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First published online 17 July 2007
doi: 10.1242/jcs.03474
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Research Article |
1 Cell and Developmental Biology Program, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
2 Hubrecht Lab/NIOB, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
* Author for correspondence (e-mail: j.pettitt{at}abdn.ac.uk)
Accepted 24 May 2007
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Summary |
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Key words: Receptor-mediated endocytosis, C. elegans, Disabled, CLASP
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). Lipoprotein receptors are an important class of endocytosis receptors, many of which have a broad ligand-binding specificity, and participate in a diverse range of biological processes (May and Herz, 2003; Nykjaer and Willnow, 2002). The endocytosis of lipoprotein receptors is dependent upon the presence of F/YxNPxY motifs located in the intracellular domains of these molecules (Chen et al., 1990; Trommsdorff et al., 1998), and members of the Disabled family of adaptor molecules have a high affinity for this motif, mediated by the highly conserved phosphotyrosine-binding (PTB) domain (Stolt et al., 2003; Yun et al., 2003).
Disabled was first identified in Drosophila melanogaster on the basis of five mutations that enhanced the phenotype caused by loss of the Abelson tyrosine kinase (Gertler et al., 1993). These mutations were recently shown to be alleles of a neighbouring gene, neurotactin (Liebl et al., 2003), so the apparent interaction between Abelson kinase and Disabled is now in doubt, and much of what is known about Disabled function has come from studies of the two mammalian Disabled proteins: Dab1 and Dab2. Dab1 is a key component in the Reelin signalling pathway that regulates neuroblast migration during mammalian brain development (Rice and Curran, 2001). Engagement of very low density lipoprotein (VLDL) and ApoER2 receptors by the extracellular ligand Reelin recruits Dab1, and leads to the mutual activation of Dab1 and Src family kinases (Arnaud et al., 2003; Bock and Herz, 2003; Howell et al., 2000). This initiates a signalling cascade that leads to modification of cell behaviour.
Dab2 has been classified as a tumour suppressor based upon studies of a variety of carcinomas (Fulop et al., 1998; Mok et al., 1998; Sheng et al., 2000; Tseng et al., 1998; Zhoul et al., 2005); however, the reason why reduced expression of Dab2 affects tumour progression is not well understood. Dab2 is more widely expressed than Dab1, but also acts as an adaptor protein associated with the endocytosis of lipoprotein receptors (Mishra et al., 2002; Morris et al., 2002a; Morris and Cooper, 2001; Oleinikov et al., 2000). Studies in mice have shown that Dab2 is required for megalin endocytosis in the visceral endoderm (Maurer and Cooper, 2005; Morris et al., 2002b); an event essential for embryonic development. Conditional Dab2 mutants also have defects in megalin-dependent transport in the proximal kidney tubules (Morris et al., 2002b). However, there is also experimental evidence implicating Dab2 in both transforming growth factor-
(TGF-) and Wnt signalling pathways (Hocevar et al., 2003; Hocevar et al., 2005; Hocevar et al., 2001; Prunier and Howe, 2005), although the mechanistic basis of these observations is not well understood.
Caenorhabditis elegans has a single Disabled gene that is predicted to encode two almost identical isoforms (Fig. 1A). The functional significance of the two isoforms is unclear, and for reasons of clarity we will refer collectively to both as DAB-1. The primary structure of DAB-1 shows more similarity to Dab2 than it does to Dab1, suggesting that it might have more functional affinity to the former. DAB-1 can bind to the intracellular domains of the lipoprotein receptors LRP-1 and LRP-2 (Kamikura and Cooper, 2003), as well as to clathrin and the AP-1 and AP-2 adaptor proteins (Kamikura and Cooper, 2006), a feature it shares with mammalian Dab2. Loss of dab-1 function, or both lrp-1 and lrp-2 together, leads to reduced secretion of the fibroblast growth factor (FGF) homologue EGL-17 (Kamikura and Cooper, 2003; Kamikura and Cooper, 2006). In addition, dab-1(RNAi) animals display an incompletely penetrant defect in cuticle moulting (ecdsysis) similar to, but less severe than, that observed by lrp-1-null mutants (Yochem et al., 1999).
