Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis Type IV orthologue

Developmental Biology 293 (2006) 382 – 391 www.elsevier.com/locate/ydbio

Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis Type IV orthologue Lara Schaheen, Hope Dang, Hanna Fares ⁎ Department of Molecular and Cellular Biology, University of Arizona, 1007 E. Lowell Street, Life Sciences South Room 531, Tucson, AZ 85721, USA Received for publication 17 November 2005; revised 30 January 2006; accepted 6 February 2006 Available online 10 March 2006

Abstract Mutations in MCOLN1, which encodes the protein h-mucolipin-1, result in the lysosomal storage disease Mucolipidosis Type IV. Studies on CUP5, the human orthologue of h-mucolipin-1 in Caenorhabditis elegans, have shown that these proteins are required for lysosome biogenesis. We show here that the lethality in cup-5 mutant worms is due to two defects, starvation of embryonic cells and general developmental defects. Starvation leads to apoptosis through a CED-3-mediated pathway. We also show that providing worms with a lipid-soluble metabolite partially rescues the embryonic lethality but has no effect on the developmental defects, the major cause of the lethality. These results indicate that supplementing the metabolic deficiency of Mucolipidosis Type IV patients may not be sufficient to alleviate the symptoms due to tissue degeneration. © 2006 Elsevier Inc. All rights reserved. Keywords: CUP-5; h-mucolipin-1; Mucolipidosis Type IV; Apoptosis

Introduction Mucolipidosis Type IV patients have two classes of symptoms (Altarescu et al., 2002; Bach, 2001). The first class, which includes corneal clouding and achlorhydria is due to the accumulation of large lipid-rich vacuoles in the respective tissues. The second class, which includes psychomotor retardation, is due to atrophy of some regions of the brain. The gene responsible for this disease encodes the protein hmucolipin-1 that belongs to the “Transient Receptor Potential” family of proteins (Bargal et al., 2000; Bassi et al., 2000; Sun et al., 2000). h-mucolipin-1 has been shown to function as a nonselective cation channel whose channel activity is modulated by pH (LaPlante et al., 2002; Raychowdhury et al., 2004). The C. elegans CUP-5 protein is the orthologue of hmucolipin-1 (Fares and Greenwald, 2001b). Expression of hmucolipin-1 in worms rescues all of the cup-5 mutant phenotypes (Hersh et al., 2002; Treusch et al., 2004). As in human patients, cup-5 mutations have two classes of symptoms. The first class is the presence of large vacuoles and the absence of lysosomal degradation in various cell types due to a defect in ⁎ Corresponding author. Fax +1 520 621 3709. E-mail address: [email protected] (H. Fares). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.02.008

lysosome biogenesis (Fares and Greenwald, 2001b; Hersh et al., 2002; Treusch et al., 2004). The second class is a maternal effect embryonic lethality and increased apoptosis in the dying embryos (Hersh et al., 2002). Significantly, the embryonic lethality is only partially rescued by mutations in effectors of cell death ced-3 or ced-4 (Hersh et al., 2002). Furthermore, the majority of eggs that do hatch in the presence of ced-3 or ced-4 mutations arrest as L1 larvae (Hersh et al., 2002). cup-5 mutants therefore provide an excellent model to determine the effects of lysosomal dysfunction on cellular metabolism and on tissue structuring, and to determine the relative contribution of each to lethality. Materials and methods C. elegans strains and methods Standard methods were used for genetic analysis (Brenner, 1974). RNAi was done by the feeding method as previously described (Timmons and Fire, 1998). The cdIs77 transgenic line of GFP∷LGG-1 was obtained by bombardment (Praitis et al., 2001). Markers used: cup-5(ar465) III (Fares and Greenwald, 2001b); cup-5(zu223) III (Hersh et al., 2002); cup-5(n3194) III (Hersh et al., 2002); unc-36(e251) III results in the worm having an uncoordinated (Unc) phenotype (Brenner, 1974) and is closely linked to cup-5 (Fares and Greenwald, 2001a, 2001b); rme-2(b1008) IV (Grant and Hirsh, 1999); rme-1(b1045) V (Grant et al., 2001); tag-283(gk378) V is a deletion within the open reading

