Sec5, a member of the exocyst complex, mediates<i>Drosophila</i>embryo cellularization

Murthy, Mala; Teodoro, Rita O.; Miller, Tamara P.; Schwarz, Thomas L.

doi:10.1242/dev.048330

Cited by 34 publications

(40 citation statements)

References 65 publications

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“…The rate of furrow formation is similar to those reported for furrow ingression during the fast, but not slow, phase of cellularization (0.8 µm/min; Lecuit et al, 2002). The fast phase of cellularization is a process that, like syncytial furrow formation, is heavily dependent on targeted membrane addition (Lecuit and Wieschaus, 2000;Pelissier et al, 2003;Murthy et al, 2010; L.M.M., Z.Z., J.T.B., unpublished). Furrow retraction rates, by contrast, are not consistent between different mitotic cycles, and retraction speeds increase with syncytial mitotic cycles.…”

Section: Discussionsupporting

confidence: 80%

“…Embryos were dechorionated in 50% bleach solution and fixed for 1 h 15 min at the interface of heptane and 3.7% formaldehyde in 0.1 M sodium phosphate buffer ( pH 7.4) before being manually devitellinized and stained with Alexa 546-phalloidin (1:200; Molecular Probes), guinea pig anti-RalA (1:500; Teodoro et al, 2013), Hoechst (1:500; Sigma), mouse anti-Lamin (1:100; DSHB) or mouse anti-Sec5 (1:35; Murthy et al, 2010). Secondary antibodies conjugated with Alexa 488 or Alexa 568 (Molecular Probes) were used at 1:500.…”

Section: Embryo Fixation and Immunostainingmentioning

confidence: 99%

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A rapid, membrane-dependent pathway directs furrow formation through RalA in the earlyDrosophilaembryo

et al. 2015

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Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. Here, we use 4D imaging to identify furrow formation, stabilization, and regression periods, and identify a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. We demonstrate that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos.

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Section: Discussionsupporting

confidence: 80%

Section: Embryo Fixation and Immunostainingmentioning

confidence: 99%

A rapid, membrane-dependent pathway directs furrow formation through RalA in the earlyDrosophilaembryo

et al. 2015

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“…S4). Interestingly, immunostaining using a well-documented Sec5 (an exocyst subunit) (Beronja et al, 2005;Langevin et al, 2005;Murthy et al, 2003;Murthy et al, 2010) antibody, also uncovered a similar front-back ratio of 1.80±0.15 (n=16) in its distribution pattern (Fig. 3E,E′,J).…”

Section: Sec 3 Is Required For Border Cell Migrationmentioning

confidence: 62%

Guidance receptor promotes the asymmetric distribution of exocyst and recycling endosome during collective cell migration

et al. 2013

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During collective migration, guidance receptors signal downstream to result in a polarized distribution of molecules, including cytoskeletal regulators and guidance receptors themselves, in response to an extracellular gradient of chemotactic factors. However, the underlying mechanism of asymmetry generation in the context of the migration of a group of cells is not well understood. Using border cells in the Drosophila ovary as a model system for collective migration, we found that the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) is required for a polarized distribution of recycling endosome and exocyst in the leading cells of the border cell cluster. Interestingly, PVR signaled through the small GTPase Rac to positively affect the levels of Rab11-labeled recycling endosomes, probably in an F-actindependent manner. Conversely, the exocyst complex component Sec3 was required for the asymmetric localization of RTK activity and F-actin, similar to that previously reported for the function of Rab11. Together, these results suggested a positive-feedback loop in border cells, in which RTKs such as PVR act to induce a higher level of vesicle recycling and tethering activity in the leading cells, which in turn enables RTK activity to be distributed in a more polarized fashion at the front. We also provided evidence that E-cadherin, the major adhesion molecule for border cell migration, is a specific cargo in the Rab11-labeled recycling endosomes and that Sec3 is required for the delivery of the E-cadherin-containing vesicles to the membrane.

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“…During lumen formation and ciliogenesis in mammalian cells, Rab8 binds to the Sec15 subunit of the exocyst complex (Bryant et al, 2010;Knödler et al, 2010;Feng et al, 2012). This function and interaction might be preserved during cellularization, as the Sec5 subunit of the exocyst complex is required for cellularization, and Sec5 localizes to the apicolateral region of the cellularizing furrow in the area where membrane addition and Rab8 localization occur (Murthy et al, 2010). Indeed, disruption of Sec5 by RNAi leads to a failure of Rab8 to transition to a cortical association.…”

Section: Discussionmentioning

confidence: 99%

“…As the exocyst complex is required for targeted membrane addition during furrow formation in the early embryo (Murthy et al, 2010;Holly et al, 2015) and Rab8 can bind to the Sec15 subunit of the exocyst during lumen formation and ciliogenesis (Bryant et al, 2010;Knödler et al, 2010;Feng et al, 2012), we asked if exocyst complex function is required for Rab8 dynamics in the early embryo. RNAi against Sec5 was performed in a YFP:Rab8 background and imaged over time.…”

Section: Sec5 and Rab11 Are Required For Targeting Of Rab8 To The Celmentioning

confidence: 99%

Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster

Mavor

Miao

Zuo

et al. 2016

Journal of Cell Science

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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, GTPlocked 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.

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Sec5, a member of the exocyst complex, mediatesDrosophilaembryo cellularization

Cited by 34 publications

References 65 publications

A rapid, membrane-dependent pathway directs furrow formation through RalA in the earlyDrosophilaembryo

A rapid, membrane-dependent pathway directs furrow formation through RalA in the earlyDrosophilaembryo

Guidance receptor promotes the asymmetric distribution of exocyst and recycling endosome during collective cell migration

Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster

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