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), indicating that even if dab-1(gk291) homozygotes produce a small amount of product that is not detectable by western blotting, it is unlikely to have any activity. Both dab-1 mutants display variable defects in egg-laying and frequently become temporarily trapped in old cuticle (Fig. 2A), or remain attached to large fragments of shed cuticle (22%; n=596); both of which are phenotypes previously associated with dab-1 loss-of function. Most dab-1(gk291) homozygotes (97%; n=596) reach fertile adulthood, and they produce approximately wild-type brood sizes: gk291 homozygotes produce an average of 225 (s.e.m.±19) progeny. Both dab-1 mutants grow more slowly than wild type, and are sluggish in their movements.
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; Mishra et al., 2002; Morris et al., 2002a; Morris and Cooper, 2001). In order to investigate whether DAB-1 also plays a role in endocytosis we examined dab-1 mutants for defects in endocytosis. In C. elegans, yolk uptake into oocytes and fluid phase endocytosis from the pseudocoelomic fluid by the macrophage-like coelomocytes are well-characterised endocytic events (Fares and Greenwald, 2001; Grant and Hirsh, 1999). We thus examined these two processes in dab-1 mutants.
Yolk synthesised by the adult intestine is secreted into the body cavity where it is selectively taken up by oocytes through receptor-mediated endocytosis. A member of the lipoprotein receptor family, RME-2, is essential for yolk endocytosis (Grant and Hirsh, 1999). Transgenic strains that express a green fluorescent protein (GFP)-tagged version of the YP170 yolk protein (YP170::GFP) can be used to assay this process (Grant and Hirsh, 1999). We crossed the YP170::GFP-expressing transgene into the two dab-1 mutant backgrounds, and examined the distribution of the fusion protein. In both mutants the oocytes failed to accumulate detectable levels of YP170::GFP, and the fusion protein instead accumulated in the body cavity (Fig. 2E; n=369). This defect in receptor-mediated endocytosis (Rme phenotype) indicates that dab-1 is essential for endocytosis of yolk protein by oocytes.
A similar assay for coelomocyte endocytosis involves monitoring the expression of a secreted form of GFP from a transgene that is expressed in body wall muscle cells (Pmyo-3::ssgfp) (Fares and Greenwald, 2001). In wild-type animals the secreted GFP is taken up from the pseudocoelomic fluid and degraded by the coelomocytes. Impaired coelomocyte endocytosis will result in animals that accumulate GFP in the pseudocoelomic cavity (Fares and Greenwald, 2001). We monitored GFP fluorescence in adult dab-1(gk291) mutants carrying the Pmyo-3::ssgfp, and observed elevated levels of accumulation in the pseudocoelomic space of all animals examined (Fig. 2B; n=119). A similar coelomocyte uptake (Cup) defect was observed in hu186 homozygotes. By contrast, dab-1(gk291) animals carrying a wild-type copy of the dab-1 gene as an extrachromosomal transgene consistently showed wild-type levels of pseudocoelomic GFP accumulation (Fig. 2C), indicating that the Cup phenotype was attributable specifically to loss of dab-1 function. We also monitored coelomocyte function by injecting Texas Red-conjugated bovine serum albumin (BSA) into the pseudocoelom, revealing similar defects in coelomocyte uptake of this molecule in dab-1 mutants (data not shown).
DAB-1 is expressed in developing oocytes
Because dab-1 was required for YP170::GFP uptake, we expected it to be expressed in developing oocytes. We investigated DAB-1 expression using a monoclonal antibody raised to the N-terminus of the DAB-1 protein (Kamikura and Cooper, 2003). We detected DAB-1 expression in a punctate pattern localised at, or close to, the membranes of oocytes (Fig. 3A,D); a pattern of immunoreactivity not present in dab-1 mutants (Fig. 3G,H). The DAB-1 expression is consistent with an involvement in yolk uptake, because the DAB-1 puncta correlated with the presence of yolk protein accumulation.
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) (Fig. 3B,E), which possesses a putative DAB-1-binding motif in its intracellular tail (890YGNPMY), raising the possibility that the two molecules act together to mediate yolk protein uptake. Surprisingly, examination of the expression of DAB-1 and RME-2 in the same oocytes revealed little colocalisation of the two proteins (Fig. 3C,F); moreover, although both proteins were found in membrane-associated puncta, the DAB-1 puncta were consistently smaller than those of RME-2, some of which resembled vesicles in their morphology.
To further investigate the relationship between DAB-1 and RME-2 we examined the distribution of RME-2 in dab-1 mutants. We were able to detect membrane-associated RME-2 in dab-1 mutant oocytes, however the prominent puncta and vesicular structures observed in wild-type oocytes were dramatically reduced (Fig. 3H,K). The correlation between absence of RME-2 puncta and YP170::GFP uptake in dab-1 mutant oocytes suggests that DAB-1 plays a role in organising RME-2 into compartments competent to endocytose yolk protein.