L. Schaheen et al. / Developmental Biology 293 (2006) 382–391 frame generated by the Worm Gene Knockout Consortium; qC1 is a balancer chromosome that suppresses recombination between cup-5 and unc-36 (Graham and Kimble, 1993); ced-4(n1162) III (Yuan and Horvitz, 1992); ced-3(n717) IV (Ellis and Horvitz, 1986); arIs37[pmyo-3∷ssGFP] I (Fares and Greenwald, 2001a); bIs1[VIT-2∷GFP; pRF4] expresses a fusion of the worm YP170 protein VIT-2 to GFP (Grant and Hirsh, 1999). cup-5(zu223) unc-36(e251) or ced-4 (n1162) cup-5(n3194) worms bearing various transgenes were isolated from qC1-balanced parent heterozygotes; the eggs from these homozygous progeny were analyzed in the various assays.

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times on each sample and the average measurement was used as a DNA concentration. The adjusted ATP measurement from each sample was then divided by the amount of genomic DNA in the sample to get the normalized values. We could not use a protein assay because cup-5(zu223) embryos are defective in protein degradation. The results represent the average of the nine normalized values for each strain grown on plates with or without methylpyruvate (three plates for each condition, three luciferase assays per plate); the bars represent standard deviations.

TUNNEL assay Methylpyruvate feeding For all methylpyruvate experiments, methylpyruvate (Sigma, St. Louis, MO) was added to the NGM medium at a concentration of 14 mM before the plates were poured. In addition, OP50 was spun down and resuspended in a 14 mM solution of methylpyruvate before seeding the plates.

Lysotracker staining Lysotracker (Invitrogen, Carlsbad, CA) was added to the NGM medium at a concentration of 2 μM before the plates were poured. In addition, OP50 was spun down and resuspended in a 2 μM solution of lysotracker before seeding the plates.

Measuring embryonic viability

The TUNNEL assays were carried out as described previously, with one deviation, using the in situ cell-death detection kit from Roche (Wang et al., 2002). We permeabilized embryos using the freeze-crack method, as described previously, instead of bleaching (Bossinger et al., 2004). This was done to eliminate false positives due to differences in susceptibility to bleach of the various strains. The negative controls for staining in which fixed embryos were incubated solely with Label Solution without the addition of Terminal Deoxynucleotidyl Transferase did not result in any labeling of embryos.

Molecular methods Standard methods were used for the manipulation of recombinant DNA (Sambrook et al., 1989). All enzymes were from New England Biolabs (Beverly, MA), unless otherwise indicated.

Statistical methods

To measure viability, adult worms were allowed to lay eggs overnight at 20°. The adults were then removed and the percentage of eggs that hatched after one day was determined at 20°. Each experiment was repeated three times and at least 90 eggs were counted in each case. The results represent the average of these three experiments and the bars represent standard deviations.

Student's t test was used to compare average measurements from two samples using a two-tailed distribution (Tails = 2) and a two-sample unequal variance (Type = 2).

