sweeping model of dynamin activity

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Sweeping Model of Dynamin Activity

VISUALIZATION OF COUPLING BETWEEN EXOCYTOSIS AND ENDOCYTOSIS UNDER AN EVANESCENTWAVE MICROSCOPE WITH GREEN FLUORESCENT PROTEINS*

Received for publication, January 25, 2002, and in revised form, February 20, 2002Published, JBC Papers in Press, February 20, 2002, DOI 10.1074/jbc.C200051200

Takashi Tsuboi, Susumu Terakawa**, Bethe A. Scalettar, Claire Fantus, John Roder,

and Andreas Jeromin

From theLaboratory of Cell Imaging, Photon Medical Research Center, Hamamatsu University School of Medicine,1-20-1 Handayama, Hamamatsu 431-3192, Japan, Department of Physics, Lewis & Clark College, Portland, Oregon97219, and Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, SLRI-860, Toronto, Ontario M5G 1X5, Canada

Vesicle recycling through exocytosis and endocytosis

is mediated by a coordinated cascade of protein-proteininteractions. Previously, exocytosis and endocytosis

were studied separately so that the coupling betweenthem was understood only indirectly. We focused on the

coupling of these processes by observing the secretoryvesicle marker synaptobrevin and the endocytotic vesi-

cle marker dynamin I tagged with green and red fluo-rescent proteins under an evanescent wave microscopein pheochromocytoma cells. In control cells, many syn-

aptobrevin-expressing vesicles were found as fluores-cent spots near the plasma membrane. Upon electrical

stimulation, many of these vesicles showed an exocy-totic response as a transient increase in fluorescence

intensity followed by their disappearance. In contrast,

fluorescent dynamin appeared as clusters increasingslowly in number upon stimulation. The clusters of flu-

orescent dynamin moved around beneath the plasmamembrane for a significant distance. Simultaneous ob-

servations of green fluorescent dynamin and red fluo-rescent synaptobrevin indicated that more than 70% of

the exocytotic responses of synaptobrevin had no imme-

diate dynamin counterpart at the same site. From thesefindings it was concluded that dynamin-mediated recy-

cling is not directly coupled to exocytosis but rathercompleted by a scanning movement of dynamin for the

sites of invaginating membrane destined to endocytosis.

Exocytosis and endocytosis are linked and regulated coordi-

nately 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 indi-

rectly (25). 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, simulta-

neously under an evanescent field microscope (68). The fluo-

rescence 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 aroundcontinuously 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 hypothe-

sis, sweeping model of dynamin, for an efficient retrieval of

superfluous membranes by endocytosis.

EXPERIMENTAL PROCEDURES

Expression Vector and TransfectionA construct of Syb fused withenhanced GFP (EGFP) was produced by subcloning the rat Syb cDNAas a HindIII/BamHI fragment into pEGFP C1 (CLONTECH, Tokyo,Japan). A construct of Syb fused with DsRed was made by shuffling theSyb cDNA from EGFP fusion construct into DsRed C1 (CLONTECH).Constructs of dynamin I-EGFP and dynamin K44A-EGFP (9, 10) wereprovided by Dr. Patrizia Okamoto and Dr. Richard Vallee (University ofMassachusetts). To transfect pheochromocytoma cells (PC12), an ex-pression vector containing (2 g/l) LipofectAMINE 2000 (Invitrogen,

Tokyo, Japan) solution was mixed with a culture medium, and the cellswere cultured in the mixture for 4 h. Then, the cells were washed withthe culture medium and stored in a fresh medium. Under this condition,many cells (70%) were successfully transfected. The cells were usedfor experiments in 2448 h after transfection.

The culture medium was Dulbeccos modified Eagles medium sup-plemented with 10% fetal bovine serum (both from Invitrogen). For thefluorescence imaging, a modified Krebs solution containing (in mM)NaCl, 135; KCl, 5.0; CaCl

2, 2.0; MgCl

2, 1.0; Na-HEPES, 10; glucose, 10

(buffered at pH 7.3 by titration with NaOH) was used.Imaging SystemsFor most fluorescence observations, we employed

a total internal reflection fluorescence microscope (TIRFM or evanes-cent field microscope) described previously by Tsuboi et al. (7). Theincident light for evanescent illumination was introduced from theobjective lens (NA 1.45, 60 magnification) installed on an inverted

microscope (IX70, Olympus, Tokyo, Japan). To observe the EGFP fluo-rescence image, we used a 473-nm laser (diode-pumped solid state, 30milliwatts; Shimadzu, Tokyo, Japan) for the evanescent wave excitationand a long pass filter (515 nm) for barrier. The laser beam was passedthrough an electromagnetically driven shutter (Uniblitz; Vincent Asso-ciates, Rochester, NY). The shutter was opened synchronously withcamera 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 werecaptured with a monochromatic ICCD camera (DAS-512, Imagista,Tokyo, Japan) combined with an image intensifier (VS41845, Video-Scope, Sterling, VA). The video images were contrast-enhanced with adigital image processor (ARGUS-20, Hamamatsu Photonics,Hamamatsu, Japan), then recorded on digital videotape (GV-D900,Sony, Tokyo, Japan) continuously, and stored finally on a computerhard disk.