RNA interference (RNAi) targeted to the clathrin heavy chain gene (chc-1) and the gene encoding the 
-adaptin subunit of the AP-2 complex (apa-2) also causes an Rme phenotype (Grant and Hirsh, 1999). We thus examined the distribution of DAB-1 in oocytes derived from animals subjected to chc-1(RNAi) and apa-2(RNAi) (Fig. 4). In both cases RNAi-treated animals displayed a clear Rme phenotype, as previously reported, in that YP170::GFP accumulated in the pseudocoelom; although significant accumulation of YP170::GFP in oocytes still occurred (Fig. 4G,J,M,P). These oocytes showed no evidence of DAB-1 puncta, although the protein could still be detected at the surface of the oocytes (Fig. 4H,K,N,Q). Thus, both clathrin and AP-2 are required for the organisation of DAB-1 into discrete structures, although not for its membrane localisation. A similar diffuse pattern of RME-2 localisation has previously been observed in animals subjected to chc-1(RNAi) (Grant and Hirsh, 1999).
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; Fig. 5B,E). DAB-1, in contrast, remains associated with the plasma membrane during the first few embryonic cleavages (Fig. 5A). In dab-1 mutant embryos RME-2 also becomes intracellular, and is similarly degraded during development (Fig. 5E). Thus, RME-2 can be internalised by distinct mechanisms: a DAB-1-dependent mechanism employed during the uptake of yolk, and one that is DAB-1-independent that operates to remove RME-2 from the surface of the fertilised embryo.
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), and thus could not use it to observe dab-1 expression in oocytes.
The dab-1::gfp transgene complements the Cup phenotype (and the ecdysis defects) displayed by the dab-1 mutants, indicating that the DAB-1::GFP fusion protein probably retains the activity and expression domains of the endogenous protein. The DAB-1::GFP in the coelomocytes formed surface-associated puncta (Fig. 6A,D), suggesting that DAB-1 is associated with clathrin-coated pits on the plasma membranes of these cells. To confirm this we examined the colocalisation of DAB-1::GFP in coelomocytes that expressed the clathrin light chain tagged with monomeric DsRed (CLIC-1::mDsRed). We found that all DAB-1::GFP puncta colocalised with CLIC-1::mDsRed (Fig. 6C,F), but also we were unable to observe any CLIC-1::mDsRed puncta that did not colocalise with DAB-1::GFP, suggesting that DAB-1 is the major adaptor protein associated with clathrin-mediated endocytosis in coelomocytes.
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To better understand the nature of the DAB-1-containing puncta we examined the behaviour of the DAB-1::GFP puncta in the epidermis and the coelomocytes in live animals using time-lapse fluorescence microscopy (Fig. 8; supplementary material Movies 1 and 2). Capturing images every five seconds in the same focal plane revealed that the puncta were highly dynamic, with puncta appearing and disappearing during the five-minute recording periods. The length of time that individual puncta persisted for was variable, with an average of approximately 40 seconds. Within the hypodermal puncta bands, we also observed regions that were devoid of puncta formation during the recording periods, as well as regions in which puncta formation was particularly frequent. However, we did not observe repeated puncta formation at the exact same points. The duration and clustering of puncta are reminiscent of those found in mammalian cells expressing GFP-tagged clathrin light chain and adaptin complex subunits (Gaidarov et al., 1999; Keyel et al., 2004; Perrais and Merrifield, 2005; Rappoport et al., 2005; Rappoport et al., 2004; Rappoport et al., 2003). In our time-lapse recordings of both the epidermis and the coelomocytes, the region of puncta formation and disappearance was tightly confined in close proximity to the plasma membrane: we did not observe any DAB-1::GFP puncta persisting or forming beyond this narrow region.
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). In order to investigate the effect of loss of dab-1 on the endosome and lysosomes, we used the coelomocyte-expressed RME-8::GFP and GFP::CUP-5 transgenes as markers of the endosomal and lysosomal compartments, respectively (Zhang et al., 2001; Treusch et al., 2004) (Fig. 9A,C). In dab-1 mutant coelomocytes RME-8::GFP-labelled vesicles were greatly reduced in size, and tended to cluster at the plasma membrane, and in association with large internal vesicles (Fig. 9B). We observed a similar reduction in the size of GFP::CUP-5-labelled vesicles (Fig. 9D). These defects are likely to be because of the reduced fluid and membrane uptake caused by reduced endocytosis.
