Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Abstract

Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

Full text

Page 1

T H E J O U R N A L O F C E L L B I O L O G Y
JCB: ARTICLE
The Journal of Cell Biology, Vol. 169, No. 3, May 9, 2005 481–489
http://www.jcb.org/cgi/doi/10.1083/jcb.200412069
JCB481
Insulin stimulates the halting, tethering, and fusion
of mobile GLUT4 vesicles in rat adipose cells
Vladimir A. Lizunov,
1,3
Hideko Matsumoto,
2
Joshua Zimmerberg,
1
Samuel W. Cushman,
2
and Vadim A. Frolov
1,3
1
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, and
Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892
A.N. Frumkin Institute of Electrochemistry, Russian Academy of Science, Moscow, 119071, Russia
G
2
Experimental Diabetes, Metabolism, and Nutrition
3
lucose transport in adipose cells is regulated by
changing the distribution of glucose transporter
4 (GLUT4) between the cell interior and the
plasma membrane (PM). Insulin shifts this distribution by
augmenting the rate of exocytosis of specialized GLUT4
vesicles. We applied time-lapse total internal reflection
fluorescence microscopy to dissect intermediates of this
GLUT4 translocation in rat adipose cells in primary culture.
Without insulin, GLUT4 vesicles rapidly moved along a
microtubule network covering the entire PM, periodically
stopping, most often just briefly, by loosely tethering to
the PM. Insulin halted this traffic by tightly tethering vesicles
to the PM where they formed clusters and slowly fused to
the PM. This slow release of GLUT4 determined the over-
all increase of the PM GLUT4. Thus, insulin initially recruits
GLUT4 sequestered in mobile vesicles near the PM. It is
likely that the primary mechanism of insulin action in
GLUT4 translocation is to stimulate tethering and fusion of
trafficking vesicles to specific fusion sites in the PM.
Introduction
Insulin regulates glucose transport in muscle and adipose cells
through the intracellular redistribution of the glucose transporter
4 (GLUT4; Cushman and Wardzala, 1980; Suzuki and Kono,
1980). GLUT4 content in the plasma membrane (PM) of adipose
cells is determined by a dynamic equilibrium between its exocy-
tosis and internalization. In basal adipose cells, the content of
GLUT4 in the PM remains low (
?
times faster than it is delivered to the PM (Slot et al., 1991; Satoh
et al., 1993; Holman et al., 1994; Malide et al., 2000; for review
see Bryant et al., 2002). Insulin considerably stimulates the rate
of GLUT4 exocytosis with relatively little effect on the rate of
internalization (Satoh et al., 1993; Karylowski et al., 2004).
Consequently,
?
50% of intracellular GLUT4 is translocated to
the PM upon insulin activation, providing a
the amount of transporter on the cell surface (Malide et al., 2000;
for reviews see Pessin et al., 1999; Bryant et al., 2002).
GLUT4 is carried to the PM by specialized tubulo-
vesicular compartments (referred to here as GLUT4 vesicles)
5%) as GLUT4 internalizes 10
?
10-fold increase in
tightly packed with the transporter (Rea and James, 1997).
GLUT4 vesicles belong to specialized exocytic compartments
actively excluding other recycling proteins (Martin et al.,
1996; Malide et al., 1997; Hashiramoto and James, 2000;
Lampson et al., 2001). In the basal state, the GLUT4 vesicles
sequester up to 95% of the total GLUT4 in adipose cells
(Martin et al., 1996; Malide et al., 1997; Lee et al., 1999). The
vesicles slowly exchange GLUT4 with the rest of the intracellular
pool and with the PM (Holman et al., 1994; Karylowski et al.,
2004; Watson et al., 2004). The dynamics of basal GLUT4
recycling indicate that a fraction of the GLUT4 vesicles is
mobile (Bryant et al., 2002). It is increasingly thought that
insulin regulates the mobility of GLUT4 vesicles, yet the details
and mechanisms of insulin’s action remain controversial.
