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Colocalization of synapsin and actin during synaptic vesicle recycling.

Bloom O, Evergren E, Tomilin N, Kjaerulff O, Löw P, Brodin L, Pieribone VA, Greengard P, Shupliakov O - J. Cell Biol. (2003)

Bottom Line: In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones.Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster.Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10021, USA. ona@chronos.med.yale.edu

ABSTRACT
It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

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Disruption of the actin-rich cytomatrix by microinjection of synapsin antibodies. (A) A reticulospinal synapse in an uninjected axon subjected to high frequency stimulation at 18 Hz for 6 min. Boxed area is shown in C at higher magnification. (B) A synapse from an axon from the same preparation injected with synapsin antibodies. Boxed area is shown in D at higher magnification. Thick arrow indcates active zone, and thin arrow indicates filamentous matrix. svc, synaptic vesicle cluster; v, vesicles in the filamentous matrix; d, dendrite; a, axoplasmic matrix. Bars, 100 nm.
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fig7: Disruption of the actin-rich cytomatrix by microinjection of synapsin antibodies. (A) A reticulospinal synapse in an uninjected axon subjected to high frequency stimulation at 18 Hz for 6 min. Boxed area is shown in C at higher magnification. (B) A synapse from an axon from the same preparation injected with synapsin antibodies. Boxed area is shown in D at higher magnification. Thick arrow indcates active zone, and thin arrow indicates filamentous matrix. svc, synaptic vesicle cluster; v, vesicles in the filamentous matrix; d, dendrite; a, axoplasmic matrix. Bars, 100 nm.

Mentions: The activity-dependent migration of synapsin to the endocytic zone and its colocalization with actin suggest that it may be involved in the organization of actin in this region. To test this possibility, antisynapsin antibodies were microinjected into giant reticulospinal axons to perturb the function of synapsin. G-304 antibodies, which disrupt the distal pool of synaptic vesicles in lamprey synapses (Pieribone et al., 1995), were used in these experiments. After antibody microinjection, control and injected axons were subjected to high-frequency action potential stimulation (18 Hz) for 6 min to induce synaptic vesicle recycling and then immediately fixed. The ultrastructure of synapses from injected and control axons was studied in serial ultrathin sections. In agreement with our previous studies, a significant decrease in the number of vesicles in injected synapses was observed, whereas large synaptic vesicle clusters were still present in control synapses (Fig. 7, A and B). In both groups of synapses, a similar number of clathrin-coated pits was observed within the endocytic zone (Fig. 7, A–D). In control synapses, a distinct filamentous cytomatrix was consistently observed in the vicinity of the clathrin-coated intermediates (Fig. 7 C). In antibody-injected synapses, however, this cytomatrix was significantly reduced and in many sections completely absent (Fig. 7 D).


Colocalization of synapsin and actin during synaptic vesicle recycling.

Bloom O, Evergren E, Tomilin N, Kjaerulff O, Löw P, Brodin L, Pieribone VA, Greengard P, Shupliakov O - J. Cell Biol. (2003)

Disruption of the actin-rich cytomatrix by microinjection of synapsin antibodies. (A) A reticulospinal synapse in an uninjected axon subjected to high frequency stimulation at 18 Hz for 6 min. Boxed area is shown in C at higher magnification. (B) A synapse from an axon from the same preparation injected with synapsin antibodies. Boxed area is shown in D at higher magnification. Thick arrow indcates active zone, and thin arrow indicates filamentous matrix. svc, synaptic vesicle cluster; v, vesicles in the filamentous matrix; d, dendrite; a, axoplasmic matrix. Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199372&req=5

fig7: Disruption of the actin-rich cytomatrix by microinjection of synapsin antibodies. (A) A reticulospinal synapse in an uninjected axon subjected to high frequency stimulation at 18 Hz for 6 min. Boxed area is shown in C at higher magnification. (B) A synapse from an axon from the same preparation injected with synapsin antibodies. Boxed area is shown in D at higher magnification. Thick arrow indcates active zone, and thin arrow indicates filamentous matrix. svc, synaptic vesicle cluster; v, vesicles in the filamentous matrix; d, dendrite; a, axoplasmic matrix. Bars, 100 nm.
Mentions: The activity-dependent migration of synapsin to the endocytic zone and its colocalization with actin suggest that it may be involved in the organization of actin in this region. To test this possibility, antisynapsin antibodies were microinjected into giant reticulospinal axons to perturb the function of synapsin. G-304 antibodies, which disrupt the distal pool of synaptic vesicles in lamprey synapses (Pieribone et al., 1995), were used in these experiments. After antibody microinjection, control and injected axons were subjected to high-frequency action potential stimulation (18 Hz) for 6 min to induce synaptic vesicle recycling and then immediately fixed. The ultrastructure of synapses from injected and control axons was studied in serial ultrathin sections. In agreement with our previous studies, a significant decrease in the number of vesicles in injected synapses was observed, whereas large synaptic vesicle clusters were still present in control synapses (Fig. 7, A and B). In both groups of synapses, a similar number of clathrin-coated pits was observed within the endocytic zone (Fig. 7, A–D). In control synapses, a distinct filamentous cytomatrix was consistently observed in the vicinity of the clathrin-coated intermediates (Fig. 7 C). In antibody-injected synapses, however, this cytomatrix was significantly reduced and in many sections completely absent (Fig. 7 D).

Bottom Line: In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones.Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster.Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10021, USA. ona@chronos.med.yale.edu

ABSTRACT
It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

Show MeSH
Related in: MedlinePlus