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Eps8, Arp2/3 and IRSp53 in TNT formation

ABSTRACT: Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How the cell controls a common pool of proteins to generate these actin-mediated protrusions spanning length scales beyond those attainable by canonical filopodia remains unknown. Through a combination of surface micropatterning, microscopy and optical tweezer-based approaches, we found that Arp2/3-dependent pathways attenuate the extent with which actin polymerizes in nanotubes, limiting the formation and attainable lengths of TNTs. Upon Arp2/3 inhibition Epidermal growth factor receptor kinase substrate 8 (Eps8) exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 resulting in increased TNTs. In these conditions, Eps8 interaction with proteins enhancing filament turnover and depolymerization were reduced. Our data reveals how common players in protrusions (Eps8 and IRSp53) drive outward linear actin extension to form TNTs, and that their interaction is enhanced when competing pathways overutilizing actin in branched Arp2/3 networks are inhibited, suggesting a shift in the equilibrium (and proteins usage) between branched and linear actin polymerization to form different cell protrusions.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Neuronal Cell Body

DISEASE(S): Brain Cancer

SUBMITTER: MARIETTE MATONDO

LAB HEAD: Michael henderson

PROVIDER: PXD035976 | Pride | 2023-11-28

REPOSITORIES: Pride

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Project description:Development and homeostasis of blood vessels critically depend on the regulation of endothelial cell-cell junctions. Perturbations in cell-cell junction organization and function results in developmental defects and vascular pathologies including chronic inflammation, edema and atherosclerosis. Although many aspects of blood vessel formation and homeostasis depend on cell-cell junctions, the molecular mechanisms that regulate their dynamic rearrangement are not fully understood. The VEcad-catenin complex, which constitute the molecular basis of the adherens junctions (AJ), is connected to the actin cytoskeleton and its function is regulated by cytoskeletal contraction and actin-driven plasma membrane protrusions. Junction-associated intermitted lamellipodia (JAIL) are small actin-driven protrusions at cell-cell junctions controlled by the actin related protein 2/3 (Arp2/3)-complex that contribute to the regulation of cell-cell junctions. JAIL drive VEcad dynamics within the cell-cell junction thereby being critical for monolayer integrity, cell migration and angiogenesis. The molecular mechanisms regulating JAIL during vessel development are not completely understood. Coronin 1B (Coro1B) is an actin binding protein that controls actin networks at classical lamellipodia via both Arp2/3 complex and cofilin-mediated pathways. The role of Coro1B in endothelial cell (ECs) is not fully understood. In this study we demonstrate that Coro1B is a novel component and regulator of cell-cell junctions in ECs. Immunofluorescence studies show that Coro1B colocalizes with VEcad at cell-cell junctions in monolayers of ECs. Live-cell imaging reveal that Coro1B is recruited to, and operated at, actin-driven membrane protrusions at the cell-cell junctions. Coro1B recruitment to cell-cell junctions is regulated by cytoskeleton tension. By analyzing the Coro1B interactome, we identify integrin linked kinase (ILK) as new Coro1B-associated protein. Coro1B colocalizes with α-parvin, an interactor of ILK, at the leading edge of lamellipodia protrusions. Finally, functional experiments reveal that depletion of Coro1B causes defects in actin cytoskeleton and cell-cell junctions. In matrigel vessel network assays, depletion of Coro1B results in reduced network complexity, vessel number and vessel length. Together, our findings point towards a critical role for Coro1B in the dynamic remodeling of endothelial cell-cell junction and the assembly of vessel network.

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Eps8, Arp2/3 and IRSp53 in TNT formation

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