Live microscopy studies performed on 3T3-L1 adipo-
cytes (3T3 fibroblast cell line that has been differentiated to
adipose-like cells) demonstrate that in the basal state most
of the GLUT4 vesicles remain relatively static (Oatey et al.,
1997; Patki et al., 2001), although long-range movements of
GLUT4 vesicles along microtubules were occasionally observed
(Fletcher et al., 2000; Semiz et al., 2003). The microtubule net-
work supports the GLUT4 translocation induced by insulin
(Patki et al., 2001; Semiz et al., 2003; Karylowski et al., 2004).
The disruption of the microtubular network prevents long-
range movements of GLUT4 vesicles and substantially inhibits
GLUT4 translocation to the PM (Fletcher et al., 2000; Patki et
al., 2001). Hence, it has been suggested that insulin stimulates
V.A. Lizunov and H. Matsumoto contributed equally to this paper.
Correspondence to Joshua Zimmerberg: joshz@helix.nih.gov
H. Matsumoto’s present address is Dept. of Molecular Biology, Saitama Medical
School, Saitama, 350-0495, Japan.
Abbreviations used in this paper: GLUT4, glucose transporter 4; PM, plasma
membrane; ROI, region of interest; TIRF, total internal reflection fluorescence;
TIRFM, TIRF microscopy.
The online version of this article includes supplemental material.

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JCB • VOLUME 169 • NUMBER 3 • 2005 482
movement of GLUT4 vesicles from the cell interior toward the
PM along microtubule tracks (Patki et al., 2001; Semiz et al.,
2003). Translocation of the new vesicles to the PM would
boost GLUT4 exocytosis because the vesicles carry essential
parts of the docking/fusion machinery (Martin et al., 1996;
Timmers et al., 1996; Grusovin and Macaulay, 2003).
However, in nonstimulated primary adipose cells, a frac-
tion of all GLUT4 vesicles is already located near the PM. Even
in 3T3-L1 adipocytes, where GLUT4 is mainly concentrated in
the perinuclear region (Patki et al., 2001), GLUT4-containing
vesicles were also reportedly found near the PM (Oatey et al.,
1997). These peripheral vesicles were suggested to be an initial
source of GLUT4 translocated to the PM upon insulin stimula-
tion (Fletcher et al., 2000; Semiz et al., 2003), although it has
never been directly demonstrated. In primary adipose cells, the
disperse cytoplasmic GLUT4 vesicles already make up a major-
ity of the GLUT4-containing compartments (Lee et al., 1999;
Malide et al., 2000). In freshly isolated primary adipose cells, the
GLUT4 vesicles are uniformly spread over the whole cytoplasm
with many of the vesicles located near the PM (Slot et al., 1991;
Malide et al., 1997, 2000). Such spatial distribution of GLUT4 in
basal primary adipose cells immediately suggests that GLUT4
vesicles proximal to the PM play an essential role in the insulin
response in these cells. However, the dynamics of these GLUT4
vesicles in primary adipose cells remain unknown.
Recently, insulin has been shown to stimulate translo-
cation of effector molecules providing specific targeting of
GLUT4 vesicles to the PM (Grusovin and Macaulay, 2003; In-
oue et al., 2003; Saltiel and Pessin, 2003). Thus, insulin may be
involved in regulating tethering of GLUT4 vesicles located near
the PM. To test this hypothesis, we designed an approach to vi-
sualize the dynamic behavior of GLUT4 vesicles located near
the PM in primary adipose cells using total internal reflection
fluorescence (TIRF) microscopy (TIRFM). Here, we report the
resolution of the movements, docking, and fusion of single
GLUT4 vesicles, in both the basal and insulin-stimulated states.
