J Cell Biol

J Cell Biol. the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless SDZ 220-581 hydrochloride, SDZ220-581, SDZ-220-581 chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered. INTRODUCTION In oviparous animals, all the nutritional requirements for embryonic development are met by the deposition of a yolk store during oogenesis. This is necessary until eclosed progeny can feed independently, as early development occurs in the absence of any maternal support. Yolk typically consists of core phosphoglycolipoproteins cached in highly concentrated (often crystalline) form within mature yolk granules or spheresmembrane-bounded intracellular organelles in the oocyte cytoplasm; yolk is the major proteinaceous constituent of unfertilized eggs. In most organisms, yolk protein precursors (vitellogenins) are not synthesized by the oocyte but are instead produced by nongermline cells in a hormonally regulated manner (Raikhel and Dhadialla, 1992 ). This makes SDZ 220-581 hydrochloride, SDZ220-581, SDZ-220-581 the uptake and storage of yolk dependent on the endocytic apparatus, and, in fact, some of the earliest images of clathrin-mediated endocytosis come from ultrastructural analysis of developing eggs (Anderson, 1964 ; Roth and Porter, 1964 ; Perry and Gilbert, 1979 ). Termed vitellogenesis, the intense accumulation of yolk by the oocyte occurs at a fixed period during oocyte maturation and, since the immediate source of yolk precursors is the extracellular space, is terminated by deposition of the eggshell enshrouding the mature egg by the somatic follicular epithelium. This places temporal restrictions on the massive burst endocytic activity necessary to sequester a yolk depot that is ultimately critical to reproductive success. In the disease vector mosquitoes and and express trephin, which is transcriptionally regulated by a blood meal (Mishra ovary develops asynchronously, with individual ovarioles each Mouse monoclonal to IgG2b/IgG2a Isotype control(FITC/PE) containing a staggered series of progressively more mature egg chambers. Still, clathrin-coated structures are abundant at the oolemma of vitellogenic-stage eggs (Mahowald, 1972 ; DiMario and Mahowald, 1987 ; Sommer yolk accumulation. First, in the germline, three clathrin heavy chainCnull alleles cause a penetrant female sterility (Bazinet mutant (van der Bliek and Meyerowitz, 1991 ), disrupts yolk uptake. Within 5 min at the restrictive temperature, the prominent cortical endocytic compartment vanishes, whereas, reciprocally, extracellular yolk precursors, the oocyte surface area, and attached spherical clathrin profiles increase markedly (Kessell is designated Yolkless (Schonbaum mutant females shrivel, and strong alleles cause 10-fold decrease in clathrin-coated profiles at the oolemma of vitellogenic egg chambers (DiMario and Mahowald, 1987 ). Yolkless is unusual in that the cytosolic FXNPXY-type sorting signal is a nonstandard 1837FQNPLA, albeit positioned at the appropriate distance from the inner leaflet of the bilayer (Schonbaum species (Clark (Awasaki (Liu and Hengartner, 1998 ; Smits femaleis composed of 15 parallel ovarioles (King, 1970 ). During oogenesis, egg chambers within an ovariole progress through 14 morphologically discrete stages as they approach the oviduct, resulting in mature eggs ready for fertilization and subsequent embryonic development. Yolk storage is a vital aspect of oocyte maturation and, in ovary. (A) Endocytic uptake by vitellogenic (asterisks) stage Canton-S egg chambers revealed by in vitro incubation with trypan blue for 15 min. (B, C) Selective oocyte internalization of soluble RAP in dissociated vitellogenic egg chambers after incubation with soluble mcRFP-RAP for 20 min. Representative confocal image counterstained with Alexa 488Cphalloidin and Hoechst 33342 for DNA (B) or with differential interference contrast (C). (D) Thin-section micrograph of the cortical oocyteCfollicle cell region (vitellogenic stage 10) highlighting the active endocytic zone and extra-oocyte space (pseudocolored). (E) Confocal section showing germline expression and oocyte-specific localization (arrowheads) of Yolkless in dissociated Canton-S egg chambers SDZ 220-581 hydrochloride, SDZ220-581, SDZ-220-581 counterstained for DNA. (FCH) Confocal images of localization of clathrin in germline triple-driver Gal4 UAS-GFP-clathrin light-chain (LC) fly egg chambers. Arrowheads indicate cortical deposition in oocytes (asterisk) while little clathrin masses on the cortical plasma membrane of nurse cells (arrows). Scale bar, 100 m; 2 m in D. Ced-6 expression in the fly ovary LDL receptor superfamily members like Yolkless are generally recognized by PTB-domain CLASPs for internalization (Traub, 2009 ), and ARH orthologues operate in yolk storage in SDZ 220-581 hydrochloride, SDZ220-581, SDZ-220-581 (Zhou species do not have an immediate ARH/trephin orthologue and that in the most closely related PTB-domain protein to these endocytic factors is Ced-6 (CG11804; Tamborindeguy CED-6 protein that participates in the engulfment of apoptotic cell corpses (Liu and Hengartner, 1998 ; Smits transcript expression in ovarioles. Dissected ovaries from Canton-S.