Sweeping Model of Dynamin Activity

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. 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. green fluorescent protein enhanced green fluorescent protein synaptobrevin Exocytosis and endocytosis are linked and regulated coordinately by a cascade of protein-protein interactions (1Bottomlet M.J. Surdo P.L. Driscoill P.C. Curr. Biol. 1999; 9: R301-R304Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) 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 (2Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (516) Google Scholar, 3Steyer J.A. Horstmann H. Almers W. Nature. 1997; 388: 474-478Crossref PubMed Scopus (363) Google Scholar, 4Schmoranzer J. Goulian M. Axelrod D. Simon S.M. J. Cell Biol. 2000; 149: 23-32Crossref PubMed Scopus (160) Google Scholar, 5Toomre D. Steyer J.A. Keller P. Almers W. Simons K. Cell Biol. 2000; 49: 33-40Crossref Scopus (137) Google Scholar). 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 (6Tsuboi T. Zhao C. Terakawa S. Rutter G.A. Curr. Biol. 2000; 10: 1307-1310Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 7Tsuboi T. Kikuta T. Warashina A. Terakawa S. Biochem. Biophys. Res. Commun. 2001; 282: 621-628Crossref PubMed Scopus (23) Google Scholar, 8Tokunaga M. Kitamura K. Saito K. Iwane A.H. Yanagida T. Biochem. Biophys. Res. Commun. 1997; 235: 47-53Crossref PubMed Scopus (279) Google Scholar). 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. 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 I-EGFP and dynamin K44A-EGFP (9Obar R.A. Collins C.A. Hammarback J.A. Shpetner H.S. Vallee R.B. Nature. 1990; 347: 256-261Crossref PubMed Scopus (286) Google Scholar, 10Vasquez R.J. Okamoto P. Wheatley S. Wang Y.L. Maxfield F. Vallee R.B. Mol. Biol. Cell. 1998; 9 (abstr.): 196Google Scholar) were provided by Dr. Patrizia Okamoto and Dr. Richard Vallee (University of Massachusetts). To transfect pheochromocytoma cells (PC12), an expression vector containing (2 μg/μl) LipofectAMINE 2000 (Invitrogen, Tokyo, Japan) solution was mixed with a culture medium, and the cells were cultured in the mixture for 4 h. Then, the cells were washed with the culture medium and stored in a fresh medium. Under this condition, many cells (>70%) were successfully transfected. The cells were used for experiments in 24–48 h after transfection. The culture medium was Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (both from Invitrogen). For the fluorescence imaging, a modified Krebs solution containing (in mm) NaCl, 135; KCl, 5.0; CaCl2, 2.0; MgCl2, 1.0; Na-HEPES, 10; glucose, 10 (buffered at pH 7.3 by titration with NaOH) was used. For most fluorescence observations, we employed a total internal reflection fluorescence microscope (TIRFM or evanescent field microscope) described previously by Tsuboi et al. (7Tsuboi T. Kikuta T. Warashina A. Terakawa S. Biochem. Biophys. Res. Commun. 2001; 282: 621-628Crossref PubMed Scopus (23) Google Scholar). The incident light for evanescent illumination was introduced from the objective lens (NA = 1.45, 60× magnification) installed on an inverted microscope (IX70, Olympus, Tokyo, Japan). To observe the EGFP fluorescence image, we used a 473-nm laser (diode-pumped solid state, 30 milliwatts; Shimadzu, Tokyo, Japan) for the evanescent wave excitation and a long pass filter (515 nm) for barrier. The laser beam was passed through an electromagnetically driven shutter (Uniblitz; Vincent Associates, Rochester, NY). The shutter was opened synchronously with camera exposure under control with a personal computer (Endeavor Pro-400, Epson, Tokyo, Japan) running a MetaMorph software package (Universal Imaging Co., West Chester, PA). Fluorescence images were captured with a monochromatic ICCD camera (DAS-512, Imagista, Tokyo, Japan) combined with an image intensifier (VS4–1845, VideoScope, Sterling, VA). The video images were contrast-enhanced with a digital image processor (ARGUS-20, Hamamatsu Photonics, Hamamatsu, Japan), then recorded on digital videotape (GV-D900, Sony, Tokyo, Japan) continuously, and stored finally on a computer hard disk. To observe fluorescence images of EGFP and DsRed simultaneously under the evanescent wave illumination, we used a single laser line (473 nm) for excitation and the W-view optical system (A-4313, Hamamatsu Photonics) for emission, as described previously (6Tsuboi T. Zhao