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dab-1 synergises with components of the adaptor protein complexes
The fact that loss of dab-1 function affected both the endosome and lysosomes in coelomocytes, coupled with the broad expression of DAB-1::GFP, raised the possibility that loss of dab-1 might have a general impact on the trafficking to these compartments. The moulting defects that we, and others, have observed in dab-1 mutants support this conclusion. In order to investigate this possibility further we examined the phenotypes of dab-1 mutants lacking the function of genes encoding adaptor proteins involved in trafficking to and from other membrane compartments, reasoning that the contribution of DAB-1 might only be visible when other trafficking pathways were perturbed.
The AP-2 complex is required for the endocytosis of many cargos, and the genes encoding this complex were thus good candidates for molecules that might show genetic interactions with dab-1. However, we were unable to examine the interaction between dab-1 and the genes encoding the AP-2 complex, as loss of this multimeric adaptor alone results in embryonic lethality (Shim and Lee, 2000). The AP-1 complex is associated with clathrin-dependent vesicles budding from internal membranes to the plasma membrane and endosomes (Robinson, 2004). We examined the effect of reducing AP-1 trafficking in dab-1 mutants by using a loss-of-function mutation in unc-101 that encodes one of two C. elegans AP-1 µ1 subunits (Lee et al., 1994; Shim et al., 2000). Approximately 50% of unc-101(sy108) homozygotes survive to adulthood, whereas only 10% (n=703) of unc-101; dab-1 double mutants reach adulthood, with most failing to hatch or arresting just after hatching (Fig. 10E).
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We also examined the interaction between DAB-1 and the AP-3 complex, which is associated with the clathrin-independent targeting of proteins to lysosomes and lysosome-related organelles. In C. elegans the AP-3 complex function, as is the case in other organisms, is not essential for viability (Hermann et al., 2005), rather such animals display defects in the formation of the lysosome-related gut granules. RNAi targeted against apb-3, which encodes the -subunit of the AP-3 complex, in dab-1 mutants resulted in 100% embryonic lethality, but no observable lethality in similarly treated wild-type worms. To confirm the RNAi phenotype we made double mutants combining a putative null mutation affecting apb-3 (Hermann et al., 2005) with the dab-1-null mutations. apb-3(ok429); dab-1(gk291) double mutants displayed an almost completely penetrant maternal-effect embryonic lethal phenotype (n>1000), whereas neither single mutant showed significant levels of embryonic or larval lethality, indicating that the double mutant phenotype results from a synthetic interaction. We examined the affected embryos under high magnification to determine the cause of the developmental arrest. Most of the embryos underwent normal morphogenesis, reaching four times their pre-morphogenetic length, and displayed normal motility within the eggshell. Although these embryos developed a lumpy surface morphology, and accumulated birefringent gut granules in the gut lumen (Fig. 10D), both phenes are observed in the viable apb-3(ok429) single mutants (Hermann et al., 2005) (Fig. 10B), and are thus not likely to be the cause of the lethality. The only observable synthetic morphological defect was that approximately 15% of double-mutant embryos displayed a single large vacuole that invariably formed in the vicinity of the excretory cell (Fig. 10D). Similar vacuoles were observed to form in unc-101; dab-1 double mutants (Fig. 10E). We observed rare `escaper' apb-3; dab-1 double mutants that were able to hatch, and these in most cases developed to become fertile adults. These gave rise to broods showing the same maternal-effect lethality as their parents, suggesting that perhaps one defect in these animals is the inability to hatch.
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Our analysis of the role of DAB-1 in oocytes indicates that it is required for the uptake of yolk protein by the lipoprotein receptor RME-2, a process that is also dependent upon clathrin and AP-2. It is likely that the RME-2 puncta and vesicles detected in oocytes are the sites of active yolk protein endocytosis, and the fact that DAB-1 is required for both yolk uptake and the formation of RME-2 puncta suggests that DAB-1 affects yolk protein endocytosis, by preventing the formation of such sites. We have also shown that the DAB-1 puncta require clathrin and AP-2, suggesting that DAB-1 might act to recruit RME-2 to nacsent clathrin-coated pits. The fact that DAB-1 colocalises with clathrin in coelomocytes, the other major cell type affected by loss of dab-1 function, provides further evidence that DAB-1 is a component of clathrin-coated pits.
