Genetic Analysis of Endocytosis in Caenorhabditis elegans: Coelomocyte Uptake Defective Mutants

Corresponding author: Hanna Fares, University of Arizona, Department of Molecular and Cellular Biology, Life Sciences South Bldg., Rm. 531, 1007 E. Lowell St., Tucson, AZ 85721., fares{at}email.arizona.edu (E-mail)

Communicating editor: B. J. MEYER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.

ENDOCYTOSIS is a basic function of eukaryotic cells that leads to the internalization of fluid from the extracellular medium, nutrient uptake, and the recycling of membrane components. In multicellular organisms, endocytosis has also been adapted by specialized cells for specific functions, including the downregulation of signaling, synaptic vesicle recycling, and antigen presentation. There are various routes by which endocytosis is accomplished, involving different cellular structures: clathrin-coated pits, non-clathrin-coated pits, caveolae, and macropinosomes (WATTS and MARSH 1992 ; ROBINSON et al. 1996 ; MUKHERJEE et al. 1997 ). The particular mechanism used to regulate these pathways depends on the ligand being internalized and the cell type examined. The multiplicity of options adds complexities that suggest that endocytosis should be studied using several different approaches and experimental systems.

Many of the steps in endocytosis have been determined through studies on mammalian tissue culture cells that are technically suited for this kind of analysis (WATTS and MARSH 1992 ; ROBINSON et al. 1996 ; MUKHERJEE et al. 1997 ). In brief, a vesicle containing the internalized medium buds from the membrane. The internalized substances then are sorted as they traverse a series of membranous compartments, called endosomes, before either reaching the lysosomes to be degraded or getting recycled to the surface. Many different protein components of these vesicles have been identified through biochemical methods using mammalian cell culture systems.

Genetic analysis has also been an important tool for dissecting endocytosis. The most extensive and informative screens have been done in the yeast Saccharomyces cerevisiae (RIEZMAN 1985 ; CHVATCHKO et al. 1986 ; MUNN and RIEZMAN 1994 ; WENDLAND et al. 1996 ; ZHENG et al. 1998 ). For example, numerous insights into the machinery involved in the trafficking of molecules between the Golgi compartment, endosomes, and the vacuole (yeast lysosome) have emerged from such investigations (LEMMON and TRAUB 2000 ). In particular, these studies have been crucial in elucidating protein complexes involved in the retrograde transport of molecules to the Golgi compartment.

Multicellular organisms utilize endocytosis in many more contexts than do unicellular organisms, raising the possibility that they exhibit differences in regulating endocytosis. One such difference between yeast cells and mammalian cells, for example, is that the adaptor complexes are not needed for clathrin function or for endocytosis in yeast (HUANG et al. 1999 ; YEUNG et al. 1999 ). Although screens for mutants defective in endocytosis have been carried out in mammalian cells (ROBBINS et al. 1983 ; KRIEGER 1986 ; COLBAUGH et al. 1988 ; HOBBIE et al. 1994 ) and in Dictyostelium discoideum (BACON et al. 1994 ), the only concerted effort to screen for endocytosis mutants in a multicellular organism has been the study of the receptor-mediated endocytosis of yolk in Caenorhabditis elegans oocytes (GRANT and HIRSH 1999 ). C. elegans is particularly suited to large-scale genetic screens, and many genes that are conserved in worms and humans are not found in yeast (LANDER et al. 2001 ; VENTER et al. 2001 ). Indeed, the molecular analysis of C. elegans endocytosis mutants so far has demonstrated that novel components involved in endocytosis unique to multicellular organisms can be identified using genetic analysis in C. elegans (see DISCUSSION).

Here, we report an approach to studying endocytic pathways in C. elegans, using an assay based on the uptake of fluid-phase markers from the pseudocoelom by coelomocytes. We show that in this coelomocyte uptake (Cup) assay many different fluid-phase markers can be endocytosed by coelomocytes. We describe the behavior of known endocytosis mutants and RNA-mediated interference (RNAi) for known endocytosis genes using the Cup assay and the results of a screen for viable cup mutants. This work establishes the Cup assay as a powerful genetic screen for endocytosis mutants in a multicellular organism.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

C. elegans methods and strains:
General methods for the handling and maintenance of C. elegans are as described previously (BRENNER 1974 ). The wild-type parent for all strains was C. elegans var. Bristol (BRENNER 1974 ), except for sequence- tagged site (STS) mapping that used var. Bergerac strain DP13 (WILLIAMS et al. 1992 ). Standard methods were used for genetic analysis (BRENNER 1974 ), DNA microinjection (FIRE 1986 ; MELLO et al. 1991 ), and double-stranded RNA synthesis and microinjection (FIRE et al. 1998 ).

Plasmid construction:
pJF25 [pmyo-3::ssGFP] (FARES and GREENWALD 2001 ) has DNA encoding the first 79 amino acids from SEL-1 (GRANT and GREENWALD 1997 ), including a signal sequence, fused to green fluorescent protein (GFP; CHALFIE et al. 1994 ), with the S65T substitution, and under the control of the myo-3 promoter.

pJF43 [phsp::ssGFP] (FARES and GREENWALD 2001 ) has DNA encoding the SEL-1 (GRANT and GREENWALD 1997 ) signal sequence-GFP fusion under the control of the hsp16-41 (JONES et al. 1986 ) heat-shock promoter.

