Sweeping Model of Dynamin Activity

Abstract

Vesicle recycling through exocytosis and endocytosis is mediated by a coordinated cascade of protein-protein interactions. Previously, exocytosis and endocytosis were studied separately so that the coupling between them was understood only indirectly. We focused on the coupling of these processes by observing the secretory vesicle marker synaptobrevin and the endocytotic vesicle marker dynamin I tagged with green and red fluorescent proteins under an evanescent wave microscope in pheochromocytoma cells. In control cells, many synaptobrevin-expressing vesicles were found as fluorescent spots near the plasma membrane. Upon electrical stimulation, many of these vesicles showed an exocytotic response as a transient increase in fluorescence intensity followed by their disappearance. In contrast, fluorescent dynamin appeared as clusters increasing slowly in number upon stimulation. The clusters of fluorescent dynamin moved around beneath the plasma membrane for a significant distance. Simultaneous observations of green fluorescent dynamin and red fluorescent synaptobrevin indicated that more than 70% of the exocytotic responses of synaptobrevin had no immediate dynamin counterpart at the same site. From these findings it was concluded that dynamin-mediated recycling is not directly coupled to exocytosis but rather completed by a scanning movement of dynamin for the sites of invaginating membrane destined to endocytosis.Exocytosis and endocytosis are linked and regulated coordinately by a cascade of protein-protein interactions (1) to ensure the highly complex spatial and temporal patterns of membrane recycling. Previous studies focused mainly on the last step of exocytosis and inferred the kinetics of endocytosis only indirectly (2-5). In the present study, using the green fluorescent protein (GFP) 1 technique, we have focused on the coupling of exocytosis and endocytosis. We observed the vesicle-associated membrane protein, which is also referred to as synaptobrevin (Syb), and the vesicle-producing protein, dynamin, simultaneously under an evanescent field microscope (6 -8). The fluorescence imaging restricted to the plasma membrane allowed us to capture the exact moment and the site of exocytosis and compare them with the foci of endocytotic activity. In many cases, dynamin clusters appeared in the void space between the sites of exocytotic responses, and then they moved around continuously beneath the membrane, as if they were searching or scanning for the proper site of membrane retrieval. Here, based on these observations, we will propose a novel hypothesis, "sweeping model of dynamin," for an efficient retrieval of superfluous membranes by endocytosis. EXPERIMENTAL PROCEDURESExpression Vector and Transfection-A construct of Syb fused with enhanced GFP (EGFP) was produced by subcloning the rat Syb cDNA as a HindIII/BamHI fragment into pEGFP C1 (CLONTECH, Tokyo, Japan). A construct of Syb fused with DsRed was made by shuffling the Syb cDNA from EGFP fusion construct into DsRed C1 (CLONTECH). Constructs of dynamin ...

Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

“…Finally, it is important to recognize that total internal reflection fluorescence microscopy of single granule fusion events was reported to be unaffected by the expression of a dominantinterfering dynamin mutant (40). In contrast to this, our data demonstrate a marked reduction in the extent of insulin secretion.…”

Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

“…The ratio images were used to calculate [Ca 2ϩ ] off line according to established methods (42). TIRF Microscopy-To assess translocation of PKC␤II⅐EGFP, we employed a TIRF (also known as evanescent wave microscopy) microscope similar to that described previously by Tsuboi et al (32)(33)(34). The incident light for total internal reflection illumination was introduced from the objective lens (Olympus, numerical aperture ϭ 1.65, 100X magnification) through a single mode optical fiber and two illumination lenses.…”

“…Retranslocation to the cytosol was clearly evident in almost half (three of seven) of the cells examined. To provide greater temporal and spatial resolution we next employed TIRF microscopy (31)(32)(33)(34). This technique involves the generation of a thin (Ͻ100 nm) field of fluorescence at the surface of the coverslip and thus at the surface of an attached cell.…”

Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells

“…These numbers are probably underestimated because dynamin staining needed to be corrected for crosstalk from the sulforhodamine channel, thus reducing our sensitivity for dynamin detection. Furthermore, it is possible that dynamin dissociates from the retrieval site after fission and thus can no longer be detected at the time of fixation (30,31).…”