The fact that we do not detect significant colocalisation of RME-2 and DAB-1 at steady state is somewhat surprising (although it is noteworthy that RME-2 and YP170::GFP also do not colocalise at steady state) (Grant and Hirsh, 1999), and suggests that DAB-1 acts only to initiate RME-2 puncta, but is not present in later stages of RME-2-containing endocytic vesicles. This is in contrast to recent work showing that the closely related autosomal recessive hypercholesterolemia (ARH) protein escorts megalin throughout the endocytic cycle in MDCK cells (Nagai et al., 2003). If DAB-1 does not maintain its association during the subsequent endocytosis of RME-2, an alternative adaptor protein, such as AP-2, must be performing this role. It is noteworthy that the C-terminus of RME-2 consists of the motif 920ENDSLL. This motif matches the consensus signal shown to mediate rapid internalisation and targeting to endosomal-lysosomal compartments, and is able to bind to the AP-2 complex in vitro (Bonifacino and Traub, 2003). However, we cannot exclude the possibility that DAB-1 has a more stable interaction with RME-2 throughout the endocytosis cycle, and that failure to detect colocalisation is because of the epitope recognised by our DAB-1 antibody being masked when the protein is associated with RME-2.
Not all aspects of RME-2 localisation are affected in dab-1 mutants: its internalisation following fertilisation is independent of DAB-1 function. Thus, RME-2 can also be internalised through a DAB-1-independent mechanism that operates after fertilisation. What regulates the decision between the two mechanisms is an interesting question: the fact that RME-2 does not become internalised in this way in dab-1 mutants indicates that it is not simply a consequence of being engaged in endocytic activity.
Although yolk uptake, as monitored by YP170::GFP, is not detectable in dab-1 oocytes, there must be sufficient yolk uptake to support normal embryogenesis, because embryonic viability is not affected in dab-1 mutants. There is precedence for this situation, as both rme-1 and rme-6 mutant oocytes show similar defects in yolk-protein endocytosis, and likewise have no effect upon embryonic viability (Grant et al., 2001; Sato et al., 2005). The viability of such mutants hints at the existence of an alternative route for the acquisition of low levels of yolk protein sufficient to support embryonic development.
A general role for DAB-1 in lipoprotein receptor endocytosis?
Given the functional link between RME-2 and DAB-1, and the fact that DAB-1 can bind to LRP-1 and LRP-2, it seems likely that lipoprotein trafficking is a unifying feature of DAB-1 function. The requirement for DAB-1 in the endocytosis of molecules from the body cavity by the coelomocytes indicates that this process might also be mediated by lipoprotein receptors. If this proves to be the case it would not be surprising given the promiscuous activity of coelomocytes, which are able to uptake most substances injected into the body cavity (Fares and Greenwald, 2001). Lipoprotein receptors, such as megalin and LRP1, are able to bind to a diverse array of molecules (Nykjaer and Willnow, 2002), in part because of the large number of complement-type repeats found in their extracellular portions. Given this broad ligand-binding capacity, the similarly sized C. elegans receptors LRP-1 and LRP-2, as well as the so-far uncharacterised family member encoded by the gene T13C2.6, are good candidates as coelomocyte endocytic receptors. However, reducing the function of these genes through RNAi has failed to reveal a Cup phenotype (A.H. and J.P., unpublished).
In the epidermis, pharynx and cells that form structures that interface between the epidermis and internal epithelia, the DAB-1::GFP puncta were almost exclusively associated with the apical plasma membrane. All of these cells secrete an extracellular cuticle from their apical membranes, and given the moulting defects we observed in dab-1 mutants it is likely that there is a functional connection between apical DAB-1::GFP puncta and cuticle processing. This connection is supported by the fact that in the intestine, a tissue that does not secrete cuticle, the puncta were enriched on the basolateral membranes. The precise role of DAB-1 in the cuticle secreting epithelia is not clear, but given their highly similar expression patterns it seems likely that DAB-1 is acting in combination with the LRP-1 receptor. Growth of C. elegans in cholesterol-depleted media can induce similar moulting defects to those we observe in dab-1 mutants (Yochem et al., 1999), suggesting that ecdysis is critically dependent upon cholesterol. Thus, DAB-1::GFP puncta might correspond to sites involved in the LRP-1-mediated uptake of cholesterol required for normal moulting: possibly cells that are involved in cycles of cuticle synthesis and degradation have a particularly acute requirement for cholesterol. Alternatively, DAB-1/LRP-1 might act to remove an extracellular inhibitor of moulting, although it is hard to reconcile this with the dynamic behaviour of DAB-1 puncta in adult animals that no longer moult.