To make a construct expressing the Diphtheria toxin A fragment (catalytic) specifically in coelomocytes, we used plasmid pcc1 (P. SENGUPTA, personal communication), which was made by subcloning a 3993-bp fragment, now known to be the upstream region of unc-122 (P. LORIA and O. HOBERT, personal communication), into plasmid pPD95.77 (A. FIRE, personal communication). pcc1 therefore has a GFP coding sequence with unc-122 5' sequences upstream and unc-54 3'-untranslated sequences downstream. When transformed into worms, GFP is almost exclusively seen in coelomocytes, albeit in a few worms, one or more coelomocytes are not fluorescent or are more weakly fluorescent. In addition, very occasional weak expression is also seen in certain neurons.

We used the polymerase chain reaction (PCR) to amplify a 0.6-kb fragment from plasmid pDT-AK51E (DELANGE et al. 1979 ) using the primers J197 (CACACAGGATCCAAAATGGGCGCTGATGATGTTGTTGATTC; BamHI site is underlined) and J198 (CACACAGAATTCTCATTAACGATTTCCTGCACAGGC; EcoRI site is underlined). The amplified PCR fragment was restriction digested with BamHI and EcoRI and subcloned into the same sites in pcc1. The resulting plasmid, pJF142, has the Diptheria toxin A fragment bearing a K51E substitution replacing GFP in plasmid pcc1. The Diptheria toxin A fragment bearing a K51E substitution has 10% of the activity of the wild-type protein (DELANGE et al. 1979 ).

Transgenes and strains:
Extrachromosomal arrays were generated by coinjecting plasmid DNA at 1–50 µg/ml along with the marker plasmids pRF4 [rol-6(su1006)] (MELLO et al. 1991 ) or pMH86 [dpy-20(+)] (HAN and STERNBERG 1991 ) into the germline and were integrated by irradiation using standard methods (MELLO and FIRE 1995 ).

arIs37[pmyo-3::ssGFP] (FARES and GREENWALD 2001 ) was made by integrating an extrachromosomal array that was made by injecting a DNA mix containing pJF25 [pmyo-3::ssGFP] (62.5 µg/ml) + pMH86 (20 µg/ml) + pBluescript SK⁺ (100 µg/ml; Stratagene, La Jolla, CA) in a dpy-20(e1282) (HOSONO et al. 1982 ) background. The isolated integrant was backcrossed eight times to dpy-20(e1282) prior to use. We checked 107 Unc F₁ progeny segregating from dpy-20(e1282); arIs37[pmyo-3::ssGFP]/unc-54(e190) hermaphrodites: all were Dpy, suggesting that arIs37[pmyo-3::ssGFP] maps close to unc-54(e190) on chromosome I. However, we were eventually able to construct an unc-54(e190) arIs37[pmyo-3::ssGFP] strain.

arIs36[phsp::ssGFP] (FARES and GREENWALD 2001 ) was made by integrating an extrachromosomal array that was made by injecting a DNA mix containing pJF43 [phsp::ssGFP] (20 µg/ml) + pMH86 (25 µg/ml) + pBluescript SK⁺ (100 µg/ml) in a dpy-20(e1282) background. The integrant was backcrossed twice to dpy-20(e1282) prior to use.

arEx218[pcc1::DT-A_K51E] was made by injecting a DNA mix containing pJF142 [pcc1::DT-A_K51E] (10 µg/ml) + pRF4 (100 µg/ml) into dpy-20(e1282); arIs37[pmyo-3::ssGFP] hermaphrodites.

All other genetic markers used in this article can be found referenced in the C. elegans II book by RIDDLE et al. 1997 , except for eat-20(nc4) (SHIBATA et al. 2000 ), nrf-4(sa528), nrf-5(sa513), and nrf-6(sa525) (CHOY and THOMAS 1999 ), and spg-(e2335) (L. AVERY, personal communication).

Molecular analysis:
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. PCR was done using the Expand Long Template PCR system (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.

Mutant screen and mapping:
Adult dpy-20(e1282); arIs37 [pmyo-3::ssGFP] hermaphrodites were mutagenized using ethyl methanesulfonate (EMS) as previously described (BRENNER 1974 ). Five mutagenized parents were placed on a single nematode growth media (NGM; BRENNER 1974 ) plate that had been previously seeded with Escherichia coli OP50 (BRENNER 1974 ). The F₂ hermaphrodites from each plate were screened for a defect in endocytosis by coelomocytes (evidenced by a change in the amount of GFP in the pseudocoelom and/or coelomocytes) using a modified dissection microscope equipped with fluorescence optics (Zeis, Thornwood, NY). Mutant F₂ worms were picked onto individual fresh NGM plates (with OP50).

We mutagenized 720 P₀ hermaphrodites and checked the progeny from 18,000 F₁ hermaphrodites. We picked 175 adult F₂ mutant hermaphrodites (not independent isolates) of which 97 did not give viable progeny. To ensure independent isolation of mutants, we chose only one mutant from each plate, except for two cases in which the mutant phenotypes were distinguishably different; in both cases, the two mutants picked from the same plate had mutations that mapped to different locations (see below). We also saw many F₂ hermaphrodites that had strong accumulations of GFP in the pseudocoelom but that were arrested at earlier larval stages; these were neither counted nor picked to separate plates. Finally, we kept 55 viable mutant hermaphrodites for further analysis.

