RESUMO
Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca2+-binding sites in Scramb1 are essential for its activity raising the possibility that Ca2+ signaling may control the assembly of SDs by impacting on Scramb1 activity.
Assuntos
Proteínas de Drosophila , Proteínas de Transferência de Fosfolipídeos , Podócitos , Animais , Podócitos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Microdomínios da Membrana/metabolismo , Junções Intercelulares/metabolismoRESUMO
The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. Here, we show that Drosophila CUBAM is a tripartite complex composed of Amnionless and two cubilin paralogues, Cubilin and Cubilin2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, which is strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Of note, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes suggest a possible unexpected conserved role for this endocytic receptor in vertebrate SD remodelling.
Assuntos
Proteínas de Drosophila/genética , Endocitose/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Receptores de Superfície Celular/genética , Proteínas rab5 de Ligação ao GTP/genética , Animais , Diafragma/crescimento & desenvolvimento , Diafragma/metabolismo , Drosophila melanogaster/genética , Junções Intercelulares/genética , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Morfogênese/genética , Complexos Multiproteicos/genética , Podócitos/metabolismoRESUMO
Zinc is a component of one-tenth of all human proteins. Its cellular concentration is tightly regulated because its dyshomeostasis has catastrophic health consequences. Two families of zinc transporters control zinc homeostasis in organisms, but there is little information about their specific developmental roles. We show that the ZIP transporter Fear-of-intimacy (Foi) is necessary for the formation of Drosophila muscles. In foi mutants, myoblasts segregate normally, but their specification is affected, leading to the formation of a misshapen muscle pattern and distorted midgut. The observed phenotypes could be ascribed to the inactivation of specific zinc-finger transcription factors (ZFTFs), supporting the hypothesis that they are a consequence of intracellular depletion of zinc. Accordingly, foi phenotypes can be rescued by mesodermal expression of other ZIP members with similar subcellular localization. We propose that Foi acts mostly as a transporter to regulate zinc intracellular homeostasis, thereby impacting on the activity of ZFTFs that control specific developmental processes. Our results additionally suggest a possible explanation for the presence of large numbers of zinc transporters in organisms based on differences in ion transport specificity and/or degrees of activity among transporters.