Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Abstract

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster. S ynaptic communication depends on local recycling of synaptic vesicles in nerve terminals. A major recycling pathway involves retrieval by means of clathrin-coated pits (1-3). The basic features of this process are shared with that of clathrin-dependent endocytosis in nonneuronal cells (4). It involves recruitment of clathrin by adaptors to the membrane followed by polymerization of the clathrin coat. A clathrin-coated pit grows progressively along with an invagination of the coated membrane. After a deeply invaginated coated pit has formed, the base of the pit is cut off resulting in formation of a free coated vesicle. A range of accessory proteins have been identified, which are thought to regulate different aspects of the endocytic process (2). Many of these proteins interact with components of the actin-regulating machinery, which has pointed to a coupling between endocytosis and the actin cytoskeleton (2, 5, 6). Such a coupling is also supported by the observation of actin tails at moving endocytic vesicles (7-10). Disruption of actin function has been shown to inhibit endocytosis in some but not all cell types (11)(12)(13)(14).In nerve terminals clathrin-mediated budding occurs in an endocytic zone of the plasma membrane, which surrounds the release site (1, 3). Several observations indicate that actin filaments are present in this synaptic compartment. Studies of the denervated frog neuromuscular junction have shown that phalloidin labeling accumulates around rather than within release sites (15). In cell-free synaptosome preparations actin was identified by immunogold labeling in regions containing endocytic intermediates a...

CART: An Hrs/Actinin-4/BERP/Myosin V Protein Complex Required for Efficient Receptor Recycling

“…Endosomes or endocytic transport vesicles likely use cytoskeletal elements for motility (Durrbach et al, 1996;Nakagawa and Miyamoto, 1998;Durrbach et al, 2000;Neuhaus and Soldati, 2000;Lanzetti et al, 2001;Lapierre et al, 2001;Zaslaver et al, 2001;Shupliakov et al, 2002;Engqvist-Goldstein and Drubin, 2003). Actinin-4 is predicted to have an actin-binding domain, and we have observed that it binds actin (our unpublished data).…”

Myosin II Regulates Activity Dependent Compensatory Endocytosis at Central Synapses

“…ponents of endocytosis are regulated by actin (Taylor et al, 2012), which motivated us to test for involvement of MII.…”

“…This possibility is supported by recent work showing that trafficking is MII dependent (Peng et al, 2012). While there are no data to support a role for MII in transition of vesicles between the resting pool and recently recycled pool, it is interesting to note that the key component controlling this transition, CDK5, is known to phosphorylate multiple presynaptic proteins including both dynamin-1 and the MIIB heavy chain (Tan et al, 2003;Jämsä et al, 2009). Defects in retrieval of docked vesicles that release neurotransmitter via a kiss-and-run mechanism may also prevent replenishment of neurotransmitter-loaded fusion-competent vesicles in the RRP if full collapse of vesicles does not occur.…”

“…In addition, dynamin binds to filamentous actin suggesting possible interdependencies between dynamin and actin during CME (Mooren et al, 2009), although the requirement for actin during CME is a point of controversy (Shupliakov et al, 2002;Sankaranarayanan et al, 2003;Bourne et al, 2006;Smythe and Ayscough, 2006;Taylor et al, 2012). Increased membrane tension partially inhibits CE, and actin appears to regulate endocytosis during changes in membrane tension (Boulant et al, 2011) suggesting that the requirement for actin during endocytosis may vary with activity (Heidelberger et al, 2002) and may be transient (Merrifield et al, 2002).…”

Powering membrane traffic in endocytosis and recycling

“…However, this observation needs to be independently confirmed. Actin is known to be crucial for synaptic-vesicle recycling 104 , and synaptic-vesicle movement at presynaptic terminals that are devoid of microtubules is dependent on myosin Va 105 . Myosin Va on synaptic vesicles directly interacts, in a Ca 2+ -dependent manner, with plasma membrane syntaxin-1a through its neck domain, and is thereby proposed to regulate stimulated exocytosis through the supply of readily releasable vesicles 106 .…”