ATP measurements

Microscopy

150 to 300 adult hermaphrodites were allowed to lay eggs for 1 day on NGM plates with or without methylpyruvate at 20°. For each growth condition, three different plates were assayed for each strain. The worms plus eggs were harvested, bleached, and the resulting egg suspensions were washed three times with 100 mM Tris, 4 mM EDTA pH 7.75 and resuspended in 200 μl of 100 mM Tris, 4 mM EDTA pH 7.75. Each egg suspension was quick-frozen in liquid nitrogen and immediately boiled for 15 min (before thawing). The suspension was then sonicated using a Fisher Sonic Dismembrator Model 300 (Hampton, NH) for 20 s (4 s pulses at 30% followed by 5 s on ice). This egg suspension was again quick frozen in liquid nitrogen, immediately boiled for 15 min (before thawing), and sonicated in a Branson 1210 water sonicator (Raleigh, NC). The suspension was then spun down for 1 min at 13,000 rpm in a microfuge and the supernatant was kept at −80° until used. The ATP Bioluminescence Assay Kit HSII (Roche, Indianapolis, IN) was used to measure ATP levels according to the manufacturer's instructions. Fifty microliters of the (sometimes diluted) sample was mixed with 50 μl of the luciferase reagent for each measurement. Three assays were done for each sample and the average measurement was used for determining ATP concentration after adjusting for background fluorescence from luciferase. Background fluorescence measurements were: (1) 50 μl of the (diluted) sample mixed with 50 μl of 100 mM Tris, 4 mM EDTA pH 7.75, and (2) 50 μl of 100 mM Tris, 4 mM EDTA pH 7.75 mixed with 50 μl of the luciferase reagent. The ATP standards were diluted in 100 mM Tris, 4 mM EDTA pH 7.75. All measurements were done in a microtiter plate using an ML3000 luminometer (Dynatech Laboratories, Bridgewater, NJ). Fifty microliters of luciferase was electronically injected onto 50 μl of the sample in the microtiter plate and the fluorescence measurement was determined by integrating for 10 s after a 1 s delay. Each sample was measured three times. All measurements were within the linear portion of the curve determined using the ATP standards. In our hands, the linear portion corresponded to [ATP] between 10−6 M and 10−8 M. To normalize for number of cells assayed from each sample, we used the Invitrogen Picogreen assay to measure fluorescence using an FLX800 Bio-Tek Instrument (Winooske, VT) as described previously (Zhu et al., 1996). The Picogreen assay was done three

Adult worms were allowed to lay eggs for one day on the NGM plates supplemented with lysotracker ± methylpyruvate. Embryos were placed in M9 buffer for imaging. Confocal images were taken with a Nikon PCM 2000, using HeNe 543 excitation for the red dye and argon 488 for the green dye. Exactly the same exposure and magnification was used to capture the embryos from different strains that are expressing the same marker. All of the confocal images of wild-type and cup-5 mutant embryos were opened using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) and analyzed without any modification. At least 30 discrete intracellular structures from the acquired images were highlighted and the number of pixels included was determined. The reported values reflect the average surface areas of the highlighted structures (1 pixel is approximately 0.01 μm2). The bars on the chart represent standard deviations. Immunofluorescence staining of embryos using the IBF-2 antibody MH33 was done as previously described (Rappleye et al., 1999). Immunofluorescence staining of embryos using antibodies against YP170 and RME-2 was done as previously described (Britton and Murray, 2004). Immunofluorescence staining of embryos using the LIN-12 antibody was done as previously described (Hermann et al., 2000). Exactly the same exposure and magnification was used to capture the embryos from different strains stained with the same antibodies.

Results There are two kinds of cup-5 alleles: (1) cup-5(ar465) is a hypomorphic mutation that disrupts lysosome biogenesis in scavenger cells called coelomocytes but has no effect on embryonic viability (Fares and Greenwald, 2001b); (2) cup-5 (zu223) and cup-5(n3194) are null alleles that result in large endosome/lysosome vacuoles in both coelomocytes and in embryonic cells, and that cause maternal effect lethality (Hersh et al., 2002; Treusch et al., 2004).