* This study was supported by Grants-in-aid for Scientific Research10557003 and 11794015 (to S. T.) and 8701 (to T. T.) from the Ministryof Education, Science, Sport, and Culture of Japan, grants from theMedical Research Council of Canada (to C. F., J. R., and A. J.) andNational Institutes of Health Grant GM61539 (to B. A. S.). The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of Research Fellow of the Japan Society for the Promotionof Science (JSPS).

** To whom correspondence should be addressed. Tel. and Fax: 81-53-435-2092; E-mail: [email protected].

1 The abbreviations used are: GFP, green fluorescent protein; EGFP,enhanced green fluorescent protein; Syb, synaptobrevin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 18, Issue of May 3, pp. 1595715961, 2002Printed in U.S.A.

This paper is available on line at http://www.jbc.org 15957


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To observe fluorescence images of EGFP and DsRed simultaneouslyunder 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 (6). Allimages were reproduced from the hard disk or from the digital video-tape using a personal computer (Power Macintosh G4/450, Apple Com-puter, Inc., Cupertino, CA).

Immunofluorescence StainingThe 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 roomtemperature. Then the cells were incubated with fluorescein isothiocya-nate-conjugated goat anti-mouse IgG (Southern Biotechnology Associ-ates, Inc., Birmingham, AL) for 12 h at 4 C. After a final washing, thecells were observed with a confocal microscope equipped with a micro-lens-attached Nipkow-disc scanner (CSU-10, Yokogawa Electric Co.,Tokyo, Japan).

RESULTS AND DISCUSSION

Imaging of Vesicles Expressing Syb-EGFPTo assess the

exocytotic response of secretory vesicles, we observed the fluo-

rescence of Syb-EGFP expressed in the vesicles under the ev-

anescent 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 linearmovements (average velocity: 3.21 0.82 m/s,n 10 cells) in

various directions in control cells. The average velocity was

significantly decreased to 0.12 0.02 m/s (n 10 cells, p

0.05) when colchicine (10 M, 30 min) was applied to the cells.

However, no change was observed when ML-9, a myosin light

chain kinase inhibitor (20 M, 30 min), was applied (average

velocity: 2.94 0.64 m/s, n 10 cells).

When electrical stimulation (1-A current pulse, 1-ms dura-

tion) was applied with a micropipette attached to the cell (11),

many of the Syb-EGFP vesicles (76 4%, n 10 cells) abruptly

disappeared after their transient increase in fluorescence in-

tensity (flash) with individual delays. During the flash re-

sponse for about 30 ms, the image of the vesicle became diffu-

sively 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 (4 6). We did not detect a significant increase in fluores-

cence of the plasma membrane after stimulation. This could beaccounted 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 inter-

nalized 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.

Imaging of Dynamin I-EGFPWe 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 cellswas visualized as punctuate fluorescent spots of variable sizes

(0.4 m). Hereafter, we will refer to the large fluorescent

spots as clusters. Electrical stimulation induced a marked in-

crease 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 activ-

ity. 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

FIG. 1. Effect of electrical stimulation on Syb-EGFP-expressing vesicles in a single PC12 cell. a, image observed by evanescent waveexcitation before (0 s) and after (1 and 5 s) stimulation.b, sequential images of a single vesicle observed after electrical stimulation (figures indicatetime after stimulation in milliseconds). The second image shows a diffuse cloud of the Syb-EGFP and the third image a disappearance of thefluorescent spot. c, evanescent wave (total internal reflection fluorescence) microscopic images of an EGFP fluorescence of a cell observed with acolor CCD camera without additional staining (left panel) and with a 5-min staining with 3 Macridine orange (middle panel). The yellow colorindicates co-localization of Syb-EGFP with acridine orange. A magnified view of the portion in a box in the middle panel is shown (right panel).Thearrowhead indicates a vesicle that was free from acridine orange (middleand right panels). The scale barindicates 5 m (a and c in center

panel) and 1 m (band c in right panel).d, fluorescence intensity measured in the center of three different vesicles. An arrowindicates the timeof electrical stimulation (single current pulse of 1-ms duration and 1 A).e, time course of the fluorescence intensity change of Syb-EGFP measuredin the whole part of a single cell. Stimulation was given at the time indicated by an arrow.

