-G'''), further supporting the role of PtdIns3P in LST-4 recruitment to phagosomes. Addition of PtdIns3P, which by itself displays limited affinity to LST-4, significantly enhances LST-4 binding to PtdIns(4,5) P 2 liposomes in vitro, suggesting that PtdIns3P may facilitate or stabilize the binding of LST-4 with PtdIns(4,5)P 2 . Our results are consistent with a recent study that shows SNX9 is selectively recruited by coincidence of PtdIns(4,5)P 2 and PtdIns3P on curved liposomal membranes (Gallop et al., 2013). Binding of SNX9 to liposomes is significantly increased by the combination of PtdIns(4,5)P 2 and PtdIns3P compared with PtdIns(4,5) P 2 alone and PtdIns3P alone (Gallop et al., 2013). We found that LST-4 interacts with MTM-1 through the lipid-binding PX-BAR domain but not the protein-protein-interacting SH3 domain, raising the possibility that binding of LST-4 to MTM-1 and lipids may be mutually regulated. However, more work is needed to uncover how PtdIns3P and/or MTM-1 may modulate LST-4 binding to PtdIns(4,5)P 2 liposomes or phagosomes.

The PtdIns(4,5)P 2 -PtdIns3P-MTM-1-LST-4 regulatory module may be used in mammals
When expressed in worms, human MTM1 localizes to plasma membranes and efficiently rescues the persistent cell corpse phenotype of mtm-1(lf), suggesting evolutionarily conserved roles of myotubularin in this process. Mutations in MTM1, BIN1 (which encodes an N-BAR and SH3 domain-containing protein), and DNM2 lead to X-linked, autosomal recessive, and autosomal dominant centronuclear myopathies, respectively, and cause similar T tubule defects in skeletal muscle, suggesting that they may act in a common pathway important for muscle function (Cowling et al., 2012). MTM1 interacts with BIN1 in skeletal muscle and enhances BIN1-mediated membrane tubulation dependent on both binding and phosphatase activity (Royer et al., 2013). A PI-binding motif, which is present only in the muscle isoform of BIN1, facilitates Pt-dIns(4,5)P 2 binding and promotes membrane tubulation (Lee et al., 2002). Thus, the PtdIns(4,5)P 2 -PtdIns3P-MTM-1-BAR regulatory module may play a conserved role to maintain muscle function and could be relevant to pathological alterations in centronuclear myopathies.
mtm-1(qx322) was isolated from a genetic screen by defective clearance of residual bodies. qx322 contains a C to T transition that results in an early stop codon after Lys99 and is probably a null mutation of mtm-1. mtm-1(gk890934), which was provided by the Caenorhabditis Genetics Center, contains the amino acid substitutions V188M and R229K. qx322 and gk890934 were out-crossed with the wild-type N2 strain six and four times, respectively, before further analysis.

Quantification of cell corpses and phagosome maturation
The number of somatic cell corpses in the head region of living embryos and the number of germ cell corpses in one gonad arm at various adult ages (24 or 48 h after L4/adult molt) were scored as described previously (Gumienny et al., 1999;Wang et al., 2002). The cell corpses were identified by their raised-button-like morphology using Nomarski optics. At least 15 animals were scored at each stage in each strain. To study apoptotic cell clearance in C. elegans germlines, gla-3 RNAi was performed to increase germ cell apoptosis without affecting cell corpse removal (Kritikou et al., 2006), thus allowing examination of more phagosomes. To induce gene expression by heat-shock treatment, L4 larvae were picked to fresh nematode growth medium plates and treated at 33°C every 12 h. The number of germ cell corpses was scored at 48 h after L4/adult molt. To examine phagosome maturation, differential interference contrast (DIC) and fluorescent images were captured using a microscope (Axioimager A1; Carl Zeiss) as described previously (Lu et al., 2008). The percentage of germ cell corpses labeled by various phagosomal markers was quantified in adult animals (36 or 48 h after L4/ adult molt) by dividing the total number of cell corpses by the number of labeled ones. Corpses were examined at multiple focal planes. At least 15 animals were scored in each strain.

Spinning-disk time-lapse recording
Embryos at the precomma or comma stage, or adult worms at 24 or 36 h after L4/adult molt, were mounted on agar pads in M9 buffer without (embryos) or with 5-mM levamisole (adult worms; levamisole prevents animals from moving but does not affect the germline or the gonad). Fluorescent images were captured using a 100× objective (CFI Plan Apochromat Lambda; NA 1.45; Nikon) with immersion oil (type NF) on an inverted fluorescence microscope (Eclipse Ti-E; Nikon) with a spinning-disk confocal scanner unit (UltraView; PerkinElmer) with 488 (emission filter 525 [W50]) and 561 (dual-band emission filter 445 [W60] and 615 [W70]) lasers. Images in 20-25 z series (1.0 µm/ section) were captured every 1 or 2 min for 1-2 h (embryos) or 2-6 h (adult worms) at 20°C and were viewed and analyzed using Volocity software (PerkinElmer). The numbers of phagosomes that were followed and quantified are indicated in the figures and figure legends. In general, at least 10 phagosomes from three independent animals were followed and quantified in each strain.