We find that without insulin GLUT4 vesicles rapidly traffic near
the PM along stationary trajectories formed by a microtubule
network covering the entire PM, although GLUT4 vesicles tran-
siently stop and loosely tether to the PM. Insulin stops this traf-
fic, tightly tethering vesicles at the PM where they form clus-
ters. After fusion, the slow diffusion of GLUT4 from clustered
vesicles determines the overall increase of PM GLUT4. We
conclude that one of the primary modes of insulin action on adi-
pose cells is to halt a hitherto unrecognized form of GLUT4
trafficking that scans the cytoplasmic surface of a cell mem-
brane, and then stimulates tethering and fusion of vesicles bear-
ing glucose transporters into the cell membrane.
Results
In isolated adipose cells, GLUT4 vesicles
are scattered near the PM
When isolated from tissue, white adipose cells are round and
their interior is filled with a large droplet of stored triglyceride
(Fig. 1, A and B), leaving a thin (1–3-
tween the lipid droplet and the PM (Cushman, 1970; Malide et
al., 2000). In our experiments, these cells, which ordinarily float
in their medium, were slightly pressed against the coverslip such
that a small flattened part of the PM and nearby cytoplasm (here-
after termed the “TIRF zone”) was illuminated by the evanescent
wave (Fig. 1 B and Fig. S1 A, available at http://www.jcb.org/
?
m) layer of cytoplasm be-
Figure 1.
adipose cells. (A) Differential interference contrast image of
an isolated cell slightly squashed between two coverslips
with nuclear region facing to the left side. (B) Flat round re-
gion of PM and adjacent thin layer of cytoplasm accessible
for TIRF imaging (TIRF zone). (inset) Under TIRF illumination
the fluorescence excitation intensity decreases exponentially
with distance from the coverslip. (C) Three-dimensional recon-
struction of confocal images of a basal adipose cell trans-
fected with GLUT4-GFP. Use red and green glasses to view
this image. (D) Randomly selected part of basal adipose cell
visualized with TIRF. Substantial amounts of GLUT4 vesicles
are located near the PM (within 400 nm; see Materials and
methods) in randomly scattered fashion. Note the variation
of fluorescence intensity of vesicles due to different positions
relative to the coverslip. Bars, 10 ?m.
Confocal microscopy and TIRFM of isolated white

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INSULIN HALTS AND FUSES VESICLES • LIZUNOV ET AL. 483
cgi/content/full.200412069/DC1). The three-dimensional distri-
bution and dynamics of GLUT4 vesicles in the cytoplasm of
basal cells was characterized by confocal and TIRFM.
The HA-GLUT4-GFP construct expressed in these cells
(Dawson et al., 2001) was seen in distinct bright fluorescent spots
(Fig. 1, C and D). This punctate pattern of GLUT4 distribution is
consistent with the previous observations that up to 95% of intra-
cellular GLUT4 is stored in vesicles (Lee et al., 1999; Malide et al.,
2000). Confocal microscopy showed that for most of the trans-
fected cells cultured overnight the punctate patterns of GLUT4
distribution (Fig. 1 C) resemble those of freshly isolated cells; i.e.,
GLUT4 predominantly resides in vesicles scattered over the
whole cytoplasm (Malide et al., 1997). However, with increasing
time in culture (
?
24 h), the bulk of the fluorescence starts to relo-
cate to the perinuclear region (unpublished data). We used cells
that were incubated overnight, but within 24 h after isolation; we
selected those cells with substantial amounts of fluorescent vesi-
cles scattered throughout the cytoplasm.
Analysis of TIRFM images showed a similar punctate pat-
tern of GLUT4 distribution (Fig. 1 D), demonstrating that the
spots mostly corresponded to single GLUT4 vesicles (see Mate-
rial and methods). In the basal state, we detected 15.7
icles per a 100-
?
m area of the PM (65 cells, SD) in the TIRF
zone. The proximity of many GLUT4 vesicles to the PM is con-
sistent with earlier papers on GLUT4 distribution in adipose
cells in primary culture (Lee et al., 1999; Malide et al., 2000).
