Biology subjects

Luschnig, S.

Publications and source records attributed to Luschnig, S..

12 recordsLinked to original sources

bioRxiv2026

TGF-β signaling regulates epithelial permeability in Drosophila ovaries by modulating adhesion independent of actomyosin contractility

Epithelial morphogenesis and homeostasis rely on dynamic remodeling of cell-cell junctions. Tricellular junctions (TCJs) at cell vertices are key sites that control epithelial permeability and plasticity, yet how TCJs are remodeled remains unclear. In the follicular epithelium (FE) in Drosophila ovaries, TCJs open transiently in a process called patency to allow passage of yolk proteins for uptake by the oocyte. We investigated how a gradient of TGF-{beta} signaling activity suppresses patency in a graded manner across the FE. We show that TGF-{beta} signaling blocks patency in a cell-autonomous manner by strengthening E-Cadherin (E-Cad)-based adhesion through inducing E-Cad transcription and preventing its removal from cell vertices. In parallel, TGF-{beta} signaling activates myosin II through Rho-Rok signaling. However, myosin II activity is dispensable for TGF-{beta}-mediated suppression of patency. We show that TGF-{beta} signaling controls TCJ remodeling in follicle cells primarily by reinforcing E-Cad-based adhesion, in part through upregulating p120-catenin. Our findings disentangle the roles of adhesion and actomyosin contractility in maintaining TCJ integrity and reveal how a tissue-scale morphogen gradient is translated into graded epithelial permeability.

developmental biology↗

bioRxiv2024

Rho/Rok-dependent regulation of actomyosin contractility at tricellular junctions controls epithelial permeability in Drosophila

Cell contacts in epithelia are remodeled to regulate paracellular permeability and to control passage of migrating cells, but how barrier function is modulated while preserving epithelial integrity is not clear. In the follicular epithelium of Drosophila ovaries, tricellular junctions (TCJs) open transiently in a process termed patency to allow passage of externally produced yolk proteins for uptake by the oocyte. Here we show that modulation of actomyosin contractility at cell vertices controls TCJ permeability. Before patency, circumferential actomyosin bundles are anchored at apical follicle cell vertices, where tension-sensing junctional proteins, Rho-associated kinase (Rok), and active Myosin II accumulate and maintain vertices closed. TCJ opening is initiated by redistribution of Myosin II from circumferential bundles to a medial pool, accompanied by decreasing tension on vertices. This transition requires activation of Cofilin-dependent F-actin disassembly by the phosphatase Slingshot and Myosin II inactivation by Myosin light chain phosphatase, and is counteracted by Rok. Accordingly, constitutive activation of Myosin or of Rho signaling prevent vertex opening, whereas reduced Myosin II or Rok activity cause excessive vertex opening. Thus, the opening of intercellular gaps in the follicular epithelium relies on relaxation of actomyosin contractility rather than active actomyosin-based pulling forces. Conversely, F-actin assembly is required for closing intercellular gaps after patency. Our findings are consistent with a force transduction model in which TCJ integrity is maintained by vertex-anchored contractile actomyosin. We propose that the cell-type-specific organization of actomyosin at cell vertices determines the mode of contractility-dependent regulation of epithelial permeability.

developmental biology↗

bioRxiv2024

Tracking exocytic vesicle movements with MSP-tracker/viewer reveals the spatial control of secretion in epithelial cells.

Understanding how specific secretory cargoes are targeted to distinct domains of the plasma membrane in epithelial cells requires analysing the trafficking of post-Golgi vesicles to their sites of secretion. We used the RUSH (retention using selective hooks) system to synchronously release an apical cargo, Cadherin 99C (Cad99C), and a basolateral cargo, the ECM protein Nidogen, from the endoplasmic reticulum and follow their movements to the plasma membrane. We also developed an interactive vesicle tracking framework, MSP-tracker and viewer, that exploits developments in computer vision and deep learning to determine vesicle trajectories in a noisy environment without the need for extensive training data. MSP-tracker outperformed other tracking software in detecting and tracking post-Golgi vesicles, revealing that Cad99c vesicles predominantly move apically with a mean speed of 1.1{micro}m/sec. This is reduced to 0.85 {micro}m/sec by a dominant slow dynein mutant, demonstrating that dynein transports Cad99C vesicles to the apical cortex. Furthermore, both the dynein mutant and microtubule depolymerisation cause lateral Cad99C secretion. Thus, microtubule organisation plays a central role in targeting apical secretion, suggesting that Drosophila does not have distinct apical versus basolateral vesicle fusion machinery. Nidogen vesicles undergo planar-polarised transport to the leading edge of follicle cells as they migrate over the ECM, whereas most Collagen is secreted at trailing edges. The follicle cells therefore bias secretion of different ECM components to opposite sides of the cell, revealing that the secretory pathway is more spatially organised than previously thought.

cell biology↗

bioRxiv2023

Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila

Tricellular junctions (TCJs) provide essential adhesive and occluding functions at epithelial cell vertices and play key roles for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka), Gliotactin (Gli), and M6 constitute tricellular occluding junctions. Aka and M6 localize in an interdependent manner to vertices and are required jointly to localize Gli, but how these proteins interact to assemble TCJs was not known. Here, we show that the tetraspan proteolipid protein M6 physically interacts with Aka and with itself. M6 is palmitoylated on a conserved juxta-membrane cysteine cluster. This modification promotes efficient vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed accumulation of M6 and Aka at vertices but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, while subsequent TCJ growth relies on different mechanisms independent of M6 palmitoylation.