We first backcrossed the 55 mutant hermaphrodites three times to dpy-20(e1282); arIs37[pmyo-3::ssGFP] males. We then mapped the backcrossed mutations to a region of the genome using STS mapping, as previously described (WILLIAMS et al. 1992 ). Individual cup mutations were tested by complementation analysis for the Cup phenotype against all cup and rme mutations that mapped to the same region and against some other mutations (see below) in the same region. We thus determined that the 55 mutations defined 14 genes. Three of these, rme-1, rme-6, and rme-8, were previously identified as mutants defective in yolk uptake by oocytes (GRANT and HIRSH 1999 ). We named the rest cup-1 through cup-11 (cup is the coelomocyte uptake defect); these were mapped further using conventional markers.

cup-1 I: We isolated 48 recombinants from unc-101(m1) glp-4(bn2) arIs37[pmyo-3::ssGFP]/cup-1(ar478) arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 48 Unc non-Glp recombinants, 31 picked up cup-1(ar478). cup-1(ar478) complemented nrf-4(sa528).

cup-2 I: We isolated 55 recombinants from dpy-24(s71) unc-101(m1) arIs37[pmyo-3::ssGFP]/cup-2(ar506) arIs37[pmyo-3::ssGFP] hermaphrodites. None of the 23 Dpy non-Unc recombinants picked up cup-2(ar506). All of the 32 Unc non-Dpy recombinants picked up cup-2(ar506). We also isolated 36 recombinants from unc-13(e51) dpy-24(s71) arIs37[pmyo-3::ssGFP]/cup-2(ar506) arIs37[pmyo-3::ssGFP] hermaphrodites. None of the 36 Dpy non-Unc recombinants picked up cup-2(ar506). Thus, cup-2(ar506) maps close to dpy-24(s71). cup-2(ar506) complemented nrf-4(sa528) and dyf-5(mn400).

cup-3 II: We isolated 38 recombinants from dpy-10(e128) unc-4(e120)/cup-3(ar496); arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 15 Dpy non-Unc recombinants, 5 picked up cup-3(ar496). Of the 23 Unc non-Dpy recombinants, 10 picked up cup-3(ar496). cup-3(ar496) complemented nrf-6(sa525) and dyf-13(mn396).

cup-4 III: We isolated 10 recombinants from sma-3(e491) unc-36(e251)/cup-4(ar494); arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 6 Sma non-Unc recombinants, 4 picked up cup-4(ar494). Of the 4 Unc non-Sma recombinants, 1 picked up cup-4(ar494).

cup-5 III: We isolated 18 recombinants from sma-3(e491) unc-36(e251)/cup-5(ar465); arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 10 Sma non-Unc recombinants, 5 picked up cup-5 (ar465). Of the 8 Unc non-Sma recombinants, 5 picked up cup-5(ar465).

cup-6 III: We isolated 23 recombinants from unc-25(e156) bli-5(e518)/cup-6(ar513); arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 20 Unc non-Bli recombinants, 19 picked up cup-6 (ar513). None of the 3 Bli non-Unc recombinants picked up cup-6(ar513). We also isolated 71 recombinants from dpy-18 (e364) unc-64(e246)/cup-6(ar513); arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 36 Dpy non-Unc recombinants, 35 picked up cup-6(ar513). None of the 35 Unc non-Dpy recombinants picked up cup-6(ar513). Thus, while cup-6 maps to the extreme right arm of chromosome III, due to the single recombinant from each mapping cross, we cannot confirm its exact location relative to the markers used. cup-6(ar513) complemented dyf-2(m160).

cup-7 IV: We isolated 15 recombinants from lin-1(e1275) unc-33(e204)/cup-7(ar492); arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 15 Lin non-Unc recombinants, 6 picked up cup-7 (ar492). cup-7(ar492) complemented unc-17(e245), dpy-13(e184), dpy-9(e2), ced-2(e1752), unc-33(e204), cha-1(p1152), eat-7(ad450), eat-10(ad606), and dyf-3(m185).

cup-8 V: We isolated three recombinants from unc-34(e566) unc-60(e677) dpy-11(e224)/cup-8(ar466); arIs37[pmyo-3::ssGFP] hermaphrodites. All three Unc-34 non-Unc-60 recombinants picked up cup-8(ar466). We then picked 252 F₁ Unc-34 hermaphrodites from unc-34(e566) cup-8(ar466)/unc-60(e723); arIs37[pmyo-3::ssGFP] hermaphrodites. Four of these were recombinants between unc-34(e566) and unc-60(e723). Two of these recombinants also picked up cup-8(ar466). cup-8(ar466) complemented nrf-5(sa513).

cup-9 V: We isolated 22 recombinants from sma-1(e30) unc-39(e257)/cup-9(ar467); arIs37[pmyo-3::ssGFP] hermaphrodites. Of the 14 Sma non-Unc recombinants, 6 picked up cup-9 (ar467). Of the 8 Unc non-Sma recombinants, 5 picked up cup-9(ar467). cup-9(ar467) complemented nrf-5(sa513) and dyf-4(m158).

cup-10 V: We isolated 47 Dpy non-Lag recombinants from lag-2(q420) dpy-11(e224)/cup-10(ar479); arIs37[pmyo-3::ssGFP] hermaphrodites: 4 picked up cup-10(ar479). We tested whether cup-10(ar479) could complement deletions in that region (D. BAILLIE, personal communication). cup-10(ar479) did not complement the deficiencies sDf26, sDf28, sDf42, and sDf50. cup-10(ar479) did complement sDf31, sDf32, sDf45, and sDf46. cup-10(ar479) complemented nrf-5(sa513).

cup-11 X: We isolated 15 Unc non-Mup recombinants from mup-2(e2346) unc-6(e78)/cup-11(ar472); arIs37[pmyo-3::ssGFP] hermaphrodites: 9 picked up cup-11(ar472). cup-11(ar472) complemented dyn-1(ky51ts).