We do not know what determines whether DAB-1::GFP puncta become localised to the apical or the basolateral membranes. The PTB domain of Disabled selectively binds to phosphatidylinositol-4,5-bisphosphate (Stolt et al., 2005; Stolt et al., 2003; Yun et al., 2003), but there is little evidence in any animal cell that there is an apical-basal asymmetry in the distribution of this moiety, so it is likely that DAB-1 puncta are localised by asymmetric recruitment through protein-protein interactions, possibly by the receptor molecules themselves.
Taken together, the experimental evidence presented here indicates that DAB-1, like its mammalian homologues, is a monomeric adaptor protein involved in receptor-mediated endocytosis. The first reported study of dab-1 showed that animals with reduced dab-1 function displayed reduced secretion of the fibroblast growth factor homologue EGL-17 from vulval epidermal cells, leading to its accumulation in these cells (Kamikura and Cooper, 2003). This led to the proposal of a model whereby DAB-1, along with LRP-1 and LRP-2, participates in the formation of vesicles that mediate the export of newly synthesised EGL-17. Our data show that DAB-1 is exclusively associated with puncta on the apical surface of vulval cells. This observation, coupled with the localisation of LRP-1 at the same location (Yochem et al., 1999), suggests that DAB-1 is likely to regulate EGL-17 secretion through its role as an endocytic adaptor protein. Thus, DAB-1 might maintain high levels of LRP-1/LRP-2 at the recycling endosomes through endocytosis, from where they could associate with EGL-17 and facilitate its export (independent of DAB-1) through the basolateral membrane of the vulval cells. Alternatively, it may be that a significant fraction of EGL-17 is routed by default to the apical surfaces of the vulval cells from where it must be endocytosed by DAB-1/LRP-1/LRP-2 for subsequent trafficking between the apical and basolateral endosomes. This latter `transcytosis' hypothesis is supported by recent data showing that in dab-1 mutants the vulval cells accumulate GFP-tagged EGL-17 at the apical surface (Kamikura and Cooper, 2006), as might be expected if the role of DAB-1 is to recover misrouted EGL-17 from this membrane compartment.
Interaction between DAB-1 and adaptor protein complexes
Our identification of genetic interactions between dab-1 and the genes encoding components of the AP-1 and AP-3 complexes suggests that DAB-1 influences the transport pathways in which these multimeric adaptors participate. The localisation of DAB-1 with plasma membrane-associated vesicles, coupled with its clear role in endocytosis in oocytes and coelomocytes, makes it unlikely that DAB-1 is functionally equivalent to AP-1 or AP-3, which are associated with trafficking between internal membrane compartments (Bonifacino and Traub, 2003; Harasaki et al., 2005). One possibility is that DAB-1 is a component of a pathway that acts in parallel to AP-1 and AP-3 to traffic molecules from the plasma membrane. Thus, in the absence of AP-1- or AP-3-dependent pathways, cargoes normally sorted by these adaptors can still reach their normal destinations through DAB-1-dependent endocytosis from the plasma membrane. However, we have also shown that, at least in coelomocytes, loss of dab-1 affects the size and distribution of endosomes and lysosomes. Thus, an alternative possibility is that reduced endocytosis caused by loss of DAB-1 has an indirect effect by reducing the flow of membrane through the endosome and lysosome compartments. This might critically enhance the trafficking defects caused by loss of AP-1 and AP-3 complex components.
The importance of endocytosis in lysosome trafficking has been emphasised by recent work showing that AP-2-mediated endocytosis of the lysosome-associated membrane proteins (Lamps) plays a significant role in their targeting to lysosomes (Janvier and Bonifacino, 2005). The genetic interaction between dab-1 and apb-3 provides the first evidence that Disabled-mediated endocytosis influences lysosomal trafficking. Although we have no direct evidence that the synthetic lethal phenotype displayed by apb-3; dab-1 double mutants is caused by defects in lysosomal transport pathways, the fact that all known instances of AP-3 function from yeast to mammals involve sorting of cargo molecules to the lysosomal compartment suggests that defects in this process are responsible for the embryonic/early larval arrest phenotype displayed by apb-3; dab-1 mutants. Given the ample evidence for AP-3-independent lysosomal sorting pathways in mammalian cells (Dell'Angelica et al., 1999; Ihrke et al., 2004), it is possible that we have uncovered a conserved function for Disabled proteins in lysosomal trafficking. It will be interesting to determine whether Dab2 also plays a role in trafficking of molecules to the lysosomal compartments in mammalian cells.