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with a clear delay of 10 60 s after stimulation (Fig. 2d). The

time course of this endocytotic response estimated with dy-

namin I-EGFP was consistent with recent observations (12

14). 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 fluores-

cence 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 dy-

namin activity of pinching endocytotic vesicles from the plasma

membrane. There were clusters linked loosely to others, form-

ing a super cluster in the shape of a ribbon (Fig. 2 c) or some-

times a quite large ring (Fig. 3c).

Many clusters showed a continuous zig-zag movement with a

maximum step length of about 1 m (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 m before they faded (Fig. 3b).

Almost the entire area of the cell under observation was cov-

ered 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 invagi-

nating pits destined to endocytosis. We observed such re-sponses 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 al-

ways 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 direc-

tions. 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 sig-

nificantly (to 124 5%, n 10 cells) by application of an

anti-mitotic agent, colchicine (10 M, 30 min). However, no

blockade was observed when an inhibitor of myosin light chain

kinase, ML-9 (20 M, 30 min), was applied (211 11%, n 10

cells).

Simultaneous Imaging of Syb-DsRed and Dynamin

I-EGFPA fate of Syb-DsRed-expressing vesicles after the exo-

cytotic event was examined in the cell co-expressing dynamin

I-EGFP, using a dual window (W-view) evanescent wave mi-

croscope. 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. In-

stead, 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 flu-

orescence, 27 1% (n 8 cells) had the appearance of dynaminI-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 m,n 8 cells). Many of

such large vesicles were also stained with acridine orange (data

not shown). These results suggested that large dense core gran-

ules 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 dif-

ferent 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 distribution of

another vesicle protein, synaptophysin (Fig. 4c). Co-localization

averaged 81 5% (n 3 cells), minimizing the possibility that

the DsRed-conjugated protein was mistargeted. We calculated

FIG. 2. Effect of electrical stimulation on dynamin I-EGFP in a single PC12 cell. a, image obtained by evanescent wave excitation in asingle PC12 cell expressing wild-type dynamin I-EGFP observed before (0 s) and after stimulation (10 and 60 s). b, single PC12 cell expressingmutant type dynamin I-EGFP observed before (0 s) and after stimulation (10 and 60 s). c, sequential images of wild-type dynamin I-EGFPfluorescence observed after electrical stimulation.Scale barsrepresent 5 m (inaandb) and 1 m (inc).d, time courses of the fluorescence changemeasured in the center of dynamin I-EGFP spots, which were relatively stable in position. The arrowindicates the time of electrical stimulation.

e, time courses of the fluorescence intensity of wild type dynamin I-EGFP (closed circle) and mutant type dynamin I-EGFP (open circle) measuredin the whole part of a cell shown in a and b. The arrow indicates the time of electrical stimulation.

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the diffusion coefficient of Syb-EGFP- and Syb-DsRed-express-

ing vesicles to be 2.5 0.5 1010 cm2/s (n 8 cells) and 2.6

0.2 1010 cm2/s (n 8 cells), respectively. No significant

difference was observed indicating that Syb-EGFP and Syb-

DsRed were targeted similarly in a non-aggregated form and

that fusion of Syb to DsRed did not alter its targeting.

Appearance and disappearance of some dynamin clusters

was assessed by measuring the fluorescence intensity at the

exact site where an exocytotic response of a Syb-DsRed vesicle

occurred (Fig. 5a). When the cell was stimulated, the fluores-

cent cluster of dynamin I appeared with an approximately 30-s

delay after an exocytotic response (Fig. 5c). After a certain

FIG. 3. Movement of dynamin clus-ters during the membrane-associatedactivity. a, sequence of video framesshowing random movements of severalclusters appearing after electrical stimu-lation. The stimulation was applied im-mediately after 0 s. The brightest clusterwas associated with the membrane fromthe beginning (before the stimulation).

This cluster and others, which appearedafter electrical stimulation disappearedfrom the plasma membrane 18 s later. b,traces of the movement of 8 of 150 200clusters transiently appearing beneaththe membrane in a cell. The shape of thecell is indicated by solid curves (outline)and dashed lines (frame). c, sweeping ac-tivity of a dynamin ring. A sequence of

video frames was taken after electricalstimulation at time 0. The figure in eachframe indicates the time in seconds (a andc). Thescale barindicates 1 m in a andcand 5 m inb.