Phenotypic analysis of mutants:
To check for defects in yolk uptake by the oocytes, the backcrossed mutant strains and their unmutagenized parent strain were grown on NGM (+OP50) plates at 20° and gravid adult hermaphrodites were checked by Nomarski microscopy for the accumulation of lipid droplets in the pseudocoelom. This test may not be sensitive enough to score mutants that have only a mild defect in oocyte uptake.

To check for defects in the uptake of substances by intestinal cells from the lumen of the gut, we placed three L4 of the backcrossed mutant strains and their wild-type parent on individual NGM (+OP50; no cholesterol) plates at 20°; these NGM plates did not have any added cholesterol and also contained rhodamine-conjugated dextran (M_r 11,000) added at 0.1 mg/ml. Adult F₁ hermaphrodites were visually checked by fluorescence microscopy for the uptake of the rhodamine-conjugated dextran by the intestinal cells. The two mutants that did show a defect were retested on regular NGM plates (+OP50) that were not lacking in cholesterol but still had the rhodamine-conjugated dextran.

Injections into the pseudocoelom:
Substances were injected into the pseudocoelom of 7–10 adult dpy-20(e1282); arIs37[pmyo-3:ssGFP] hermaphrodites and kept at 20° on NGM (+OP50) plates. The injected worms were then checked at various times up to 24 hr.

Chemicals:
All chemicals used in this research were purchased from Sigma (St. Louis), except for Lucifer Yellow, Bodipy-conjugated cholesterol, and Transferrin Red, which were purchased from Molecular Probes (Eugene, OR).

Heat-shock experiment:
Hermaphrodites grown at 20° were synchronized by collecting the eggs after bleaching and allowing them to hatch on NGM plates. The starved L1 larvae were put on NGM (+OP50) plates and incubated at 20° for 2 more days until the worms were at the L4 stage. The L4 larvae were then heat-shocked at 33° for 30 min before being returned to 20° and checked at different times.

Microscopy:
Worms were mounted on a 2% agarose pad in 3.5 µl of 1% formaldehyde. Compound microscope images were taken on a Zeiss (Thornwood, NY) Axioplan.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Coelomocytes endocytose fluid from the pseudocoelom:
There are six coelomocytes in the pseudocoelom of adult hermaphrodites; four are present at hatching, and two are generated in the first larval stage (CHITWOOD and CHITWOOD 1974 ). The coelomocytes are full of membrane-bound compartments of different sizes that can be visualized in the light microscope (Fig 1D). By electron microscopy, the membranes of the coelomocytes appear to have multiple invaginations indicating that they are actively internalizing fluid from the pseudocoelom (WHITE 1988 ; D. HALL, personal communication). Indeed, GFP secreted into the pseudocoelom from many tissues is taken up predominantly by coelomocytes (FITZGERALD and GREENWALD 1995 ; GRANT and GREENWALD 1997 ; FARES and GREENWALD 2001 ; A. FIRE, personal communication). We made transgenic worms in which GFP is secreted into the pseudocoelom when it is attached to a signal sequence and expressed in body wall muscles. The GFP is endocytosed and subsequently degraded, primarily by the coelomocytes. Such arIs37[pmyo-3::ssGFP] worms (MATERIALS AND METHODS) display diffuse fluorescence in the pseudocoelom and bright fluorescent vesicles in coelomocytes (Fig 1, A–D).

View larger version (64K): In this window In a new window Download PPT slide   Figure 1. Coelomocyte uptake of substances introduced into the pseudocoelom. Schematic drawing (A), epifluorescence (B and C), and Nomarski (D) micrographs of a dpy-20(e1282); arIs37[pmyo-3::ssGFP] hermaphrodite. (E and F) Epifluorescence micrographs of a coelomocyte from an N2 hermaphrodite injected with FITC-conjugated BSA and rhodamine-conjugated dextran (Mr 40,000), respectively. (G) Epifluorescence micrograph of a dpy-20(e1282); arIs37[pmyo-3::ssGFP]; arEx218 [pcc1::DT-AK51E] hermaphrodite in which the coelomocytes have been ablated by expression of Diphtheria toxin. B and G are low magnification images of whole worms (anterior is to the left). Note the presence of some GFP in the pseudocoelom and in the coelomocytes (five are seen) in B and the accumulation of GFP in the pseudocoelom in G. (C and D) Higher magnification fluorescence and Nomarski images of the same coelomocyte, respectively. Arrows indicate the coelomocytes in B and vesicles filled with GFP in C and D. The shading represents GFP in A. B, C, and D have already been published (FARES and GREENWALD 2001 ). Bar, 3.7 µm in C.

To assay whether there is any specificity in uptake by coelomocytes, we injected various substances into the pseudocoelom. Many different substances proved to be endocytosed efficiently by coelomocytes when injected into the pseudocoelom: india ink (WHITE 1988 ; J. KIMBLE, personal communication), rhodamine-dextran (M_r 40,000; C. WINTER and D. HIRSH, personal communication), fluorescein isothiocyanate (FITC)-BSA (M_r 71,000), FITC-lipopolysaccharide (LPS) from Salmonella typhimurium, FITC-dextran (M_r 35,600), Transferrin Red (M_r 80,500), FITC (M_r 389.4), and rhodamine (M_r 536.1) (Fig 1E and Fig F; data not shown).