?
3.2 ves-
2
GLUT4 vesicles near the PM are highly
mobile
Time-lapse videos made using TIRFM demonstrated a high
basal trafficking of GLUT4 vesicles in the vicinity of the
PM (Video 1, available at http://www.jcb.org/cgi/content/
full.200412069/DC1). To quantify this traffic, we used a projec-
tion algorithm producing distinct traces (Fig. 2 A) for all moving
vesicles in the TIRF zone. In basal cells, we detected on average
10.0
?
1.4 traces/100
?
m/min (22 cells, SD) representing tra-
jectories of moving vesicles. The vesicles often underwent long-
range (
?
10
?
m) lateral movements, presumably approaching
the PM along the way (as deduced from periodic increases in
vesicle fluorescence). Vesicles tended to stop for a period of time
(varying from a fraction of a second to 100 s) at dedicated places,
and sometimes two or more vesicles stopped at the same loca-
tion. Ultimately, vesicles exited the TIRF zone laterally or in a
direction perpendicular to the coverslip (for a detailed descrip-
tion, see the section Kinetic analysis of GLUT4 recycling in pri-
mary adipose cells in the online supplemental material).
Moving vesicles took predefined trajectories. Often, vesi-
cles followed exactly the same trajectories one after another (Fig.
2 B and Video 2, available at http://www.jcb.org/cgi/content/
full.200412069/DC1l). In adipose cells transfected with fluores-
cent tubulin, we observed an extensive microtubular network
(Fig. S1 B; Malide and Cushman, 1997). Long microtubular
pathways (
?
30
?
m) that cover the entire cytoplasm (Fig. S1 B)
2
Figure 2.
cells in the basal and insulin-stimulated states.
Time-lapse TIRF images were projected onto a
single plane to visualize vesicle movements.
(A) Projection image of a cell in the basal state
made from a 1-min-long recording (Video 1,
available at http://www.jcb.org/cgi/content/
full/jcb.200412069/DC1). (B) Sequential
frames show three vesicles (arrows) taking the
same pathway (white line). See Video 2. (C)
Projection image of a cell taken 10 min after
insulin application. Note that both the number
of traces and the average running distance for
GLUT4 vesicles are significantly reduced
compared with the basal state. Fluorescence
intensity is shown in pseudocolor. (D) Quantifi-
cation of traffic for cells in the basal state and
cells 5 and 10 min after insulin application.
Data represent mean number of traces de-
tected per 100 ?m2/min from at least 20 cells
in each state.
Traffic of GLUT4 vesicles in adipose

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JCB • VOLUME 169 • NUMBER 3 • 2005484
could very well account for the observed movements of GLUT4
vesicles. Indeed, the GLUT4 vesicles closely follow the paths
marked by fluorescent tubulin (Video 3, available at http://
www.jcb.org/cgi/content/full.200412069/DC1; Semiz et al.,
2003). The distribution of vesicle velocity exhibits two distinct
peaks at 0.6 and
?
2
?
m/s (Fig. S1 C), both falling into the range
of velocities characteristic for fast kinesin or dynein motors
(Higuchi and Endow, 2002; Ma and Chisholm, 2002).
Insulin stimulates tethering of mobile
GLUT4 vesicles to the PM
In stimulated cells we observed a drastic reduction in the traf-
fic (Video 4, available at http://www.jcb.org/cgi/content/
full.200412069/DC1). Only 1.4
cells, SD) were detected 10 min after the insulin treatment, ap-
proximately eightfold less compared with the basal state (Fig. 2,
C and D). Our analysis indicates that the reduction of the traffic
is due to immobilization of GLUT4 vesicles at the PM. First,
upon insulin stimulation, GLUT4 vesicles attach to the PM much
tighter. Fig. 3 A shows two GLUT4 vesicles that attached to the
PM shortly after appearance in the TIRF zone. After attachment,
the vesicles do not “wiggle” near the point of attachment (Li et
al., 2004) as much in the presence of insulin as they do in the
basal state; the amplitude of wiggling has two peaks (42
and 88
?