FIG. 4. Dual imaging of dynamin I-EGFP- and Syb-DsRed-expressingvesicles. a, sequential images of Syb-DsRed (top panels) and dynamin I-EGFP(bottom panels) fluorescence observed si-multaneously in a single cell. b,left panel,the yellow color indicates co-localizationof dynamin I-EGFP (green) and Syb-DsRed (red). Time points of Syb-DsRedand dynamin I-EGFP images were 0 and10 s after electrical stimulation, respec-tively. b, right panel, high magnification

view of the boxed portion in theleft panel.Arrowsindicate co-localization (yellow) ofdynamin I-EGFP (green) and Syb-DsRed(red). Arrowheads indicate vesicles freefrom Syb-DsRed. c, distribution of synap-tophysin marked by green fluorescence(left panel) and of Syb-DsRed (center

panel) in a cell, showing their co-localiza-tion in secretory vesicles. An overlap ofthe two colors is shown in yellow (right

panel). Thescale bar indicates 5 (aand b,left panel, and c) a n d 2 m (b, right

panel).

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period, the dynamin cluster disappeared in 30 s. Because the

stable period and disappearing period of dynamin fluorescence

were clearly distinguishable, the disappearance was judged notto be a result of photobleaching. An enhanced photobleaching of

the dynamin fluorescence with a stronger illumination led to

an exponential fading and not to a stepwise fading. The fluo-

rescence intensity of a cluster was more than a dozenfold

higher than the background intensity of dynamin fluorescence.

These findings suggested that many dynamin molecules were

involved in a single cluster.

In conclusion, using an evanescent wave microscope, we

demonstrated here dynamics of exocytosis and endocytosis of

secretory vesicles in PC12 cells. The fluorescent probes, Syb-

DsRed and dynamin I-EGFP, made it possible to distinguish

the exocytotic process from the endocytotic process and to ob-

serve the sequence of two responses in the same cell. Thus the

present study provided insight into the recycling process of

exocytosis and endocytosis in a neuronal-like cell.

The increased fluorescence of dynamin I-EGFP in stimulated

cells (Fig. 3) suggests a recruitment of dynamin I molecules

from the cytoplasm to the plasma membrane for promotion of

the membrane recycling. The increased number in clusters of

dynamin I near the plasma membrane in stimulated cells (Fig.

4) suggests a Ca2-dependent enhancement of the endocytotic

activity. The formation of clusters of dynamin may be a micro-

tubule-related response, as it was affected by colchicine at low

concentration.

Only a portion (27%) of the exocytotic responses showed a

corresponding formation of the dynamin I cluster coupled or

targeted directly to the membrane-fusion site. The rest of the

exocytotic responses probably are coupled to a membrane re-

trieval process at some distance. Our important observation isthat dynamin I clusters sweep the plasma membrane. Taken

together, it is highly likely that the major fraction of clathrin-

coated pits are formed away from sites of exocytotic response

marked by Syb-DsRed, and the pits are swept up by dynamin

clusters. Some secretory vesicles expressing phogrin (phospha-tase on the granule of insulinoma cells) remain in a vesicular

shape even after a complete exocytotic response (15), indicating

the kiss and glide of vesicles (6). Such a gliding of empty

vesicles together with the sweeping activity of dynamin would

bring about a frequent collision, thus facilitating the mem-

brane retrieval process. The present study provides evidence

that membrane recycling is mediated by dual activities of dy-

namin,i.e. sweeping-up and pinching-out the pits under the

plasma membrane.

AcknowledgementsWe thank Drs. P. Okamoto and R. B. Vallee forproviding the dynamin I-EGFP construct.

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FIG. 5. Dynamics of dynaminI-EGFP- and Syb-DsRed-expressingsingle vesicles.a, dual evanescent waveimage of a dynamin I-EGFP fluorescence(left) and Syb-DsRed fluorescence (right)observed simultaneously before stimula-

tion. b, sequential images of a single dy-namin I-EGFP and Syb-DsRed vesicle(circle 1 in a) observed after electricalstimulation.c, time courses of the fluores-cence intensity of dynamin I-EGFP(closed circle) and Syb-DsRed (open circle)measured in the center of vesicles in thecircles shown in a. The arrows indicatethe time of electrical stimulation. Thescale bars represent 5 (a) and 1 m (b).

Sweeping Model of Dynamin Activity 15961

sweeping model of dynamin activity

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