We found two substances that were not efficiently endocytosed [FITC-LPS from E. coli and Lucifer Yellow (M_r 521.56)] and two other substances [Bodipy-cholesterol and FITC-conjugated dextran (M_r 2,000,000)] that appeared to be completely excluded. Three of these substances, FITC-LPS (E. coli), Bodipy-cholesterol, and Lucifer Yellow, may not be available for uptake because they appear to adhere to yolk granules (BOSSINGER and SCHIERENBERG 1996 ; MATYASH et al. 2001 ), which are rarely taken up by the coelomocytes (D. HALL, personal communication). However, the failure of FITC-conjugated dextran (M_r 2,000,000) to be taken up by coelomocytes suggests that the coelomocytes nonspecifically endocytose soluble material below a certain size from the pseudocoelom, as FITC-dextran of M_r 35,600 is efficiently endocytosed.

Fate of GFP endocytosed by coelomocytes:
To determine the fate of GFP secreted into the pseudocoelom, we did an in vivo pulse chase using a strain carrying a transgene that expresses ssGFP from a heat-shock promoter (phsp::ssGFP). GFP is not produced at the normal growth temperature of 20°, but a brief heat shock at 33° results in a finite pulse of GFP secreted into the pseudocoelom. The fate of the GFP can then be followed as a function of time by returning the worms to 20° (Fig 2). We found that, after the worms are returned to 20°, we could observe the GFP first appearing in the pseudocoelom, then getting successively less bright in the pseudocoelom and more obvious in vesicles in the coelomocytes, before finally disappearing altogether (Fig 2). This suggests that the GFP is taken up and gets degraded, or is rendered otherwise nonfluorescent, by the coelomocytes.

View larger version (58K): In this window In a new window Download PPT slide   Figure 2. Behavior of ssGFP after its expression and secretion by heat shock in dpy-20(e1282); arIs36[phsp::ssGFP] hermaphrodites. Worms were grown at 20° (no heat shock), heat-shocked at 33° for 30 min, and put back at 20° for the indicated times shown above the micrographs. The top row of micrographs is at a higher magnification than those in the bottom row. Arrows indicate coelomocytes.

Toxin-mediated ablation of coelomocytes:
Coelomocytes are difficult to kill by laser ablation: apparently coelomocytes reported (via a personal communication in WHITE 1988 ) to have been eliminated by laser ablation had actually recovered and retained some function (J. KIMBLE, personal communication). To assess the importance of coelomocyte function, we used a coelomocyte-specific promoter (P. SENGUPTA, personal communication; P. LORIA and O. HOBERT, personal communication) to express a variant diphtheria toxin, DT-A_K51E (DELANGE et al. 1979 ). We verified the absence of the coelomocytes by examining the anatomy of dpy-20; arIs37[pmyo-3::ssGFP]; arEx218[pcc1::DT-A_K51E] hermaphrodites and the fate of the secreted GFP (MATERIALS AND METHODS). Most hermaphrodites lacked all six coelomocytes and accumulated GFP in the pseudocoelom (Fig 1G), confirming that GFP in the pseudocoelom is removed primarily by coelomocytes. We did not observe any effect on the viability or motility of this strain and were easily able to maintain the extragenic array for several generations, suggesting there is not a strong selection against it. We conclude that coelomocyte function, and in particular endocytosis by coelomocytes, is not necessary for viability and fertility of worms under normal laboratory conditions.

Involvement of genes known to function in membrane trafficking in endocytosis by coelomocytes:
We tested whether proteins that are thought to function in membrane trafficking in other systems, directly or indirectly, play a major role in endocytosis by coelomocytes. We reduced or eliminated the activity of these genes in the arIs37[pmyo-3::ssGFP] worms, either by using characterized mutant alleles or through the use of RNA-mediated interference (MATERIALS AND METHODS). We assayed the effect on coelomocytes by qualitatively examining the level of GFP in the pseudocoelom and the number and size of vesicles containing GFP in the coelomocytes.

Conventional mutations tested: Because in the arIs37 [pmyo-3::ssGFP] hermaphrodites we could reliably score only older larvae and adults for endocytosis by coelomocytes, we used only mutant alleles that did not result in zygotic lethality. We tested the effect on coelomocyte endocytosis of several genes that are known to affect membrane trafficking (snt-1, unc-26, unc-11, unc-13, unc-64, unc-101, dyn-1, pod-1, unc-104, ced-2, ced-5, nrf-5, nrf-6) and others that might influence the actin cytoskeleton (unc-60, unc-115, cyk-1, mig-2, unc-73; Table 1), as well as various miscellaneous mutations used for strain constructions (Table 1 legend). None of the mutations tested, except for dyn-1(ky51ts) (see below), resulted in a noticeable Cup phenotype. Where null alleles were used, we can conclude that the activity of the gene tested is not required for coelomocyte endocytosis. Where hypomorphic alleles were used, it remains possible that the lack of a Cup phenotype reflects residual gene activity.

dyn-1(ky51ts) is a temperature-sensitive mutation in dynamin, a large GTPase that regulates an early step in endocytosis and is involved in other cellular processes (CLARK et al. 1997 ). At the permissive temperature, 20°, dpy-20(e1282); dyn-1(ky51ts); arIs37[pmyo-3::ssGFP] worms appear indistinguishable from dpy-20(e1282); arIs37 [pmyo-3::ssGFP] worms. However, dpy-20(e1282); dyn-1 (ky51ts); arIs37[pmyo-3::ssGFP] worms that were shifted to 25° soon start displaying a Cup phenotype, characterized by accumulation of GFP in their pseudocoelom and a greatly reduced number of vesicles in coelomocytes (Fig 3).