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Embryos lacking CUP-5 have a defect in yolk processing A defect in lysosome biogenesis in embryos lacking CUP-5 should lead to the inability of embryonic cells to degrade yolk. Starvation is a likely consequence of this defect. In C. elegans, yolk is endocytosed and stored in vesicles by oocytes. During development, the yolk is degraded to provide energy and basic metabolites (for example, lipids and amino acids) for the developing cells (Britton and Murray, 2004; Grant and Hirsh, 1999). We first looked in vivo at the fate of the C. elegans yolk protein YP170 using a GFP fusion (Grant and Hirsh, 1999). YP170∷GFP is normally endocytosed by wild-type oocytes and is largely degraded over the course of the development of the embryos (Britton and Murray, 2004; Grant and Hirsh, 1999). Loss of CUP-5 did not affect the uptake and processing of yolk by oocytes: the sizes of YP170∷GFP compartments in wild-type and cup-5(zu223) oocytes were very similar (P = 0.4) (Fig. 1). In early embryos, there was a slight (P = 0.1) increase in the average size of YP170∷GFP compartments in cup-5(zu223) compared to wild type (Fig. 1). The lysosomal defect was more obvious in older embryos which showed an increase in the size and in the intensity of YP170∷GFP-containing granules in most cells of cup-5(zu223) relative to the same granules in embryonic cells of wild-type embryos at the same stage of development; cup-5(ar465) embryos at the same stage of development were slightly but significantly larger than wildtype YP170∷GFP granules (P = 5.7 × 10− 4 compared to wild type) (Figs. 2A, B). Co-staining oocytes and embryos with the lysosomal marker lysotracker red showed almost complete overlap with the YP170∷GFP granules (Figs. 1 and 2A). This result indicates that older cup-5-null embryos are defective in the processing of yolk. Embryos lacking CUP-5 have a defect in receptor degradation We wanted to determine whether cup-5(zu223) embryos also have defects in the degradation of cell surface receptors. RME2, the C. elegans yolk receptor, is found at the surface of oocytes, appears in intracellular vesicles in one and two-cell stage embryos and disappears soon after (Britton and Murray, 2004; Grant and Hirsh, 1999). Consistent with the lysosomal defect, cup-5(zu223) embryos retained RME-2 in intracellular compartments later in development where it co-localized with yolk proteins (Fig. 2C). Furthermore, ced-3(n717); cup-5 (zu223) double-mutant embryos also did not degrade RME-2 or YP170, indicating that the partial suppression of the lethality by ced-3 mutations is not due to rescue of the lysosomal defect (Fig. 2C). At later stages of embryogenesis, RME-2 staining is lost indicating that there is a reduction in the rate of lysosomal processing as opposed to a complete block (L. Schaheen and H. Fares, data not shown). LIN-12 is a C. elegans Notch that is required for the proper morphogenesis of the developing intestine in embryos (Hermann et al., 2000). LIN-12 localizes asymmetrically to the surface of specific intestinal cells on one side of developing embryos (Fig. 3) (Hermann et al., 2000). This LIN-12 staining disappears at later stages of embryogenesis

Fig. 1. cup-5 mutant oocytes and early embryos have normal yolk processing. (A) Confocal micrographs of oocytes and “4-cell” embryos in wild-type or cup-5 (zu223) hermaphrodites carrying the YP170∷GFP transgene (green in overlay) and grown on plates containing lysotracker red (red in overlay). Scale bar is approximately 10 μm. (B) Quantitation of the surface area of the YP170∷GFP granules shown in panel A.

(Fig. 3). cup-5(zu223) embryos showed a similar staining pattern to wild type at early stages of development. Contrary to wild type, we could still detect punctate staining inside cells of cup-5(zu223) embryos at later stages, indicating that there is a delay in the degradation of LIN-12 in the absence of CUP-5 (Fig. 3). Embryos lacking CUP-5 have reduced levels of ATP To ascertain that the reduced rate of yolk degradation was leading to starvation, we measured ATP levels in the various strains (Fig. 2D). We indeed found approximately a ten-fold reduction in the levels of ATP in cup-5(zu223) or ced-3(n717);

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Fig. 2. cup-5 null embryos have a defect in yolk processing. (A) Confocal micrographs of “two-fold” embryos laid by wild-type, cup-5(ar465), or cup-5(zu223) hermaphrodites carrying the YP170∷GFP transgene (green in overlay) and grown on plates containing lysotracker red (red in overlay) with or without methylpyruvate (mPvy). All images of the same marker were taken with the same exposure and at the same magnification. Scale bar is approximately 12.5 μm. (B) Quantitation of the surface area of the YP170∷GFP granules shown in Fig. 1A. (C) Confocal images of gastrulating embryos laid on regular medium and stained with antibodies against RME-2 (green in overlay) and YP170 (red in overlay). All images of the same marker were taken with the same exposure and at the same magnification. (D) Relative ATP concentrations in a mixed population of eggs laid by the indicated strains. n717 is a mutation in ced-3.

