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Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis.

ABSTRACT: Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42 which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negative curvature sensing protein, abolishes CG endocytosis. Furthermore, the identification of ARP2/3 complex at CG endocytic sites, maintained in an inactive state reveals a function for PICK1, an ARP2/3 inhibitor. The spatio-temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin-independent endocytic process. Coincident with the loss of PICK1 by GBF1-activated ARF1, CDC42 recruitment leads to the activation of IRSp53 and the ARP2/3 complex, resulting in a burst of F-actin polymerisation potentially powering scission.

SUBMITTER: Sathe M

PROVIDER: S-EPMC5943408 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis.

Sathe Mugdha M Muthukrishnan Gayatri G Rae James J Disanza Andrea A Thattai Mukund M Scita Giorgio G Parton Robert G RG Mayor Satyajit S

Nature communications 20180509 1

Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42 which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negativ ...[more]

PMID: 29743604

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Project description:Several studies have suggested crosstalk between different clathrin-independent endocytic pathways. However, the molecular mechanisms and functional relevance of these interactions are unclear. Caveolins and cavins are crucial components of caveolae, specialized microdomains that also constitute an endocytic route. Here we show that specific caveolar proteins are independently acting negative regulators of clathrin-independent endocytosis. Cavin-1 and Cavin-3, but not Cavin-2 or Cavin-4, are potent inhibitors of the clathrin-independent carriers/GPI-AP enriched early endosomal compartment (CLIC/GEEC) endocytic pathway, in a process independent of caveola formation. Caveolin-1 (CAV1) and CAV3 also inhibit the CLIC/GEEC pathway upon over-expression. Expression of caveolar protein leads to reduction in formation of early CLIC/GEEC carriers, as detected by quantitative electron microscopy analysis. Furthermore, the CLIC/GEEC pathway is upregulated in cells lacking CAV1/Cavin-1 or with reduced expression of Cavin-1 and Cavin-3. Inhibition by caveolins can be mimicked by the isolated caveolin scaffolding domain and is associated with perturbed diffusion of lipid microdomain components, as revealed by fluorescence recovery after photobleaching (FRAP) studies. In the absence of cavins (and caveolae) CAV1 is itself endocytosed preferentially through the CLIC/GEEC pathway, but the pathway loses polarization and sorting attributes with consequences for membrane dynamics and endocytic polarization in migrating cells and adult muscle tissue. We also found that noncaveolar Cavin-1 can act as a modulator for the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. This work provides new insights into the regulation of noncaveolar clathrin-independent endocytosis by specific caveolar proteins, illustrating multiple levels of crosstalk between these pathways. We show for the first time a role for specific cavins in regulating the CLIC/GEEC pathway, provide a new tool to study this pathway, identify caveola-independent functions of the cavins and propose a novel mechanism for inhibition of the CLIC/GEEC pathway by caveolin.

Dataset Information

Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis.

Publications

Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis.

Similar Datasets

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