26 nm,
n
?
15, SD) in the basal state, whereas in the
insulin-stimulated state, only the first peak (49
SD, statistically indistinguishable from the first “basal” peak) is
present (Fig. 3, B and C). Thus, insulin stimulation almost com-
pletely eliminates the long-range “wiggling” of the attached ves-
icles. Moreover, these vesicles rarely move again and so the mo-
bile vesicles all remain in the TIRF zone. Second, insulin
diminished the distance traveled by the vesicles in the TIRF zone
(from 15
?
6 to 5
?
2
?
m,
n
?
?
1.4 traces/100 m
2
/min (18
?
11
?
20 nm,
n
?
15,
70, SD; Fig. 3 D). Thus, insulin
stimulates tighter and quicker attachment of mobile GLUT4 ves-
icles to the PM of adipose cells (see Fig. S4, available at http://
www.jcb.org/cgi/content/full.200412069/DC1, and the section
Kinetic analysis of GLUT4 recycling in primary adipose cells in
the online supplemental material).
Insulin immobilizes GLUT4 vesicles into
clusters on the PM
Although the initial distribution of GLUT4 vesicles around the
cell surface is relatively uniform (Fig. 1, C and D), we observed
that insulin stimulates formation of fluorescent domains alternat-
ing with dark regions (Fig. 4, A and C). To characterize the de-
gree of nonuniformity of the spatial distribution of GLUT4 vesi-
cles near the PM, we applied a pair-distance correlation analysis.
Insulin-stimulated cells showed a distinct peak on the pair-dis-
tance correlation curve (Fig. 4 B), demonstrating that a signifi-
cant percentage of vesicles are clustered. The width of the main
peak gives an estimate of average cluster size (
tance correlations obtained with cells in the basal state exhibited
no significant deviation from a randomly scattered point distri-
bution (all points lie inside the 99% confidence interval), as
tested by simulation. These observations are consistent with pre-
vious studies that insulin targets GLUT4 vesicles to specific
places of the PM of 3T3-L1 adipocytes (Patki et al., 2001; Semiz
et al., 2003). Moreover, insulin signaling in adipose cells has
been proposed to be associated with specific domains of the PM
(Saltiel and Pessin, 2003). If insulin promotes tethering of
GLUT4 vesicles to the PM, then to complete the insulin response
docked vesicles must fuse to release GLUT4 to the PM. We fur-
ther confirmed that insulin stimulates tethering and fusion of
GLUT4 vesicles to the PM within the characteristic time of the
appearance of GLUT4 in the PM reported previously (Dawson et
al., 2001; Tengholm and Meyer, 2002).
?
5
?
m). Pair-dis-
Figure 3.
to the PM of primary adipose cells. (A) Sequen-
tial frames show two GLUT4 vesicles (arrows)
approaching and quickly tethering to the PM.
(B) Examples of trajectories (projected onto the
x-y “coverslip” plane) of GLUT4 vesicles tether-
ing to the PM in the basal (blue) and insulin-
stimulated (red) states; each point corresponds
to the center of the vesicle ROI. (C) Histograms
of SD of vesicle position from the point of
tethering in the x-y plane (amplitude of “wig-
gling” ?
frames were used for the SD calculation for
each vesicles; blue, vesicles in the basal state;
red, vesicles in the insulin-stimulated state (10
min after insulin treatment). (D) Histograms of
the distances a GLUT4 vesicle travels until the
first stop in the basal (blue) and insulin-stimu-
lated (red) states.
