title: Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster.
abstract: One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminate in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events are unclear. Here, we show that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. We additionally use CRISPR/Cas9 technology to N-terminally tag Rab8, which is then used to address endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. We also demonstrate that Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome protein Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, whereas GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through interactions with the exocyst tethering complex.
authors.affiliation: Department of Biological Sciences, University of Denver, Denver, CO 80208, USA.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Mavor
authors.affiliation: Department of Biological Sciences, University of Denver, Denver, CO 80208, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Miao
authors.affiliation: Department of Biological Sciences, University of Denver, Denver, CO 80208, USA.
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: Department of Biological Sciences, University of Denver, Denver, CO 80208, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Holly
authors.affiliation: Department of Biological Sciences, University of Denver, Denver, CO 80208, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Department of Physics, University of Denver, Denver, CO 80208, USA.
authors.forename: Dinah
authors.initials: D
authors.lastname: Loerke
authors.affiliation: Department of Biological Sciences, University of Denver, Denver, CO 80208, USA todd.blankenship@du.edu.
authors.forename: J Todd
authors.initials: JT
authors.lastname: Blankenship
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D002462:Cell Membrane / Q000378:metabolism; D003433:Crosses, Genetic; D003593:Cytoplasm / Q000378:metabolism; D029721:Drosophila Proteins / Q000502:physiology*; D004331:Drosophila melanogaster / Q000502:physiology*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism*; D005089:Exocytosis; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D008297:Male; D008565:Membrane Proteins / Q000502:physiology; D018613:Microscopy, Confocal; D017434:Protein Structure, Tertiary; D020691:rab GTP-Binding Proteins / Q000502:physiology; D000099193:rab11 GTP-Binding Proteins
chemical_list: D000199:Actins; D029721:Drosophila Proteins; D020558:GTP Phosphohydrolases; D006160:Guanosine Triphosphate; D008565:Membrane Proteins; D020691:rab GTP-Binding Proteins; C502950:Sec5 protein, Drosophila; C559123:Rab11 protein, Drosophila; D000099193:rab11 GTP-Binding Proteins; C000596969:Rab8 protein, Drosophila
keywords: Cellularization; Drosophila melanogaster; Furrow formation; Rab8
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