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The endocytic mechanisms underlying apical surface morphogenesis during epithelial development in Drosophila

Fabrowski, Piotr

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Cell shape changes are of fundamental importance during morphogenesis. These changes are often initiated by the contraction or expansion of plasma membrane domains. During differentiation the plasma membrane also undergoes more complex functional re-organization that brings about specialized function such as absorption, secretion and photo-transduction. While the role of cytoskeleton elements in controlling the structure and dynamics of the plasma membrane is well-established, little is known about the contribution of membrane trafficking in this process. The aim of this thesis was to address the contribution of membrane trafficking in controlling cell shape changes during tissue morphogenesis. More specifically I have investigated the role of endocytosis in controlling the remodeling of the plasma membrane during cellularization, the transformation of the syncytial Drosophila embryo is 6000 mononucleated cells. By following the early endocytic regulator Rab5, I identified two pools of endosomes. Early during cellularization endosomes accumulate at the invaginating furrows. Towards the end of cellularization a second pool of endosomes appeared at the apical surface. This increase in apical endosomes coincides with changes in apical morphology. Blocking endocytosis by inhibiting dynamin function prevented the re-absorption of apical protrusions and subsequent membrane flattening. Using a novel genetically-encoded cargo uptake assay I discovered that during apical surface flattening endocytosis is up-regulated of approximately five-fold. Strikingly this assay also revealed that the primary entry route for soluble extracellular cargo is through long tubular intermediates that serve as platform for the generation of Rab5 vacuolar endosomes. Blocking dynamin activity resulted in the complete inhibition of both tubular endocytosis as well as in the disappearance of Rab5 endosomes. These data collectively support a role for membrane trafficking in morphological remodeling. Surface flattening is thus an endocytosis-dependent morphogenetic process driven by the rapid internalization of large quantities of plasma membrane through tubular invagination and up-regulation of Rab5 endosome production. To further characterize the molecular machinery controlling apical endocytosis during cellularization a biochemical approach was undertaken. I performed large-scale affinity purification from 0-4 h embryos in order to identify Rab5 effectors operating during these early stages of embryonic development. This experiment led to the identification of Rabankyrin-5. Using a combination of live imaging and correlative light-electron microscopy I could show that Rabankyrin-5 controls the budding and processing of apical vacuoles from tubular plasma membrane invagination. In conclusion, in this thesis I have identified a novel endocytic pathway and linked its function to the remodeling of the apical surface during epithelial morphogenesis.

Document type: Dissertation
Supervisor: De Renzis, Dr. Stefano
Date of thesis defense: 20 February 2013
Date Deposited: 26 Mar 2013 07:41
Date: 2013
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > European Molecular Biology Laboratory (EMBL)
DDC-classification: 500 Natural sciences and mathematics
570 Life sciences
Controlled Keywords: Endocytose, Morphogenese, Taufliege
Uncontrolled Keywords: Drosophila development, cellularization
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