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); unc-101(sy108) I (Lee et al., 1994); arIs37 [pmyo-3::ssgfp] I (Fares and Greenwald, 2001); dab-1(hu186, gk291) II; mIn1 II (Edgley and Riddle, 2001); mnDf88 II; bIs1 [vit-2::gfp; rol-6(su1006)] X (Grant and Hirsh, 1999); bIs34 [rme-8::gfp] (Zhang et al., 2001); cdIs40[pcc1::GFP::CUP-5; unc-119(+)-myo-2::GFP] (Treusch et al., 2004). The dab-1(gk281) allele was detected by single-worm polymerase chain reaction (PCR) using primers DAB1A-RTL (5'-CATCACCAAAGTCCCGAC-3') and DAB13 (5'-CGTGATGCAATGATGACTGC-3') to detect the gk281 deletion, and primers DAB1B-RTL and DAB-RTR (5'-CGTGTTTTGTGAGCTCCAG-3') to detect the wild-type allele. The allelic status of dab-1(hu186) was determined by monitoring the presence of a DraI restriction enzyme site created by this mutation. Single-worm PCR was performed using the primers DAB1B-RTL and DAB13, followed by DraI digestion of the resulting product.
We verified that the mnDf88 chromosomal deficiency deletes the dab-1 locus by performing single-embryo PCR on arrested embryos derived from unc-4(e120) mnDf88/mnC1 [dpy-10(e128) unc-52(e144)] hermaphrodites, using primers DAB15 (5'-CGCTGATCTTCTCGACTTGG-3') and DAB13. A control primer pair, which amplifies the hmp-1 gene, was included in the same PCR reaction as a positive control. In all cases, putative mnDf88 embryos were negative for the dab-1-specific PCR product.
Generation and analysis of transgenic strains
We generated a GFP-tagged version of DAB-1 using the PCR fusion-based approach (Hobert, 2002). The dab-1 gene, including 4374 bp of upstream sequence between the SL1 acceptor splice site of DAB-1A and the 3' end of the upstream gene M110.4, was amplified from genomic DNA using the primers DAB-1A (5'-GGTCCCGAGAACATTTCTTG-3') and DAB-1B (5'-AGTCGACCTGCAGGCATGCAAGCTGAAGAAATCATCACCAAATG-3'). The gfp gene and the unc-54 3' untranslated region (UTR) was amplified from plasmid pPD95.75 using primers GFPC (5'-AGCTTGCATGCCTGCAGGTCGACT-3') and GFPD (5'-AAGGGCCCGTACGGCCGACTAGTAGG-3'). The dab-1 and gfp-unc-54 3' UTR amplicons were fused together by PCR using the primers DAB-1AN (5'-AAGAGACGGTCGAAAAGTCG-3') and GFPE (5'-GGAAACAGTTATGTTTGGTATATTGGG-3'). The resulting fusion amplicon was injected into the germline of N2 hermaphrodites at a concentration of 30 ng/µl along with 100 ng/µl of the pRF4 dominant selectable marker. Multiple transformed lines were obtained, all of which displayed the same expression pattern. A single line, PE140, carrying the dab-1::gfp transgenic array (designated feEx43) was used in the experiments reported. The dab-1::gfp expression pattern that we obtained differs significantly from that derived from a previous study (Kamikura and Cooper, 2003). These workers did not detect expression of plasma membrane-associated puncta, and reported a more restricted range of dab-1::gfp-expressing cells. We do not know the reasons for these discrepancies.
To determine the ability of the dab-1::gfp to rescue the dab-1 ecdysis phenotype, we established a line homozygous for gk291 carrying the feEx43 transgene. Approximately 40% of the progeny of gk291; feEx43 animals failed to inherit the feEx43 extrachromosomal array, and thus lacked dab-1 function; we screened the broods of gk291; feEx43 for larvae trapped in unshed cuticle, and scored for the presence of DAB-1::GFP expression. None of the ecdysis-defective larvae were found to carry the feEx43 array (n=75), suggesting that the presence of the dab-1::gfp was able to rescue the dab-1 ecdysis defect.
To assay the effect of the dab-1::gfp transgene on the dab-1 Cup phenotype, we generated a gk291 strain carrying both feEx43 and the arIs37 transgene. Because both transgenes resulted in expression of GFP in the coelomocytes we used the presence of GFP in the body cavity of young adults as an indicator of coelomocyte activity.
