Coordinated Actions of Actin and BAR Proteins Upstream of Dynamin at Endocytic Clathrin-Coated Pits

Abstract

SUMMARY The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin nucleating proteins, actin and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wildtype cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin, and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin’s endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.

Endosomal clathrin drives actin accumulation at the immunological synapse

“…It is well established that clathrin-mediated endocytosis in yeast requires actin rearrangement (Engqvist-Goldstein and Drubin, 2003). However, the situation in mammalian cells is less clear: whereas many authors show data involving actin in clathrin-dependent endocytosis (Chen and Brodsky, 2005;Engqvist-Goldstein et al, 2004;Ferguson et al, 2009;Toshima et al, 2005;Yarar et al, 2005), others show that actin depletion has no effect on the internalization dynamics of clathrin-coated vesicles (Boucrot et al, 2006). Studies with bacterial and fungal pathogens show that clathrin and actin both participate in pathogen internalization (Moreno-Ruiz et al, 2009;Veiga and Cossart, 2005;Veiga et al, 2007), and it has been proposed that mammalian cells support internalization of large membrane plaques in a process involving clathrin and actin that is very similar to 'classical' clathrin-mediated endocytosis (Saffarian et al, 2009).…”

Dynamin 2 homozygous mutation in humans with a lethal congenital syndrome

“…DNM2 deletion in mice causes embryonic lethality (8-12 days post coitum), suggesting that DNM2 is essential for embryonic development. 17 This is probably linked to the broad cellular function of DNM2 and the fact that DNM2 is ubiquitously expressed in contrast to dynamins 1 and 3. 18 In addition, the perinatal syndrome described in our patients, our in situ Dnm2 hybridization observations and knockdown experiments in zebrafish are consistent with Dnm2 having a role during embryonic development.…”

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

“…The assembling clathrin coat formed from mixed pentagonal and hexagonal arrays of clathrin triskelia provides a scaffold 6,8,14,55 that serves three main purposes. First, it stabilizes the deformed membrane patch 58 , thereby regulating synaptic vesicle size; second, clathrin (and/or adaptor scaffolds) concentrates synaptic vesicle cargo; and third, the assembled clathrin lattice organizes the dynamic flux of endocytic proteins needed for clathrincoated pit maturation, dynamin-mediated fission and uncoating (FIG. 2).…”

“…2), a mechanochemical enzyme recruited to endocytic sites by interaction with SH3 domain proteins 58,59 , including intersectin [60][61][62] , amphiphysin 63 , endophilin 64,65 and syndapin 66 , suggesting a tight interplay between BAR-SH3 protein-mediated membrane deformation (FIG. 2) and dynamin-catalyzed fission 58,67 . In agreement with this, dynamin 1 knockout mice exhibit a striking, activity-dependent accumulation of tubular coated endocytic intermediates 48 .…”

“…Tentative evidence also suggests putative Ca 2+ -dependent interactions between dynamin and calcineurin 87,88 , and between dynamin and the synaptic vesicle protein synaptophysin 89 . Based on these data it is possible to speculate that dynamin may directly or indirectly contribute to the clearance of release sites at or near the active zone, perhaps through membrane fission, regulation of fusion pore collapse, actin-based processes 58 or interactions with active zone proteins (FIG. 4c).…”