C. Terakawa S. Rutter G.A. Curr. Biol. 2000; 10: 1307-1310Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). All images were reproduced from the hard disk or from the digital videotape using a personal computer (Power Macintosh G4/450, Apple Computer, Inc., Cupertino, CA). The Syb-DsRed expressing PC-12 cells were fixed with 99.5% ethanol for 10 min at 4 °C. After fixation, the cells were incubated with monoclonal anti-synaptophysin antibody (Roche Molecular Biochemicals, Tokyo, Japan) for about 1 h at room temperature. Then the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Inc., Birmingham, AL) for 12 h at 4 °C. After a final washing, the cells were observed with a confocal microscope equipped with a microlens-attached Nipkow-disc scanner (CSU-10, Yokogawa Electric Co., Tokyo, Japan). To assess the exocytotic response of secretory vesicles, we observed the fluorescence of Syb-EGFP expressed in the vesicles under the evanescent field microscope. Many fluorescent spots of a quite uniform size were distributed all over the bottom area of the cell (Fig.1a). These fluorescent spots showed short linear movements (average velocity: 3.21 ± 0.82 μm/s, n = 10 cells) in various directions in control cells. The average velocity was significantly decreased to 0.12 ± 0.02 μm/s (n = 10 cells, p < 0.05) when colchicine (10 μm, 30 min) was applied to the cells. However, no change was observed when ML-9, a myosin light chain kinase inhibitor (20 μm, 30 min), was applied (average velocity: 2.94 ± 0.64 μm/s, n = 10 cells). When electrical stimulation (1-μA current pulse, 1-ms duration) was applied with a micropipette attached to the cell (11Manivannan S. Terakawa S. J. Neurosci. 1994; 14: 5917-5928Crossref PubMed Google Scholar), many of the Syb-EGFP vesicles (76 ± 4%, n = 10 cells) abruptly disappeared after their transient increase in fluorescence intensity (flash) with individual delays. During the flash response for about 30 ms, the image of the vesicle became diffusively larger (Fig.1b, middle panel). After their abrupt disappearance, the fluorescence could no longer be detected on the plasma membrane (Fig. 1, d and e). The abrupt disappearance of fluorescent vesicles indicated their exocytotic responses. The flash could be ascribed to a lateral diffusion of the Syb-EGFP from a vesicle membrane to the plasma membrane in the exponentially rising evanescent field (4Schmoranzer J. Goulian M. Axelrod D. Simon S.M. J. Cell Biol. 2000; 149: 23-32Crossref PubMed Scopus (160) Google Scholar, 5Toomre D. Steyer J.A. Keller P. Almers W. Simons K. Cell Biol. 2000; 49: 33-40Crossref Scopus (137) Google Scholar, 6Tsuboi T. Zhao C. Terakawa S. Rutter G.A. Curr. Biol. 2000; 10: 1307-1310Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). We did not detect a significant increase in fluorescence of the plasma membrane after stimulation. This could be accounted for by lateral diffusion of the Syb-EGFP-carrying vesicles also, because EGFP would be diluted at least 10-fold by the surrounding plasma membranes after the exocytotic fusion. A lack of increase in background fluorescence of the plasma membrane even after many exocytotic responses suggested that Syb-EGFP on the plasma membrane was efficiently internalized after exocytotic membrane fusion. Alternatively, Syb-EGFP transferred to the plasma membrane might be quenched by some unknown mechanism or by enzymatic degradation for recycling. We observed the dynamic activity of dynamin I-EGFP near the plasma membrane of PC12 cells by using the same evanescent wave microscope. Against a faint background, dynamin I-EGFP in control cells was visualized as punctuate fluorescent spots of variable sizes (∼0.4 μm). Hereafter, we will refer to the large fluorescent spots as clusters. Electrical stimulation induced a marked increase in number of these clusters near the plasma membrane (224 ± 5% on average, n = 10 cells; Fig.2a). Stimulation induced a significant increase in background fluorescence of the plasma membrane as well (Fig. 2, a and e). The number of clusters formed was larger in the high background areas. It is possible that dynamin molecules first translocated from the cytoplasm to the plasma membrane and then they assembled into clusters in an initial stage of the membrane-related activity. Once the clusters appeared, they did not grow later. In contrast to the rapid response of the vesicles expressing Syb-EGFP, the fluorescent clusters of dynamin I-EGFP appeared with a clear delay of 10–60 s after stimulation (Fig.2d). The time course of this endocytotic response estimated with dynamin I-EGFP was consistent with recent observations (12Sankaranarayanan S. Ryan T.A. Nat. Neurosci. 