cup-5(zu223) embryos compared to wild-type embryos. We did not observe a statistically significant difference in the levels of ATP between cup-5(ar465) and wild-type embryos. Embryos lacking CUP-5 activate autophagy Cells that are starved activate a process called autophagy where they sequester some of their own cytoplasm and

organelles in specialized compartments called autophagosomes and subsequently fuse these with lysosomes to provide themselves with nutrients (Dunn, 1994). To visualize autophagy in embryos, we used transgenic worms that express a GFPtagged LGG-1, the orthologue of yeast Apg8p and mammalian MAP-LC3 (Melendez et al., 2003). We clearly saw an increase in GFP∷LGG-1 fluorescence in cells of cup-5(zu223) embryos compared to those laid by wild-type or cup-5(ar465) embryos at

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Fig. 3. LIN-12 staining in cup-5 mutant embryos. Confocal micrographs of embryos laid by the indicated hermaphrodites stained with antibodies to detect LIN-12. Embryos are at the E16 or the “1.5-fold” stage of development. Arrows indicate LIN-12 staining of the intestinal primordial cells. Note that only cells in the right primordium express LIN-12 at this stage. Furthermore, the pattern of LIN-12 expression changes markedly during the E16 stage due to LIN-12 downregulation in some cells. All strains also carry the pmyo-3∷ssGFP array. Scale bar is approximately 15 μm.

the same stage of development (Fig. 4A). The GFP∷LGG-1 fluorescence in cells of cup-5(ar465) embryos was slightly more pronounced than those laid by wild-type embryos (Fig. 4A). This result indicates that autophagy is activated in many cup-5(zu223) embryonic cells. The GFP∷LGG-1 often colocalized with lysotracker red (Fig. 4A). The yolk degradation defect of cup-5 mutant embryos is likely the cause of the activation of autophagy since reducing the amount of yolk available to wild-type embryonic cells after RNAi of RME-2, the yolk receptor, or RME-1, an EH-domain protein required for the recycling of RME-2 to the plasma membrane, resulted in more punctate GFP∷LGG-1 fluorescence (Fig. 4B) (Grant and Hirsh, 1999; Grant et al., 2001). One prediction from these results is that cup-5(ar465) worms would rely more on autophagy for their survival. We reasoned that the slight reduction in CUP-5 activity may result in a partial defect in lysosome biogenesis and hence a requirement for autophagy to provide enough nutrients for development. We

therefore used RNAi to reduce the levels of two autophagy proteins, worm ORF T22H9.2 that is the homologue of the conserved yeast Atg9p/Apg9p, and worm ORF F41E6.13/TAG283 that is the homologue of the yeast Atg18p and human WIPI-1α (Melendez et al., 2003; Noda et al., 2000; ProikasCezanne et al., 2004). Studies in yeast have shown that Atg18p is required for the recycling of Atg9p from a pre-autophagosomal compartment (Reggiori et al., 2004). Indeed, we found that cup-5(ar465) embryonic viability is slightly, but reproducibly, hypersensitive to RNAi of these two genes (Fig. 4C). We confirmed these results using an existing null allele of tag-283: the average embryonic lethality was 7.14 ± 2.78 for tag-283 (gk378) and 18.66 ± 8 for cup-5(ar465); tag-283(gk378). Significantly, and contrary to the cup-5(ar465) single mutant, the ced-3(n717); cup-5(ar465) double mutant did not show increased embryonic lethality, compared to ced-3(n717) single mutants, after RNAi of these autophagy genes (Fig. 4C). This suggests that the main consequence of starvation is a CED-3-

