Traffic of Dynamin within Individual<b><i>Drosophila</i></b>Synaptic Boutons Relative to Compartment-Specific Markers

Estes, Patricia S.; Roos, Jack; Bliek, Alexander van der; Kelly, Regis B.; Krishnan, K. S.; Ramaswami, Mani

doi:10.1523/jneurosci.16-17-05443.1996

Cited by 166 publications

(147 citation statements)

References 65 publications

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“…Perhaps this is the explanation for so-called perforated PSDs observed by electron microscopy (64). Evidence exists for other endocytic hot spots where clathrin (66,67) and dynamin (68,69) have been found to be concentrated, in the latter case in association with DAP160, a member of the intersectin family (70). Because some members of the Shank/ProSAP family, such as Shank2/ProSAP1, are not completely restricted to the PSD, we suggest, finally, that the interactions identified in the current study may serve a more general dynamin-sequestering role.…”

Section: Dynamin-2 Associates With Shank/prosapsmentioning

confidence: 50%

Dynamin Isoform-specific Interaction with the Shank/ProSAP Scaffolding Proteins of the Postsynaptic Density and Actin Cytoskeleton

Okamoto

Gamby

Wells

et al. 2001

Journal of Biological Chemistry

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Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.Dynamin is a 100-kDa GTPase (1, 2) that controls a variety of vesicular budding events including synaptic vesicle recycling, receptor-mediated endocytosis, caveolae internalization, phagocytosis, and secretory vesicle budding from the transGolgi network (3-9). It forms long spiral polymers around the necks of coated pits (10) and on lipid tubules (11), suggesting that the protein may directly function in membrane scission. Alternatively, dynamin has also been postulated to act as a GTPase switch by recruiting other endocytic factors to the neck and then activating them to sever the coated vesicle (12).Dynamin contains an amino-terminal GTPase domain, followed by a central coiled-coil assembly domain (13), a pleckstrin homology domain, which binds to phosphoinositides and the ␤␥ subunits of heterotrimeric GTPases (14,15), and a carboxyl-terminal coiled-coil region (also called the assembly or GTPase effector domain) that is involved in self-association (13,16,17). At the extreme carboxyl terminus is a basic, prolinerich domain to which a number of Src homology 3 (SH3) 1 domain-containing proteins, acidic phospholipids, and microtubules have been shown to bind (18 -20).Considerable insight into dynamin function at the synapse has come from genetic and morphological studies on the temperature-sensitive mutants of shibire, the dynamin ortholog in Drosophila (21,22). Single point mutations in the GTPase domain of shibire cause paralysis at elevated temperatures, and ultrastructural analysis of nerve terminals under these conditions has revealed a depletion of synaptic vesicles, along with an accumulation of collared pits (23,24).In mammals, three closely related dynamin genes are expressed in a tissue-specific manner. Dynamin-1 is almost exclusively expressed in neurons (25). Dynamin-2 is found in the brain but is also widely expressed among other tissues (26 -28). Dynamin-3 was initially identified in testis (29) but is also found in brain, lung, and heart (30). Differences in the subcellular distribution of the dynamin gene products and their alternative splice forms have been reported (30). Because of its restriction to neurons, dynamin-1 has been assumed to be the synaptic isoform. The function of dynamin-2 is less well understood, and a role in neurons has not been identified. Overexpression of a dominant inhibitory mutant form of dynamin-2 in cultured hippocampal neurons was recen...

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Section: Dynamin-2 Associates With Shank/prosapsmentioning

confidence: 50%

Dynamin Isoform-specific Interaction with the Shank/ProSAP Scaffolding Proteins of the Postsynaptic Density and Actin Cytoskeleton

Okamoto

Gamby

Wells

et al. 2001

Journal of Biological Chemistry

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“…This region has been defined as a distinct synaptic compartment, the endocytic zone, on the basis of light microscopic studies in Drosophila. The dynamin-binding protein Dap160͞intersectin was shown to be accumulated in this region (32), and other endocytic proteins including dynamin and endophilin have been localized to this area in stimulated Drosophila synapses (33,34). Our recent immunogold studies in lamprey synapses have shown that dynamin can be detected in the endocytic zone after stimulation (E. Evergren, N.T., H. Gad, P.L., L.B., and O.S., unpublished observations).…”

Section: Discussionmentioning

confidence: 98%

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

Shupliakov

Bloom

Gustafsson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…demonstrate that syt-eGFP is specifically present in SVs, we took advantage of a dynamin mutant shi ts to drive fusion in the absence of vesicle recycling (Estes et al, 1996). shi ts mutants block SV endocytosis at restrictive temperature (35°C), causing SV proteins to trap in the plasma membrane.…”