2001; 4: 129-136Crossref PubMed Scopus (248) Google Scholar, 13Sankaranarayanan S. Ryan T.A. Nat. Cell Biol. 2000; 2: 197-204Crossref PubMed Scopus (344) Google Scholar, 14Li Z. Murthy V.N. Neuron. 2001; 31: 593-605Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). All clusters and the background fluorescence soon faded to restore the initial fluorescence level in the entire field of cell observed. In individual clusters, the large increase in fluorescence of dynamin I-EGFP was followed by a sharp decrease in fluorescence in 10–20 s (Fig. 2d) without a detectable change in shape. The decrease in fluorescence reflected a movement of clusters away from the evanescent field, possibly showing dynamin activity of pinching endocytotic vesicles from the plasma membrane. There were clusters linked loosely to others, forming a super cluster in the shape of a ribbon (Fig. 2c) or sometimes a quite large ring (Fig.3c).Figure 3Movement of dynamin clusters during the membrane-associated activity. a, sequence of video frames showing random movements of several clusters appearing after electrical stimulation. The stimulation was applied immediately after 0 s. The brightest cluster was associated with the membrane from the beginning (before the stimulation). This cluster and others, which appeared after electrical stimulation disappeared from the plasma membrane 18 s later. b, traces of the movement of 8 of 150–200 clusters transiently appearing beneath the membrane in a cell. The shape of the cell is indicated by solid curves(outline) and dashed lines (frame).c, sweeping activity of a dynamin ring. A sequence of video frames was taken after electrical stimulation at time 0. The figure in each frame indicates the time in seconds (a andc). The scale bar indicates 1 μm ina and c and 5 μm in b.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Many clusters showed a continuous zig-zag movement with a maximum step length of about 1 μm (per s) without altering their fluorescence intensity, suggesting their lateral movement under the plasma membrane (Fig. 3a). Some drifted laterally for final distances longer than 3 μm before they faded (Fig. 3b). Almost the entire area of the cell under observation was covered by traces of some clusters. Some ribbons of dynamin moved sideways for a few micrometers. The ring structures grew in diameter to scan a large area of the plasma membrane (Fig. 3c), enlarging like a wave made by a stone thrown into a pond. More precisely, rings fragmented into several arcs during enlargement, keeping their shape and intensity for a certain traveling distance. These various modes of gliding suggested that dynamin clusters scan the membrane to meet the invaginating pits destined to endocytosis. We observed such responses reproducibly in 5 of 7 cells tested in a series. We also observed the dynamics of mutant type dynamin I-EGFP (Fig.2b). A marked difference between the wild type and mutant dynamin was noticed in the rates of disappearance from the plasma membrane after their stimulation-induced appearance. The mutant dynamin did not disappear for more than 10 min of the observation period (Fig.2e, bottom panel). When compared before stimulation, mutant clusters were always larger in number than wild type clusters. The ribbons or the rings of clusters did not appear after stimulation in mutant cells, although individual clusters of a small size were observed similarly at a high density moving randomly in lateral directions. All these properties of mutant dynamin are probably pertinent to the impeded endocytosis. In normal cells, the appearance of fluorescent clusters of dynamin I-EGFP after electrical stimulation was blocked significantly (to 124 ± 5%, n = 10 cells) by application of an anti-mitotic agent, colchicine (10 μm, 30 min). However, no blockade was observed when an inhibitor of myosin light chain kinase, ML-9 (20 μm, 30 min), was applied (211 ± 11%, n = 10 cells). A fate of Syb-DsRed-expressing vesicles after the exocytotic event was examined in the cell co-expressing dynamin I-EGFP, using