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Fig. 4. Autophagy and apoptosis in cup-5 embryos. The mutations tested were cup-5(ar465), cup-5(zu223), and ced-3(n717). (A) Confocal micrographs of “two-fold” embryos laid by wild-type, cup-5(ar465), or cup-5(zu223) hermaphrodites carrying the GFP∷LGG-1 transgene (green in overlay) and grown on plates containing lysotracker red (red in overlay) with or without methylpyruvate (mPvy). This transgene also carries a marker that expresses GFP in pharyngeal cells (large arrows). Small arrows indicate intestinal cells in cup-5(zu223) embryos. All images of the same marker were taken with the same exposure and at the same magnification. Scale bar is approximately 12.5 μm. (B) Confocal micrographs of “1.5-fold” GFP∷LGG-1 embryos after control, rme-1, or rme-2 RNAi. All images were taken at the same exposure and magnification. Scale bar is approximately 10 μm. (C) Bar graph showing percentage of viable worms (adults divided by total eggs laid) from different strains after the indicated RNAi. The RNAi vector pPD129.36 was used as a control. (D) Confocal micrographs of embryos of the indicated genotypes stained using the TUNNEL assay to detect DNA strand breaks. Eggs were laid on medium with or without methylpyruvate (14 mM mPvy). Arrows indicate Tunnel-staining cells. These strains also carried the pmyo-3∷ssGFP array. (E) Confocal micrographs of rme-1(b1045) and rme-2(b1008) embryos stained using the TUNNEL assay to detect DNA strand breaks. Scale bar is approximately 10 μm in panels D and E.

mediated apoptotic response leading to lethality of some of the embryos. Suppression of starvation defects by elevating ATP levels in embryos lacking CUP-5 We provided the cells with methylpyruvate, a membranepermeable TCA substrate. Adding methylpyruvate to the

medium eliminates the requirement for the endo-lysosomal system for uptake and processing of nutrients/yolk for energy production. We first measured the ATP levels in embryos grown on media containing 14 mM methylpyruvate, the highest concentration we could provide that was not detrimental to wild-type worms. Addition of methylpyruvate raised ATP levels in cup-5(zu223) and ced-3(n717); cup-5(zu223) embryos to wild-type levels (Fig. 2D). As expected, methylpyruvate did not

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restore the ability of cup-5(zu223) embryos to degrade yolk, RME-2, or LIN-12 (Figs. 2A and 3; L. Schaheen and H. Fares, data not shown). Furthermore, cells in cup-5 mutant embryos grown on methylpyruvate showed reduced activation of autophagy (small arrows in Fig. 4A). cup-5 null embryos contain extra apoptotic cells; this is suppressed by mutations in ced-3 (Fig. 4D) (Hersh et al., 2002). If this extra apoptosis is due to starvation, then we expected that addition of methylpyruvate would reduce apoptosis to wild-type levels. Indeed, “comma” to “1 1/2 fold” stage embryos of cup-5(zu223) worms grown on methylpyruvate showed the same level of apoptosis as in wild-type embryos. That is, the majority of embryos did not show Tunnel staining, while occasional embryos had one Tunnel-staining nucleus (Fig. 4D). We note that this is expected since we did not use mutations to retard engulfment of apoptotic cells. Wild-type or cup-5(ar465) embryos grown with or without methylpyruvate showed a wild-type staining pattern (Fig. 4D). Furthermore, mutations in rme-1 and rme-2 did not result in increased apoptosis even though autophagy is activated in these embryos (Fig. 4E). Elevating ATP levels in embryos lacking CUP-5 only partially rescues their viability We tested the extent of the rescue we could achieve if we artificially increased ATP levels in cells. If starvation is the major cause of lethality, then we would expect that methylpyruvate would completely rescue the viability of cup-5 null embryos. We saw a clear increase in viability of cup-5(zu223) embryos grown on methylpyruvate plates such that an average of 14% of the embryos hatched (Fig. 5A). Furthermore, and similar to ced-3(n717); cup-5(zu223) embryos grown on regular media, less than 5% of the embryos that hatched developed beyond L1 larvae and all arrested before they became adults (Fig. 5A) (Hersh et al., 2002). Consistent with the model that starvation leads to CED-3-mediated apoptosis, we did not see an enhancement of embryonic or larval viability of ced-3(n717); cup-5(zu223) or of ced-4(n1162); cup-5(n3194) embryos due to methylpyruvate (Fig. 5A). Methylpyruvate's enhancement of the viability of cup-5(zu223) embryos to a larger extent than ced-3 or ced-4 mutations is likely due to slight growth defects due to the presence of these mutations (Fig. 5A). The starvation-induced apoptosis is not the major cause of the lethality since methylpyruvate, while it restores ATP levels, only partially rescues the embryonic lethality and does not rescue the larval lethality of cup-5(zu223) embryos. Fig. 5. Methylpyruvate partially suppresses viability but not the developmental defects of cup-5 null embryos. The mutations tested were cup-5(ar465), cup-5 (zu223), cup-5(n3194), ced-3(n717), and ced-4(n1162). (A) Quantitation of the percentage of embryos of the indicated genotypes that hatched on normal media or on media that included 14 mM methylpyruvate (grey). The bottom graph is of strains carrying a null mutation in cup-5. (B) Confocal micrographs of “1.5-fold” embryos of the indicated genotypes immunostained for the detection of IFB-2 with the MH33 antibody. All strains also carried the pmyo-3∷ssGFP array. Scale bar is approximately 15 μm.