The C. elegans clathrin light chain gene (clic-1) was tagged with monomeric DsRed (mDsRed) by amplifying the coding region of clic-1, together with 1135 bp of the associated upstream region, using primers CLIC-1A (5'-CATTCGAACTCGTACCAATCG-3') and CLIC-1B (5'-AGCTTGCATGCCTGCAGGTCGACTTTTTCCAGCATGTTTCAGACCAGC-3'). To generate the mDsRed amplicon we swapped the GFP coding region of pPD95.75 for the mDsRed open reading frame derived from pDsRed-Monomer vector (Clontech Laboratories) and amplified from the resultant template using GFPC and GFPD primers. The clic-1 and mDsRed amplicons were fused using the primers CLIC-1A* (5'-CCAGTCCATTTTTAGTCCACCCAA-3') and GFPE. Transgenic lines were generated by injecting the resultant clic-1::gfp transgene at 40 ng/µl, together with 200 ng/µl pRF4. Multiple, independent lines were obtained, all of which gave the same expression pattern. The clic-1::mDsRed extrachromosomal array from one of these lines was integrated into the genome by mutagenesis with ethane methyl sulphonate, selecting for animals that segregate 100% Rol progeny. The resultant integrated transgenic array (feIs6) was outcrossed ten times.
Microscopy and immunofluorescence
Microscopic analysis was performed using two systems: a Zeiss Axioplan 2 microscope equipped with DIC and epifluorescence optics, from which images were captured through a Hamamatsu Orca AG camera and Improvision Openlab software, and a Deltavision wide-field microscope (Applied Precision). For time-lapse analysis of embryonic development, embryos were mounted on 5% agar pads in egg salts, under a cover slip with Vaseline-sealed edges. Larvae and adults were mounted on 5% agar pads in M9 buffer. Where necessary, a 0.5% solution of 1-phenoxy-2-propanol was used as an anaesthetic.
Immunofluorescent detection of DAB-1 in the early embryo was performed with the PIF4-E5 monoclonal primary antibody (a generous gift from Darren Kamikura and Jonathan Cooper, Fred Hutchinson Cancer Research Center, Seattle, WA), and Cy2-conjugated goat anti-mouse or Texas Red-conjugated donkey anti-mouse secondary antibodies (Jackson ImmunoResearch). RME-2 was detected with anti-RME-2-INT primary rabbit antibody (Grant and Hirsh, 1999), and Cy2-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch). Embryos were freeze-cracked, fixed in methanol and stained essentially as described previously (Williams-Masson et al., 1997). Gonads were extruded from gravid hermaphrodites by decapitation of worms mounted on polylysine-treated slides, and fixed using the same protocol used for embryos.
The coelomocyte uptake assay using Texas Red-conjugated BSA was performed essentially as described previously (Zhang et al., 2001).
Western blotting
Protein extracts were prepared from frozen worm pellets by denaturing in Laemmli sample buffer for 5 minutes at 100°C, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto nitrocellulose by electroblotting, and probed with PIF4-E5 (mouse anti-DAB-1) or mouse anti-tubulin (Sigma) antibodies. Immunoreactive proteins were detected using the ECL system (Amersham).
RNA interference
L4-stage DH1033 animals were picked to plates seeded with HT115 bacteria expressing double-stranded RNA derived from the clathrin heavy chain gene (chc-1). Control animals were placed on HT115 bacteria transformed with the empty RNAi feeding vector. After 24 hours animals were examined to determine the localisation of YP170::GFP. chc-1(RNAi) resulted in a highly penetrant Rme phenotype not observed in control animals, as observed previously (Grant and Hirsh, 1999). For apa-2, bacterial-mediated interference did not give an obvious Rme phenotype. The insert of the cDNA clone yk132a1 (kindly supplied by Y. Kohara, National Institute of Genetics, Mishima, Japan) was PCR amplified, and the resulting product used as a template for T3 and T7 Megascript in vitro transcription kits (Ambion). The single-stranded RNAs were purified using RNeasy columns (Qiagen), combined and annealed to form double-stranded RNA (dsRNA). The syncitial germlines of DH1033 hermaphrodites were injected with dsRNA and allowed to recover for 24 hours, before being assayed for the presence of an Rme phenotype. Double-stranded RNA derived from the mDsRed transgene was used as a control for the RNAi injections.
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