View larger version (68K): In this window In a new window Download PPT slide   Figure 3. Effect of dyn-1(ky51ts) on coelomocyte endocytosis. All images are of dpy-20(e1282); dyn-1(ky51ts); arIs37[pmyo-3::ssGFP] hermaphrodites grown at 20° (A–C) or shifted for 8 hr to 25° (D–F). (B and C; E and F) Higher magnification fluorescence and Nomarski images of the same coelomocytes, respectively. Arrows indicate coelomocytes (not visible in D due to the decreased uptake of GFP). Note the presence of fewer and smaller vesicles in the coelomocytes shown in E as compared to those shown in B.

RNAi: We tested several genes (Table 2) that encode proteins that are homologous, or have domains homologous, to proteins known to function in membrane trafficking. For rme-1 and cup-5, RNAi was very effective and caused a phenotype that is essentially identical to that seen for null mutations in those genes (see below). vps-34, sec-5, and sec-8 also showed a very strong accumulation of GFP in the pseudocoelom of F₁ worms, while the accumulation of GFP in worms injected with vps-54 and clh-15 double-stranded RNA (dsRNA) was much weaker, but noticeably higher than in mock-injected worms.

When Cup RNAi phenotypes were observed, it was only in the F₁ progeny of parents that had been injected with double-stranded RNA. The injection method appeared to work best for several genes tested: when we tried soaking worms in double-stranded RNA (TABARA et al. 1998 ), we saw little or no effect (data not shown). Feeding worms bacteria expressing double-stranded RNA (TIMMONS and FIRE 1998 ) had a similar effect to RNA injection, but was less efficient (data not shown). We therefore could not use RNAi to assay several genes (e.g., clathrin heavy chain, RAB-5, and RAB-11) that are known to be involved in endocytosis but that resulted in lethality in the F₁ progeny of the injected worms and/or in sterility in the injected worms.

Screen for new mutants defective in endocytosis by coelomocytes:
We mutagenized dpy-20(e1282); arIs37[pmyo-3::ssGFP] worms with EMS and identified 55 viable mutants that displayed differences in endocytosis by coelomocytes compared to the parent strain. Of these mutations, 53 are recessive and define 14 complementation groups (Table 3; MATERIALS AND METHODS). We found that 3 of these complementation groups correspond to rme-1 rme-6 and rme-8, genes defined in a screen for receptor-mediated endocytosis of yolk (GRANT and HIRSH 1999 ). The two dominant mutations identified proved to be dominant-negative alleles of rme-1 (GRANT et al. 2001 ). On the basis of mapping data, inter se complementation tests, and complementation tests with other selected genes in the same region (see MATERIALS AND METHODS), the remainder of the 11 complementation groups appear to define new genes, which we have named cup.

The coelomocyte phenotype of cup mutants:
In all the mutants isolated, except cup-5(ar465), the gross phenotype we observed was the accumulation of GFP in the pseudocoelom, suggesting a decrease in, or the absence of, endocytosis in coelomocytes. cup-5(ar465) worms had the opposite phenotype: reduced GFP in the pseudocoelom and greatly intensified GFP accumulation in coelomocytes, suggesting enhanced uptake and decreased degradation of GFP by coelomocytes (FARES and GREENWALD 2001 ).

We examined more closely the phenotype of the coelomocytes in mutant worms grown at 20° (Fig 4). Interestingly, the coelomocytes exhibit a whole range of phenotypes, suggesting that the endocytic pathway is perturbed at several steps and/or in several ways. We show some examples in Fig 4, including almost no uptake of GFP by coelomocytes in cup-10(ar479) worms (Fig 4B), the accumulation of small peripheral GFP-filled compartments in cup-1(ar478) worms (Fig 4C), the accumulation of several vesicles that are full of GFP (indistinguishable from the wild-type profile at this level of analysis) in cup-11(ar472) worms (Fig 4D), the appearance of large vacuoles with little GFP in cup-8(ar466) worms (Fig 4E), and the accumulation of GFP in large vacuoles in cup-5(ar465) worms (Fig 4F).

View larger version (108K): In this window In a new window Download PPT slide   Figure 4. Phenotype of coelomocytes in the various cup mutant hermaphrodites. Shown are the fluorescence (1) and corresponding Nomarski micrographs (2) of wild-type worms (A) and worms carrying the following mutations: (B) cup-10(ar479), (C) cup-1(ar478), (D) cup-11(ar472), (E) cup-8 (ar466), and (F) cup-5(ar465). All worms also were dpy-20 (e1282); arIs37[pmyo-3::ssGFP]. F has already been published (FARES and GREENWALD 2001 ).

Additional phenotypes:
We checked all of the mutants for phenotypes that might indicate defects in endocytosis by other cell types (Table 3). On the basis of our limited analysis, seven of the complementation groups were deficient only in endocytosis by coelomocytes, and seven also affected endocytosis by other tissues.

Uncoordinated phenotype: An Unc phenotype might be indicative of defects in synaptic vesicle recycling (for example, NONET et al. 1993 , NONET et al. 1999 ; HARRIS et al. 2000 ). The only cup mutants that display an Unc defect are the two alleles of cup-7, which move very slowly and have a slight tendency to curl. All of the other mutants have normal movement.