FM4-64, Annexin V, and LysoSensor staining
Adult worms (36 or 48 h after L4/adult molt) were dissected to expose their gonads in 200 µl gonad dissection buffer with or without 2 µl Alexa Fluor 488-conjugated Annexin V (Invitrogen; Wang et al., 2007). 155 µl gonad dissection buffer was removed, and 5 µl FM4-64 (200 µg/ml; Invitrogen) was added to make a final concentration of 20 µg/ml. Staining was performed at 4°C for 5 min before examination by fluorescence microscopy. At least 10 animals were scored in each strain. The fluorescence intensity of Annexin V conjugates varied on different apoptotic cells, which is likely the result of differences in phosphoserine levels on cell corpses that persist for different periods of time. LysoSensor staining was performed as described previously with some modifications (Guo et al., 2010). In brief, adult animals (48 h after L4/adult molt) were dissected in gonad dissection buffer with 1-µM LysoSensor green DND-189 (Invitrogen) and examined by fluorescence microscopy.

Visualization of plasma membrane labeling by 2xFYVE in gonadal sheath cells
Adult worms (48 h after L4/adult molt) were dissected in gonad dissection buffer to expose their gonads. The distal gonadal sheath cells near the gonad loop were imaged at the top focal plane using a microscope (Axioimager A1). Three circular regions (3 µm in radius) along the gonad arm were chosen in each image, and the mean intensity per unit area was measured with Axiovision Rel. 4.7 software. At least 10 worms were imaged under the same conditions and quantified in each strain.
(Avanti Polar Lipids, Inc.) in the required molar ratio was prepared in chloroform and dried under nitrogen. Lipid films were hydrated in a buffer containing 50-mM Tris-HCl, pH 7.5, and 150-mM NaCl with a 1-mM total lipid concentration. Unilamellar vesicles (100 nm) were generated using a mini extruder (Avanti Polar Lipids, Inc.). For sedimentation assays, 2.5-30 µg proteins were incubated with 4-50 µl (1 mM) liposomes in a 60-µl reaction at room temperature for 10 min before sedimentation at 60,000 rpm for 40 min in a rotor (TLA-100; Beckman Coulter). Both supernatant and pellet were resolved with SDS-PAGE and revealed by Coomassie blue staining or Western blotting. At least three independent experiments were performed for all liposome sedimentation assays.

RNAi
The bacteria-feeding protocol was used in RNAi experiments. In each experiment, 3-10 L4 larvae (P0) were cultured on the RNAi plate, and F1 progeny at the L4 stage were transferred to fresh RNAi plates and aged for 24, 36, or 48 h before examination of germ cell corpses.

Phosphatase assay
The phosphatase activity of MTM-1 was assayed using a Malachite green assay kit (Echelon Biosciences, Inc.) by measuring the liberated free phosphate after the phosphatase reaction. Experiments were performed according to the manufacturer's instructions with some modifications. In brief, 500 ng His-tagged MTM-1 or MTM-1(C378S) was incubated with 1,000 pmol of substrate (DiC8 PtdIns3P, DiC8 PtdIns(3,5)P 2 , DiC8 PtdIns(4,5)P 2 , DiC8 PtdIns(3,4)P 2 , and DiC8 Pt-dIns(3,4,5)P 3 ; Echelon Biosciences, Inc.) in a 25-µl reaction containing 50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 2-mM CaCl 2 , 2-mM DTT, and 5% glycerol overnight at 20°C. The substrate-only controls were prepared in the same way to measure the background phosphate. Phosphate standards were prepared by diluting the phosphate standard solution provided in the kit according to the instructions. 100 µl Malachite green solution was added to each reaction, control, and phosphate standard for 20 min at room temperature, and absorbance was measured at 620 nm. Averages of triplicate samples were used to draw the phosphate standard curve and determine the free phosphate in each reaction and control.
GST pull-down assay GST::FLAG, GST::MTM-1::FLAG, or GST::MTM-1(C378S)::FLAG immobilized on glutathione-agarose beads was incubated with BL21 bacterial lysates containing His-tagged LST-4 for 2 h at 4°C. The beads were washed extensively with PBS, and the bound proteins were resolved with SDS-PAGE and revealed by Western blotting with an antibody against the hexahistidine tag.

Coimmunoprecipitation assay in worms
Mix-staged worms expressing both LST-4::GFP and mCHER-RY::MTM-1(C378S), or mCHERRY::MTM-1(C378S) alone, were lysed with a tissue grinder in worm lysis buffer containing 50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 1% Triton X-100, and protease inhibitor cocktail (EDTA free; Roche). The homogenized solutions were kept on ice for 15 min and centrifuged at 14,000 rpm for 20 min at 4°C to remove the debris. 400 µl of precleared solution was incubated with 1 µl GFP antibody (polyclonal antibody raised in rabbits against recombinant GFP; C. Yang laboratory, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for 2 h at 4°C, followed by incubation with 20 µl BSA-blocked protein G-Sepharose (Invitrogen) for 2 h. After extensive washing with 50-mM Tris-HCl, pH 7.5, and 250-mM NaCl, the bound proteins were resolved with SDS-PAGE and revealed by Western blotting with an antibody against mCHERRY (mouse, HX1810; Huaxingbio, Inc.).

Statistical analysis
The SD was used as y axis error bars for bar charts plotted from the mean value of the data. Data derived from different genetic backgrounds were compared by Student's two-way unpaired t test, one-way analysis of variance (ANOVA) followed by Tukey's post-test, or two-way ANOVA followed by Bonferroni post-test, as indicated in the figure legends. Data were considered statistically different at P < 0.05; *, P < 0.05; **, P < 0.0001 (0.001 in a two-way ANOVA analysis).