Section: Figmentioning

confidence: 99%

“…Scale ϭ 5 m. C: Syt-eGFP and endogenous syt localizes to the peripheral of NMJ synapses when exocytosis was stimulated with high [K ϩ ] of 90 mM and endocytosis is blocked under restrictive temperature (35°C, 5 min) in the dynamin mutant background of shi 1 . For a detailed procedure of high [K ϩ ] stimulation of exocytosis and high temperature blockage of endocytosis of NMJ terminals, see Estes et al (1996). The punctate staining nature (shown by arrow) of ␣-syt (in green) or ␣-CSP (in red) is evident and indicated by arrows.…”

Section: Figmentioning

confidence: 99%

Living synaptic vesicle marker: Synaptotagmin‐GFP

Zhang

Rodesch

Broadie

2002

Genesis

234

192

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Synapses are the site of chemical communication between neurons and between neurons and muscles. The synaptic vesicle (SV) is a prominent presynaptic organelle which contains chemical neurotransmitters and fuses with the plasma membrane to mediate neurotransmission. There are about 50 or so synaptic proteins which are either integral vesicle membrane proteins (e.g., synaptotagmin, syt; and synaptobrevin, syb) or vesicle-associated proteins (e.g., cysteine string protein, CSP; Fernandez-Chacon and Sudhof, 1999). We have transformed Drosophila with a novel syt-eGFP (enhanced GFP) fusion protein, the fluorescence pattern of which colocalizes with native SV proteins at synapses, suggesting that the syt-eGFP fusion protein is correctly localized as an integral SV protein and therefore a good SV marker in living synapses. We demonstrate that the syt-eGFP line can be used to study SV dynamics in vivo by fluorescence recovery after photobleach (FRAP).The syt-eGFP fusion was constructed as shown in Figure 1. The eGFP carries double substitution of Phe 64 to Leu and Ser 65 to Thr and fluoresces 35-fold more intensely than wild-type GFP when excited at 488 nm, based on spectral analysis of equal amounts of soluble protein (Cormack et al., 1996). Four syt-eGFP transgenic lines were generated; one with insertion on the X chromosome, two on the second chromosome, and one on the third chromosome. All of these lines produced clear fluorescence when crossed to a pan-neuronal GAL4 driver (elav-GAL4, see Fig. 2) or a subset neuronal GAL4 driver 4G-GAL4 (data not shown). 4G-GAL4 is identified from an enhancer trap screen for neuronal-specific genes; it starts expression at late embryogenesis panneuronally, but in a subset of motor neurons in the third instar larvae, and enriched in mushroom body in adult brain. The eGFP-positive animals, from embryos to adults, can be readily recognizable under a fluorescence dissecting scope. Stocks with expression of syt-eGFP in all neurons (recombinant chromosome carrying both elav-GAL4 and syt-eGFP on the X chromosome) or subset of neurons (recombinant chromosome carrying both 4G-GAL4 and syt-eGFP on the second chromosome) were established.Multiple lines of evidence indicate that syt-eGFP is present in SVs, with expression similar to the native syt (Fig. 2). First, syt-eGFP is highly enriched in the neuropil region of the ventral nerve cord (VNC) of the embryo (data not shown) and larva ( Fig. 2A, left), as well as in the axonal lobes of the larval mushroom body ( Fig. 2A, right). These neuropil regions are densely packed with neuronal synapses. Second, at neuromuscular junction (NMJ) synapses, where we have higher resolution of single synaptic boutons, the syt-eGFP pattern perfectly matches the staining pattern seen with antibodies against SV-associated proteins (Fig. 2B) FIG. 1.Map of syt-eGFP and syb-eGFP fusion constructs. Syt (accession number M55048) or syb (neuronal synaptobrevin, accession number S66686) coding region was fused to the N-terminal of eGFP (enhanced GFP, catalog number 6...

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Traffic of Dynamin within IndividualDrosophilaSynaptic Boutons Relative to Compartment-Specific Markers

Cited by 166 publications

References 65 publications

Dynamin Isoform-specific Interaction with the Shank/ProSAP Scaffolding Proteins of the Postsynaptic Density and Actin Cytoskeleton

Dynamin Isoform-specific Interaction with the Shank/ProSAP Scaffolding Proteins of the Postsynaptic Density and Actin Cytoskeleton

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

Living synaptic vesicle marker: Synaptotagmin‐GFP

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