Oocyte uptake of yolk: Accumulation of yolk in the pseudocoelom may be visualized as bright birefringence using Nomarski microscopy (KIMBLE and SHARROCK 1983 ). Using this assay, hermaphrodites bearing cup-1(ar478), cup-3(ar496), cup-6(ar513), or cup-7(ar492) accumulated more yolk granules in the pseudocoelom than the wild-type parent strains.

Intestinal uptake: Rhodamine-dextran (M_r 40,000) present in the growth medium is endocytosed by intestinal cells. We examined the cup mutants for intestinal accumulation of rhodamine-dextran in the presence or absence of cholesterol, a membrane component that must be added to the growth medium for continued growth of C. elegans in culture (BRENNER 1974 ) and that is required for some endocytic pathways (HOEKSTRA and VAN IJZENDOORN 2000 ). In the presence of supplemented cholesterol, only rme-8(ar486) was defective for uptake of rhodamine-dextran by intestinal cells. However, in the absence of supplemented cholesterol, both cup-1(ar478) and rme-8(ar486) showed a severe defect in rhodamine-dextran uptake in intestinal cells. The defect appeared to fall in an anterior-posterior gradient, with cells in the posterior half showing almost no uptake of the rhodamine-dextran. This gradient might reflect the concentration of the ingested substance, which may progressively decrease during its passage through the lumen. Thus, in nonmutant worms, endocytosis is efficient enough so that posterior cells may still scavenge detectable rhodamine-dextran from the lumen, whereas in cup-1 and rme-8 mutants, endocytosis is sufficiently impaired that rhodamine-dextran accumulation cannot be seen.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have shown that the coelomocytes continuously and efficiently endocytose fluid from the pseudocoelom and are not essential for the survival of the animal. We used the ability of coelomocytes to endocytose the fluid-phase marker GFP from the pseudocoelom as the basis for identifying mutants defective in endocytosis. A reverse genetic approach, using RNA-mediated interference, established that certain known genes are required for coelomocyte endocytosis and that blocking endocytosis leads to a Cup phenotype. A forward genetic screen for mutants identified 14 cup genes, 11 of which appear to be new. Here, we discuss what we have learned about the process of endocytosis in coelomocytes from our genetic analysis and evaluate the potential of the cup mutant screen for identifying new endocytosis genes.

We found that many soluble compounds injected into the pseudocoelom are taken up by the coelomocytes. The compounds that did not get endocytosed can be divided into two categories. One category comprises compounds that appeared to associate with yolk granules, which appear to be excluded from coelomocytes (D. HALL, personal communication) due to their size, hydrophobicity, or some other, unknown, property. The other category has as yet a single member, FITC-dextran (M_r 2,000,000), which did not appear to associate with yolk granules; because a smaller dextran polymer was very efficiently endocytosed by coelomocytes, size may be an important factor in determining the ability of a compound to be taken up. One reason that size may be important is that there is an electron-dense glycocalyx or basement membrane surrounding each coelomocyte (WHITE 1988 ; D. HALL, personal communication), which may in effect act as a sieve. Analysis of uptake at the electron microscopic level may help resolve this issue.

The large number of compounds that were efficiently endocytosed raises the question of whether coelomocytes take up material from the pseudocoelom by constitutive pinocytosis and/or by scavenger receptors displaying a broad spectrum of ligand recognition (PEARSON 1996 ). The results of our genetic analysis in C. elegans so far favor the possibility of a receptor-mediated mechanism.

First, the reduction or elimination of dynamin function led to an endocytosis defect in coelomocytes. Dynamin is known to function in the maturation and/or scission of the clathrin pit (SEVER et al. 2000 ; HILL et al. 2001 ) and in endocytosis through caveolae (HENLEY et al. 1998 ). Clathrin itself is very strongly expressed in coelomocytes (Y. ZHANG and D. HIRSH, personal communication) and numerous coated pits and vesicles are found on the surface of these cells (WHITE 1988 ). This suggests that clathrin-mediated endocytosis may be the major pathway in coelomocytes. An alternative explanation is that in the coelomocytes of C. elegans, dynamin might also be used in other pathways of endocytosis that do not involve receptors.

Second, rme-1 was identified in our genetic screen for cup mutants. RME-1 has been shown to be involved in membrane (and membrane protein) recycling to the plasma membrane (GRANT et al. 2001 ; LIN et al. 2001 ). In rme-1 null mutants, the Cup defect is more visible in older worms, as might be the case if scavenger receptors must be depleted from the plasma membrane before a Cup defect is seen. Alternatively, the Cup defect of rme-1 mutant worms could be due to a general depletion of bulk membrane from the surface or due to some function of RME-1 in these cells other than recycling.

Some genes encoding other proteins with characterized roles in vesicle trafficking were also identified as playing roles in coelomocyte endocytosis by virtue of a Cup phenotype in RNAi experiments. vps-34 encodes phosphoinositide 3-kinase (PI-3 kinase), a protein that regulates multiple steps in endocytosis (BACKER 2000 ). sec-5 and sec-8 encode homologs of the yeast Sec5p and Sec8p, members of the exocyst complex. S. cerevisiae sec mutants, including sec5 and sec8, which accumulate proteins in secretory vesicles, are all defective in endocytosis (RIEZMAN 1985 ). Vps54p in S. cerevisaie is involved in vacuolar trafficking (CONBOY and CYERT 2000 ). clh-15 encodes a chloride channel that in human cells is involved in receptor-mediated endocytosis (WANG et al. 2000 ); the reduction in endocytosis in clh-15(RNAi) worms is less severe than for other genes that affect endocytosis by coelomocytes, possibly because of the existence of multiple genes that encode chloride channels in C. elegans (NEHRKE et al. 2000 ).