Tethering of mobile GLUT4 vesicles
); ?100 consecutive
SDx
SDy
+
()

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INSULIN HALTS AND FUSES VESICLES • LIZUNOV ET AL. 485
GLUT4 vesicles fuse with the PM shortly
after docking in clusters
Formation of clusters was already seen after 2 min of insulin
treatment (Fig. 4 C, top). Shortly after, we saw an increase of
GLUT4 fluorescence between the clusters, where no vesicles
were detected (Fig. 4 C, bottom). The time lag between the in-
crease of fluorescence integrated over the cluster region (Fig. 4
D, red curve) and over the dark region (Fig. 4 D, black curve)
was several minutes. That fluorescence redistributes from clus-
ters to the dark regions of the PM strongly indicates that vesi-
cles in the clusters fuse to the PM. We confirmed the appear-
ance of GLUT4 on the PM by antibody labeling; moreover,
wortmannin, known to interfere with insulin signaling, ablated
the translocation of GLUT4 to the PM (Fig. S2, A and B, avail-
able at http://www.jcb.org/cgi/content/full.200412069/DC1;
Dawson et al., 2001). Next, we analyzed the dynamics of
GLUT4 release from individual vesicles tethered to the PM.
In insulin-stimulated cells, GLUT4 vesicles lost their
fluorescence shortly after attachment; the vesicles approached
the PM and stopped, and then the vesicle fluorescence dimmed.
Fig. 5 A demonstrates the fluorescence spreading from the
same GLUT4 vesicle indicated by the green arrow in Fig. 3 A
Figure 4.
PM after application of insulin in adipose cells.
(A) TIRF image of a cell 5 min after insulin
stimulation. (B) Pair-distance correlation func-
tions of vesicle distributions for cells in the
basal and insulin-stimulated states. With insulin
(red curve) this function exhibits a peak in the
range of 1–5 ?m, and shows that a significant
fraction of vesicles are clustered. The corre-
lation function for cells in the basal state
(black) revealed no statistically significant
deviation from the random distribution. Green
curves represent the 99% confidence interval
for spatially random distribution acquired by
computer simulation. (C) Sequential images
show arrival of new vesicles to a cluster (top)
and subsequent redistribution of GLUT4 to
adjacent regions outside the cluster (bottom).
(D) Time course of fluorescence integrated
over (red) and outside (black) the cluster.
Clustering of GLUT4 vesicles near
Figure 5.
adipose cells. (A) Image sequence (top) showing
fusion of the vesicle from Fig. 3 A (green arrow)
detected by the spreading of GLUT4 (Video 5,
available at http://www.jcb.org/cgi/content/
full/jcb.200412069/DC1). Gaussian fit (bot-
tom) of the vesicle fluorescence radial intensity
profile shows a decrease of peak intensity and
simultaneous radial widening of the profile
(? represents full-width half-maximum). (B)
Time course of peak width (black) and peak
intensity (red) acquired from the Gaussian fit,
and total fluorescence integrated over a circular
region (4-?m-diam) surrounding the vesicle
(green); the total fluorescence (green) remains
constant as the vesicle diameter (40 nm) is
significantly smaller than the characteristic
penetration length of the evanescent wave
(Loerke et al., 2002). All intensities are nor-
malized to their initial values. (C) Histogram of
the time of vesicle fluorescence decay (?) for
insulin-stimulated (red) and basal (blue) cells.
Fusion of single GLUT4 vesicles in
The values of ? were obtained by exponential fit of fluorescence time course. In basal cells, the average time of fluorescence decay (77 ? 13 s) was
statistically indistinguishable from the rate of bleaching (80 ? 10 s). (D) Vesicle fluorescence before (blue, 23 ? 14 AU, SD, n ? 423) and 15 min after
(red, 48 ? 18 AU, SD, n ? 398) insulin application. The increased brightness indicates closer proximity to the PM as the vesicle fluorescence decreases
exponentially with distance from the PM (Fig. 1 B, inset).

