Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization

Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris F-75015, France.
The Journal of Cell Biology (Impact Factor: 9.83). 10/2011; 195(3):525-36. DOI: 10.1083/jcb.201105152
Source: PubMed


Bacterial pathogens recruit clathrin upon interaction with host surface receptors during infection. Here, using three different infection models, we observed that host-pathogen interactions induce tyrosine phosphorylation of clathrin heavy chain. This modification was critical for recruitment of actin at bacteria-host adhesion sites during bacterial internalization or pedestal formation. At the bacterial interface, clathrin assembled to form coated pits of conventional size. Because such structures cannot internalize large particles such as bacteria, we propose that during infection, clathrin-coated pits serve as platforms to initiate actin rearrangements at bacteria-host adhesion sites. We then showed that the clathrin-actin interdependency is initiated by Dab2 and depends on the presence of clathrin light chain and its actin-binding partner Hip1R, and that the fully assembled machinery can recruit Myosin VI. Together, our study highlights a physiological role for clathrin heavy chain phosphorylation and reinforces the increasingly recognized function of clathrin in actin cytoskeletal organization in mammalian cells.

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Available from: Matteo Bonazzi, Oct 09, 2015
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    • "Receptor ubiquitination induces the recruitment of clathrin and other components of the endocytic machinery, leading to assembly of clathrin-coated pits, whose reduced size precludes bacterial compartmentalization into those pits. Instead, it is proposed that the clathrin lattices function as platforms for the recruitment of actin via the actin-binding protein Hip1R and for the recruitment of myosin VI, which drives bacterial internalization (Bonazzi et al., 2011). Together, InlA and InlB exploit E-cadherin-and Metmediated signalling pathways, as well as their recycling pathways, to cause actin polymerization at bacterial entry sites (Fig. 1). "
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    ABSTRACT: Entry into host cells and intracellular persistence by invasive bacteria are tightly coupled to the ability of the bacterium to disrupt the eukaryotic cytoskeletal machinery. Herein we review the main strategies used by three intracellular pathogens to harness key modulators of the cytoskeleton. Two of these bacteria, namely Listeria monocytogenes and Salmonella enterica serovar Typhimurium, exhibit quite distinct intracellular lifestyles, and therefore, provide a comprehensive panel for the understanding of the intricate bacteria-cytoskeleton interplay during infections. The emerging intracellular pathogen Vibrio parahaemolyticus is depicted as a developing model for the uncovering of novel mechanisms used to hijack the cytoskeleton. This article is protected by copyright. All rights reserved.
    Cellular Microbiology 12/2014; 17(2). DOI:10.1111/cmi.12399 · 4.92 Impact Factor
    • "Based on these observations, we propose that underneath a phagocytic cup, CHC-1 oligomerizes into a scaffolding structure that facilitates actin remodeling. This structure might resemble the flat clathrin patches that coat specific membrane domains on endosomes or the trans-Golgi network (Young, 2007; Williams and Urbé, 2007), or the clathrin plaques that facilitate the entry of bacterial pathogens into non-phagocytic mammalian cells through organizing actin polymerization (Bonazzi et al., 2011). Our observations further suggest that, unlike in endocytosis, where actin primarily facilitates the invagination of clathrin-coated membrane and the generation of relatively small endocytic vesicles, during the engulfment of apoptotic cells, which are much larger (at least 3 μm), the clathrin-actin crosstalk not only induces membrane curvature, but, more importantly, directs actin polymerization and drives pseudopod extension around apoptotic cells. "
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    ABSTRACT: The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.
    Development 08/2013; 140(15):3230-43. DOI:10.1242/dev.093732 · 6.46 Impact Factor
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    • "Thus, having tandem repeats in proteins such as clathrins might be a way of providing the flexibility to accommodate varied volumes in the cage yet conserving the protein-protein interactions that provide strength to the lattice. In a few instances the clathrin heavy chain get phosphorylated at Y1477 and Y1487, which is implicated in actin remodeling and movement of the clathrin vesicle in cell [48]. Although the residues are not directly involved in any interactions reported in the present analysis, they are very close to the interface. "
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    ABSTRACT: In eukaryotic organisms clathrin-coated vesicles are instrumental in the processes of endocytosis as well as intracellular protein trafficking. Hence, it is important to understand how these vesicles have evolved across eukaryotes, to carry cargo molecules of varied shapes and sizes. The intricate nature and functional diversity of the vesicles are maintained by numerous interacting protein partners of the vesicle system. However, to delineate functionally important residues participating in protein-protein interactions of the assembly is a daunting task as there are no high-resolution structures of the intact assembly available. The two cryoEM structures closely representing intact assembly were determined at very low resolution and provide positions of Cα atoms alone. In the present study, using the method developed by us earlier, we predict the protein-protein interface residues in clathrin assembly, taking guidance from the available low-resolution structures. The conservation status of these interfaces when investigated across eukaryotes, revealed a radial distribution of evolutionary constraints, i.e., if the members of the clathrin vesicular assembly can be imagined to be arranged in spherical manner, the cargo being at the center and clathrins being at the periphery, the detailed phylogenetic analysis of these members of the assembly indicated high-residue variation in the members of the assembly closer to the cargo while high conservation was noted in clathrins and in other proteins at the periphery of the vesicle. This points to the strategy adopted by the nature to package diverse proteins but transport them through a highly conserved mechanism.
    PLoS ONE 02/2012; 7(2):e31445. DOI:10.1371/journal.pone.0031445 · 3.23 Impact Factor
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