Many genes, some of which are known to function in membrane trafficking, did not seem to be involved in endocytosis by coelomocytes. For example, null alleles of snt-1 Synaptotagmin, unc-26 Synaptojanin, unc-11 AP180, and unc-101 AP1 perturb membrane trafficking in neurons and other tissues in C. elegans but do not cause a Cup phenotype. While it is possible that some of the genes tested may not be expressed in coelomocytes, it is also possible that they are expressed in coelomocytes but only affect one pathway used by coelomocytes to internalize fluid. Furthermore, several of the genes tested have relatives in the C. elegans genome that may afford functional redundancy.

We screened for and identified mutations involved in the uptake of GFP by coelomocytes and identified alleles of 11 new cup genes and 3 previously identified rme genes. In this initial screen, we selected for homozygous viable and fertile Cup mutants. It is likely, therefore, that we have not identified genes that function in endocytosis and that regulate essential processes, like cell-cell signaling during embryonic development. Indeed, during the course of this screen, we saw some indication that there may be a category of endocytosis mutants that are also associated with zygotic or maternal-effect lethality. For several of the cup genes, we identified only single alleles, so we have not achieved saturation for this screen. Some of the alleles we recovered may prove to be non-null alleles of essential genes.

The cup screen described here and the rme screen described by B. GRANT and D. HIRSH (1999) are both powerful screens for endocytosis mutants and thus far have yielded largely complementary sets of genes. We have tested the available cup genes for yolk accumulation, and B. GRANT and D. HIRSH (personal communication) have tested most of the rme genes for a Cup phenotype. Although neither screen has been conducted to saturation, the data so far suggest that roughly one-third to one-half of the genes affect both yolk uptake by oocytes and pseudocoelomic GFP uptake by coelomocytes.

Both the Rme and the Cup screens have identified genes that play important roles in endocytosis in animal cells that do not appear to have homologs in yeast. As mentioned above, rme-1, which was identified in both the Rme and Cup screens, appears to be involved in membrane (and membrane protein) recycling to the plasma membrane (GRANT et al. 2001 ; LIN et al. 2001 ). rme-8, also identified in both the Rme and Cup screens, appears to be involved in endocytic vesicle trafficking (ZHANG et al. 2001 ). Finally, cup-5, which was identified solely by the Cup screen, encodes the C. elegans homolog of human mucolipin-1 (FARES and GREENWALD 2001 ). Loss of human mucolipin-1 underlies Mucolipidosis Type IV, a lysosomal storage disease that results in severe developmental neuropathology (BARGAL et al. 2000 ; BASSI et al. 2000 ; SUN et al. 2000 ). As we report elsewhere (FARES and GREENWALD 2001 ), our analysis has revealed that there are striking parallels between the cellular phenotypes and endocytosis defects of cup-5 mutant C. elegans and cells from patients with Mucolipidosis Type IV. We expect that at least some other cup genes will also prove to be components of the endocytosis machinery that are unique to animal cells.

Finally, what is the function of the coelomocytes of C. elegans? In many invertebrates, coelomocytes/hemocytes mediate the cellular immunity of the organism (PANCER et al. 1999 ). We have not found evidence that the coelomocytes of C. elegans provide a potent defense against bacterial infection; as few as six bacterial cells (OP50) injected into the pseudocoelom can multiply and kill the worm (H. FARES and I. GREENWALD, unpublished observations). This function for coelomocytes may be better explored by comparing the susceptibility of wild type vs. worms lacking coelomocytes to infection; to date, however, the only microorganism reported to invade the pseudocoelom of worms is the fungus Drechmeria coniospora, which extends its hyphae into the pseudocoelom (JANSSON 1994 ). However, it may be that in C. elegans, the coelomocyte functions more like a liver. The pseudocoelomic fluid serves as the circulatory system for nutrients that are taken up, ingested, degraded, and secreted into the pseudocoelom by the intestinal cells. It is possible that the coelomocytes reprocess that fluid to detoxify it of harmful ingested substances. In that event, it is likely that the absence of coelomocytes would not be harmful to worms grown under laboratory conditions. It is now possible to test this by comparing the susceptibility of wild type vs. worms lacking coelomocytes to toxic material introduced into the growth medium or secreted by bacterial pathogens (TAN and AUSUBEL 2000 ). With the availability of worms that lack coelomocytes and worms that are defective in endocytosis by coelomocytes, we are now in a better position to determine the biological function of these cells in C. elegans.

	ACKNOWLEDGMENTS

We are grateful to David Hirsh for inspiring our interest in coelomocytes, Barth Grant and Yinhua Zhang for helpful reagents and discussions throughout the project, Piali Sengupta for the coelomocyte-specific promoter and Paula Loria and Oliver Hobert for additional information about it, David Hall for communicating unpublished observations, and John Collier for the Diphtheria toxin clones. We also thank Alan Coulson and Yuji Kohara for providing cosmids, strains, and cDNA clones, Richard Ruiz and Ilya Temkin for excellent technical assistance, Barth Grant, Oliver Hobert, and Sophie Jarriault for critical reading of the manuscript, and past and present members of the Greenwald, Hirsh, and Hobert laboratories for helpful discussions. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources (NCRR). I.G. is an Investigator of the Howard Hughes Medical Institute. H.F. was a Postdoctoral Associate of the HHMI and was also supported by an award from the Metropolitan Life Foundation to I.G.

Manuscript received May 1, 2001; Accepted for publication May 25, 2001.

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