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Developmental defects in embryos lacking CUP-5 We asked whether there were other defects in cup-5(zu223) embryos that could contribute to lethality. We noticed premature activation of a pharynx-specific myo-2 promoter fused to GFP in the presumptive pharyngeal cells of transgenic cup-5(zu223) embryos (large arrows in Fig. 4A). We therefore checked for other developmental defects. The intestinal cells in cup-5 (zu223) embryos showed a severely disorganized intestinal terminal web when checked by immunofluorescence microscopy using antibodies against the intermediate filament protein IFB-2 (Fig. 5B) (Bossinger et al., 2004). This indicates that embryos lacking CUP-5 are also defective in establishing the architecture of some tissues/organs. Significantly, none of these developmental defect are rescued by methylpyruvate and/or by ced-3(n717) (Figs. 4A and 5B). Discussion Our analysis indicates that cup-5(zu223) embryonic cells show a retardation in the degradation of yolk proteins and of cell surface receptors. One consequence of this defect is a decrease in nutrient and biosynthetic compound availability that leads to the activation of autophagy. However, autophagosomes also require lysosomes to break down their contents. Thus, cells in cup-5(zu223) embryos are unable to utilize autophagy to compensate for the decrease in nutrient and biosynthetic compound availability. This is in contrast to mutations in rme-1 and rme-2 where there is less yolk available to embryonic cells but where there is normal lysosomal function. One consequence of the degradation defect is starvation leading to ectopic apoptosis. Two results indicate that the ectopic apoptosis due to starvation is mediated by CED-3/CED4. First, we show that inactivation of two autophagy genes slightly increases the embryonic lethality of cup-5(ar465) embryos, but not of ced-3(n717); cup-5(ar465) embryos. Second, we show that increasing ATP levels in cells partially rescues the embryonic lethality of cup-5(zu223) embryos but has no effect on the viability of cup-5(zu223) embryos homozygous for mutations in ced-3 or ced-4. A recent study demonstrated that there is a physical and developmental link between autophagy and apoptosis in C. elegans embryos (Takacs-Vellai et al., 2005). These results are also consistent with a study that showed that Bax−/−Bak−/− mouse cells that are defective in apoptosis require autophagy for cell survival upon nutrient depletion (Lum et al., 2005). Inactivation of autophagy results in cell death only in the absence of growth factors. Furthermore, another recent study has shown that inactivation of the lysosomal-associated protein LAMP2 in HeLa cells blocks the fusion of autophagosomes with lysosomes; starving these LAMP2-negative cells results in cell death with all the hallmarks of apoptosis (Gonzalez-Polo et al., 2005). cup-5(zu223) embryos laid on methylpyruvate do not have defects associated with starvation: [ATP] is restored to wildtype levels and there is a dramatic reduction in Tunnel-staining cells. However, there is only a modest increase in embryonic viability and no effect on larval viability. The persistent lethality

