Alex Holmes & Jonathan Pettitt. The mammalian Disabled proteins are cargo-specific adaptor proteins that facilitate the endocytosis of lipoprotein receptors. We, and others, have obtained data implicating the sole C. elegans Disabled, DAB-1, in the trafficking of the lipoprotein receptors LRP-1/-2 (Kamikura & Cooper, 2003; Holmes & Pettitt, IWM, 2005), suggesting that this is a conserved function of Disabled proteins. We have shown that DAB-1 is expressed in oocytes, early embryos, and in all epithelia of late stage embryos and postembryonic stages. It forms membrane-associated puncta that may correspond to clathrin-coated pits or vesicles. DAB-1 puncta are largely confined to the apical membranes of cuticle-secreting epithelia, such as the hypodermis and pharynx: in contrast they are enriched on the basolateral membranes of intestinal cells. Strong punctate expression is also seen on the surfaces of coelomocytes, consistent with the coelomocyte uptake (Cup) phenotype observed in
dab-1 null mutants. DAB-1::GFP expression during endocytosis in epithelial tissues and coelomocytes is particularly dynamic, and we are currently investigating whether DAB-1 puncta dynamics in the hypodermis are subject to developmental regulation, such as during moulting. We are also examining the role of clathrin and the AP-2 complex on puncta turnover. We have previously identified a synergistic genetic interaction between AP-1-mediated trafficking and DAB-1 mediated endocytosis. Similarly, loss-of-function mutations affecting components of the AP-3 complex are synthetic lethal in combination with
dab-1 mutations. This suggests 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, some cargoes normally sorted by these adaptors can still reach their normal destinations via DAB-1-dependent endocytosis from the plasma membrane.. We want to identify receptors participating in DAB-1-dependent endocytosis. Coelomocytes endocytose a broad range of substrates suggesting that the receptor(s) involved can bind to a wide range of ligands. Lipoprotein receptors, such as megalin, are known to recognise a broad range of ligands making these good candidates for endocytic receptors acting in coelomocytes. We have identified 16 transmembrane proteins containing the DAB-1-binding motif, [F/Y]xNPxY, in their intracellular domain. Five of these are members of the lipoprotein receptor family: LRP-1, LRP-2, RME-2, T13C2.6 and APL-1. It is likely that not all identified receptors bind only to DAB-1, since NUM-1 and CED-6 have also been shown to bind to NPxY motifs. We are carrying out systematic RNAi knockdown of each of these genes to determine whether this will phenocopy the
dab-1 null phenotype. Combined RNAi of
lrp-1 and
lrp-2 results in a high level of embryonic/larval lethality with a greatly increased frequency of moulting defects, indicating that these molecules have overlapping functions, however coelomocyte endocytosis is not noticeably defective.