a dual window (W-view) evanescent wave microscope. Syb-DsRed was monitored in the red-filtered window and dynamin I-EGFP in the green-filtered window. In the resting cell, most of vesicular images appeared only in the red window (Fig. 4a, top panels). When a depolarizing pulse was applied to the cell, many of Syb-DsRed-expressing vesicles in this window disappeared in a period of several seconds. Instead, many of the fluorescent spots (clusters) of dynamin I-EGFP appeared in the green window (Fig. 4a,bottom panels). The decrease in fluorescence intensity of Syb-DsRed ended earlier than the onset of the increase in intensity of dynamin I-EGFP fluorescence. We obtained such reproducible results of simultaneous imaging in 7 of 12 cells we tested. Failure was mainly due to a low frequency of responses. We next examined the coincidence of the dynamin I-EGFP response with the Syb-DsRed response by overlaying the two images of pseudocolor representing both responses separately (Fig. 4b). Among all disappearing responses of Syb-DsRed fluorescence, 27 ± 1% (n = 8 cells) had the appearance of dynamin I-EGFP in exactly the same site (Fig. 4b, arrows). The Syb-DsRed vesicles that showed such successive responses were mostly large in diameter (0.43 ± 0.1 μm, n = 8 cells). Many of such large vesicles were also stained with acridine orange (data not shown). These results suggested that large dense core granules are more likely to attract dynamin I at active sites of endocytosis. The dual window evanescent wave microscopy described here will allow one to investigate the modes of different types of secretory vesicles in further detail. Syb-DsRed was present exclusively on secretory vesicles in PC-12 cells as judged by comparison with the of vesicle protein, (Fig. ± 5% (n = 3 the that the protein was We the diffusion of and Syb-DsRed-expressing vesicles to be ± (n = 8 cells) and ± (n = 8 significant difference was observed that Syb-EGFP and Syb-DsRed were similarly in a and that fusion of Syb to DsRed did not and disappearance of some dynamin clusters was by the fluorescence intensity at the exact site an exocytotic response of a Syb-DsRed vesicle When the cell was the fluorescent cluster of dynamin I appeared with an delay after an exocytotic response After a certain the dynamin cluster disappeared in s. the period and disappearing period of dynamin fluorescence were the disappearance was judged not to be a of enhanced of the dynamin fluorescence with a illumination to an and not to a The fluorescence intensity of a cluster was more than a than the background intensity of dynamin fluorescence. These findings suggested that many dynamin molecules were in a single In using an evanescent wave we here dynamics of exocytosis and endocytosis of secretory vesicles in PC12 cells. The fluorescent Syb-DsRed and dynamin I-EGFP, made it possible to the exocytotic from the endocytotic and to observe the sequence of two responses in the same cell. the present provided into the recycling of exocytosis and endocytosis in a cell. The fluorescence of dynamin I-EGFP in cells a of dynamin I molecules from the cytoplasm to the plasma membrane for of the membrane recycling. The number in clusters of dynamin I near the plasma membrane in cells (Fig. a of the endocytotic activity. The of clusters of dynamin be a as it was by colchicine at low a of the exocytotic responses showed a of the dynamin I cluster coupled or directly to the site. The of the exocytotic responses probably are coupled to a membrane retrieval at some distance. observation is that dynamin I clusters the plasma membrane. it is highly likely that the of pits are formed away from sites of exocytotic response marked by and the pits are by dynamin clusters. Some secretory vesicles expressing on the of cells) in a vesicular shape even after a exocytotic response Zhao C. C. Rutter G.A. Biochem. J. 1998; PubMed Scopus Google Scholar), the and of vesicles (6Tsuboi T. Zhao C. Terakawa S. Rutter G.A. Curr. Biol. 2000; 10: 1307-1310Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). a gliding of vesicles with the sweeping activity of dynamin would about a the membrane retrieval The present that membrane recycling is mediated by dual of dynamin, and the under the plasma membrane. We P. Okamoto and Vallee for the dynamin I-EGFP

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