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is likely due to pleiotropic defects stemming from the retardation in the rate of degradation of signaling receptors and/or in the availability of biosynthetic building blocks. These lead to developmental defects that include the disorganization of intestinal tissue and premature activation of a pharynx reporter. We should emphasize that embryos laid by cup-5 null hermaphrodites only show a clear defect at late stages of development. The most obvious defect is in developing intestinal cells during the endocytosis of yolk that is secreted by all other embryonic cells into the periviteline space (Bossinger and Schierenberg, 1996). This is in contrast to mutations in worm Cathepsin L that show a lysosomal defect as early as one-cell stage embryos (Britton and Murray, 2004). It is possible that lack of CUP-5 only retards lysosome biogenesis/ function. In these lysosomes, the slow accumulation of substrates over time further inhibits lysosomal degradation, which is consistent with the phenotype appearing at later stages of development. Tissues with high endocytic rates and transport to lysosomes show the most severe defects, for example, developing intestinal cells and adult coelomocytes, scavenger cells found in the body cavity (Fares and Greenwald, 2001b; Treusch et al., 2004). In addition to endocytosis rates, CUP-5 function may be redundant with unrelated proteins in some tissues and/or there may be specialized CUP-5-independent pathways of lysosome biogenesis in some tissues and/or stages of development. For example, while CUP-5 is expressed in most tissues in adult worms, only coelomocytes show an obvious defect in their lysosomes (Fares and Greenwald, 2001b; Treusch et al., 2004).

Fig. 6. Model of lethality in the absence of CUP-5. The absence of CUP-5 leads to a defect in lysosomal degradation. The inability to degrade nutrients results in reduced ATP production, which activates CED-3-mediated apoptosis and hence in approximately a 10–20% drop in embryonic viability. The main reasons for embryonic and larval lethality are the developmental defects, exemplified by a thicker arrow, which lead to cell death that is mediated by unknown factors X.

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We favor a model in which the lysosomal defect due to the absence of CUP-5 results in starvation, due to the inability to degrade yolk, and to pleiotropic developmental defects because of a general defect in the degradation of endocytosed material (Fig. 6). Lysosomal defects have previously been linked to developmental abnormalities. For example, studies of the late endosomal protein Hrs in Drosophila melanogaster have shown the importance of lysosomal degradation of growth factor receptors for normal embryonic development (Jekely and Rorth, 2003; Lloyd et al., 2002). Furthermore, mutating the mouse lysosomal acid phosphatase gene results in disruption of the cytoarchitecture of cerebellar neurons and of hair follicles (Mannan et al., 2004). The starvation defect results in CED-3/ CED-4-mediated cell death. Both defects contribute to embryonic lethality. Furthermore, the larval lethality is almost completely due to the developmental defects such that when embryos lacking CUP-5 hatch, either due to the presence of ced3 or ced-4 mutations or by adding methylpyruvate to the medium, they do not develop further. These results indicate that supplementing the energy requirements is not sufficient to ensure the proper development of cells and the modeling of tissues. The contribution of starvation and developmental defects to the cell and neuron degeneration in Mucolipidosis Type IV patients is not known and may vary among cell types. We propose that supplementing the diet of these Mucolipidosis Type IV or other “lysosomal storage disease” patients (or of pregnant mothers with affected fetuses) with metabolites that can be pumped directly into the cytoplasm or that are membrane-permeable may rescue some of the cell death phenotypes and give partial suppression of the symptoms (Vellodi, 2005). Such a treatment would not alleviate the symptoms due to viable cells with defective lysosome biogenesis or to cell death due to developmental defects. The latter require a better understanding of the exact function of hmucolipin-1/CUP-5 in this process and the identification of suppressors/treatments that restore lysosomal function. Acknowledgments We are grateful for Barth Grant, Peg MacMorris, Stuart Kim, and James McGhee for antibodies to RME-2, YP170, LIN-12, and IFB-2, respectively. We also thank Alicia Melendez for the GFP∷LGG-1 construct and the C. elegans Gene Knockout Consortium for the deletion alleles of tag-283. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. The authors declare that they have no competing financial interests. This work was funded by a March of Dimes grant (#6-FY04-60) to H.F. References Altarescu, G., Sun, M., Moore, D.F., Smith, J.A., Wiggs, E.A., Solomon, B.I., Patronas, N.J., Frei, K.P., Gupta, S., Kaneski, C.R., Quarrell, O.W., Slaugenhaupt, S.A., Goldin, E., Schiffmann, R., 2002. The neurogenetics of mucolipidosis type IV. Neurology 59, 306–313.

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