cell biology↗

bioRxiv2022

Src42A is required for E-cadherin dynamics at cell junctions during Drosophila axis elongation

Src kinases are important regulators of cell adhesion. Here we explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular and tricellular junctions in germband cells and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and by CRISPR-Cas9-induced germline mosaics. We find that during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knock down. Furthermore, we provide evidence that Src42A acts in parallel to Abl, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes, one affecting tension generated by the planar polarity of MyoII and secondly, it can act as a signalling factor at tAJs in controlling E-cadherin residence time.

developmental biology↗

bioRxiv2022

Acute manipulation and real-time visualization of membrane trafficking and exocytosis in Drosophila

Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but tools for investigating dynamics of membrane trafficking have been limited to cultured cells. Here we present a system that enables acute manipulation and real-time visualization of membrane trafficking through reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER-retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualize and manipulate membrane trafficking in diverse cell types in vivo.

cell biology↗

bioRxiv2021

Tracheal tube fusion in Drosophila involves release of luminal exosomes from multivesicular bodies

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. Despite extensive work on the formation and functions of EVs in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We defined two distinct EV subpopulations, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion events. The formation of Stac-positive luminal EVs depends on the tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. We propose that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4.

developmental biology↗

bioRxiv2021

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

cell biology↗

bioRxiv2020

The transmembrane proteins M6 and Anakonda cooperate to initiate tricellular junction assembly in epithelia of Drosophila

Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1, 2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained and remodeled is poorly understood. In Drosophila the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembrane-domain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8, 9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10, 11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here we show that M6 is essential for the assembly of tricellular, but not bicellular occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a low-mobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices.

cell biology↗

bioRxiv2020

Transient opening of tricellular vertices controls paracellular transport through the follicle epithelium during Drosophila oogenesis

Paracellular permeability is regulated to allow solute transport or migration of cells across epithelial or endothelial barriers. However, how occluding junction dynamics controls paracellular permeability is poorly understood. Here we describe patency, a developmentally regulated process in Drosophila oogenesis, during which cell vertices in the follicle epithelium open transiently to allow paracellular transport of yolk proteins for uptake by the oocyte. We show that the sequential removal of E-Cadherin, N-Cadherin, NCAM/Fasciclin-2 and Sidekick from vertices precedes their basal-to-apical opening, while the subsequent assembly of tricellular occluding junctions terminates patency and seals the paracellular barrier. E-Cadherin-based adhesion is required to limit paracellular channel size, whereas stabilized adherens junctions, prolonged NCAM/Fasciclin-2 expression, impeded endocytosis, or increased actomyosin contractility prevent patency. Our findings reveal a key role of cell vertices as gateways controlling paracellular transport, and demonstrate that the dynamic regulation of adhesion and actomyosin contractility at vertices governs epithelial barrier properties.

developmental biology↗

bioRxiv2019

Matrix metalloproteinase 1 modulates invasive behavior of tracheal branches during ingression into Drosophila flight muscles

Tubular networks like the vasculature extend branches throughout the bodies of animals, but how developing vessels interact with and invade tissues is not well understood. We investigated the underlying mechanisms using the developing tracheal tube network of Drosophila indirect flight muscles (IFMs) as a model. Live imaging revealed that tracheal sprouts invade IFMs directionally with growth-cone-like structures at branch tips. Ramification inside IFMs proceeds until tracheal branches fill the myotube. However, individual tracheal cells occupy largely separate territories, possibly mediated by cell-cell repulsion. Matrix metalloproteinase 1 (MMP1) is required in tracheal cells for normal invasion speed and for the dynamic organization of growth-cone-like branch tips. MMP1 remodels the Collagen IV-containing matrix around branch tips and promotes degradation of Branchless FGF in cultured cells. Thus, tracheal-derived MMP1 may play dual roles in sustaining branch invasion by modulating ECM properties as well as by shaping the distribution of the FGF chemoattractant.

cell biology↗

bioRxiv2017

Smg5 is required for multiple nonsense-mediated mRNA decay pathways in Drosophila

The nonsense-mediated mRNA decay (NMD) pathway is a cellular quality control and post-transcriptional gene regulatory mechanism and is essential for viability in most multicellular organisms. A complex of proteins has been identified to be required for NMD function to occur, however the individual contribution of each of these factors to the NMD process is not well understood. Central to the NMD process are two proteins Upf1 (SMG-2) and Upf2 (SMG-3), which are found in all eukaryotes and are absolutely required for NMD in all organisms in which it has been examined. The other known NMD factors, Smg1, Smg5, Smg6, and Smg7 are more variable in their presence in different orders of organisms, and are thought to have a more regulatory role. Here we present the first genetic analysis of the NMD factor Smg5 in Drosophila. Surprisingly, we find that unlike the other analyzed Smg genes in this organism, Smg5 is essential for NMD activity. We found this is due at least in part to a role for Smg5 in the activity of two separable NMD-target decay mechanisms: endonucleolytic cleavage and 5'-to-3' exonucleolytic decay. Redundancy between these degradation pathways explains why some Drosophila NMD genes are not required for all NMD-pathway activity. We also found that while the NMD component Smg1 has only a minimal role in Drosophila NMD during normal conditions, it becomes essential when NMD activity is compromised by partial loss of Smg5 function. Our findings suggest that not all NMD complex components are required for NMD function at all times, but instead are utilized in a context dependent manner in vivo.

genetics↗