Biology subjects

Schejter, E. D.

Publications and source records attributed to Schejter, E. D..

6 recordsLinked to original sources

bioRxiv2026

Vesicular pseudopodia define the fusion site on large secretory vesicles of the Drosophila salivary glands

Large secretory vesicles (LSVs) pose a scaling problem for regulated exocytosis. Their micron-scale dimensions greatly increase the vesicular membrane surface area, making productive engagement between the vesicular and target membrane fusion machinery unlikely. Here, we show that vesicular pseudopodia define the fusion sites of LSVs in Drosophila larval salivary glands. Focused ion beam scanning electron microscopy revealed that most LSVs project polarized pseudopodia that interconnect neighboring vesicles and orient toward the apical membrane. Exposed pseudopodia were frequently observed at the apical surface and associated with narrow fusion pores, indicating that fusion occurs at these structures. Three-dimensional correlative light and electron microscopy showed that the I-BAR protein Missing in Metastasis (MIM) selectively localizes to exposed pseudopodia. Proteomic analysis based on a MIM pull-down assay identified exocyst components, including Sec15, which localizes to pseudopodia and persists at fusion sites throughout secretion. Finally, the tetraspanin Tsp42Ee marked complementary apical fusion domains and was required for efficient exocytosis. Our findings support a model in which prepatterned vesicular and apical membrane domains coordinate efficient exocytosis. SummaryRegulated exocytosis of large secretory vesicles is facilitated by vesicular pseudopodia and an apical fusion domain that spatially organizes membrane tethering and fusion during secretion.

cell biology↗

bioRxiv2023

Structured RhoGEF recruitment drives myosin II organization on large exocytotic vesicles

The Rho family of GTPases plays a crucial role in cellular mechanics, by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, that in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 diffuse to the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.

cell biology↗

bioRxiv2023

Fusion pore dynamics of large secretory vesicles define a distinct mechanism of exocytosis

Exocrine cells utilize large secretory vesicles (LSVs) up to 10 m in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. We observe that the fusion pore of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. We identify several Bin-Amphiphysin-Rvs (BAR) homology domain proteins that regulate fusion pore expansion and stabilization. We show that the I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. We conclude that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis that facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion.

cell biology↗

bioRxiv2021

Generation and timing of graded responses to morphogen gradients

Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here we examine whether morphogens can also induce a graded response within such domains. To this end we explore the role of the Dorsal protein nuclear gradient along the dorso-ventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of critical zygotic target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions where the levels are lower, due to a Dorsal-dependent priming probability of transcription sites. Thus, sites that are activated earlier will lead to more mRNA accumulation. Second, once the sites are primed, the rate of Pol II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, that provide a delay in production of the mature mRNAs.

developmental biology↗

bioRxiv2018

Dynamics of Spaetzle morphogen shuttling in the Drosophila embryo shapes pattern

Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible extracellular milieu, and rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, we followed the generation of graded nuclear Dorsal (Dl) protein, the hallmark of pattern formation along the dorso-ventral axis, in live embryos. We show that a sharp gradient is formed through extracellular, diffusion-based morphogen shuttling that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dl into the nuclei at each cycle refines the signaling output, by guiding graded accumulation of the T48 transcript that drives patterned gastrulation. We conclude that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos.

bioinformatics↗

bioRxiv2017

Formation and disassembly of a contractile actomyosin network mediates content release from large secretory vesicles

Secretion of adhesive glycoproteins to the lumen of Drosophila larval salivary glands is carried out by contraction of an actomyosin network that is assembled around large secretory vesicles, following their fusion to the apical membranes. We have identified a cycle of actin coat nucleation and disassembly that is independent of myosin. Recruitment of active Rho1 to the fused vesicle triggers activation of the formin Diaphanous and nucleation of linear actin. This, in turn, leads to actin-dependent localization of a RhoGAP protein that locally shuts off Rho1, promoting disassembly of the actin coat. Recruitment of the branched actin nucleation machinery is also required for effective Rho1 inactivation. Interestingly, different blocks to actin coat disassembly arrested vesicle contraction, indicating that actin turnover is an integral part of the actomyosin contraction cycle. The capacity of F-actin to trigger a negative feedback on its own production may be utilized in a variety of scenarios, to coordinate a succession of morphogenetic events or maintain homeostasis.\n\nSummaryThis work identified a cycle of actin assembly and disassembly in large secretory vesicles of Drosophila salivary glands. Actin disassembly is triggered by actin-dependent recruitment of a RhoGAP protein, and is essential for the contractility of the vesicle leading to content release to the lumen.

cell biology↗