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JCB • VOLUME 169 • NUMBER 3 • 2005486
to the PM; the corresponding radially symmetric Gaussian fits
of the fluorescence intensity are placed below each frame
(Video 5, available at http://www.jcb.org/cgi/content/full.
200412069/DC1). The fits demonstrate that while the peak
fluorescence decayed (Fig. 5 B, red curve), the profile of vesi-
cle fluorescence widened (Fig. 5 B, black curve) but the total
amount of fluorescence in the circular region (4
ter) surrounding the vesicle remained constant (Fig. 5 B, green
curve), verifying that the vesicle underwent fusion rather than
moved away from the PM (Loerke et al., 2002).
The coefficient of lateral diffusion of GLUT4 from such
immediate fusion events can be estimated as 2
from the kinetics of the fluorescence profile widening (Fig. 5
B, black curve; Schmoranzer et al., 2000). Using FRAP, we ob-
tained a similar estimate of the diffusion coefficient of GLUT4
in the PM, 1.4
?
10 cm/s (Fig. S3 A, available at http://
www.jcb.org/cgi/content/full.200412069/DC1), thus pointing
out that GLUT4 vesicles can quickly release the transporter to
PM. However, the quick unrestricted release of GLUT4 corre-
sponding to detectable widening of the fluorescence profile, as
in Fig. 5 A, was detected only rarely. We estimated the charac-
teristic times of GLUT4 release from individual fusing vesicles
by an exponential fit of the decay of vesicle fluorescence (as in
Fig. 5 B, red curve). Fig. 5 C demonstrates that most of the ves-
icles release GLUT4 much slower than 1 s, the estimated char-
acteristic time of unrestricted release calculated from the above
diffusion constant (Fig. 5 B). Yet, Fig. 5 C shows that the de-
cay of the vesicle fluorescence was indeed due to insulin
stimulation: insulin-stimulated vesicles lose fluorescence much
faster (in 20
?
12 s) than vesicles in the basal state (in 77
A decay time slower than 70 s was statistically indistin-
guishable from the characteristic time of vesicle bleaching un-
der constant laser illumination (80
ments performed on cells in the basal state using intermittent
illumination, no fluorescence decay was detected for most of
the vesicles, confirming that bleaching was the reason for the
slow decay (
?

?
70 s). Because fluorescence is not lost but re-
distributes in times substantially faster than 70 s, these data fur-
ther confirm that insulin-stimulated tethering of GLUT4 vesi-
cles in clusters is followed by fusion of the vesicles with the
PM. Fig. 5 C also shows that the fluorescence of a small frac-
tion of vesicles in nonstimulated cells (Fig. 5 C, blue bars) de-
cayed quickly, indicating fusion events in the basal state. These
rare exocytic events can account for slow basal recycling of
GLUT4 (Holman et al., 1994; Karylowski et al., 2004).
Insulin changes both the kinetic behavior and the spatial
distribution of GLUT4 vesicles in the TIRF zone. Within 15
min of insulin treatment, the vesicles moved into clusters (Fig.
4 A) and became closer to the PM (Fig. 5 D). Thus, insulin ini-
tially mobilized vesicles in the TIRF zone, relocating them to
fusion sites on the PM. New GLUT4 vesicles that are being
constantly formed to replenish the vesicle population provide
GLUT4 to the PM (Bryant et al., 2002). Fig. 2 D demonstrates
that vesicle movements are infrequently detected 10 min after
insulin stimulation, when the new nontrafficking distribution of
GLUT4 is established (Holman et al., 1994). We suggest that
these short movements of GLUT4 vesicles in the steady-state
?
m in diame-
?
10
?
9
cm
2
/s
?
92
?
13 s).
?
10 s). In control experi-
after insulin treatment represent the same pathways that vesi-
cles take in the basal state. However, the average vesicle dis-
placement is much shorter because the probability of irrevers-
ible attachment to the PM, mostly leading to fusion (Fig. S4
and the section Kinetic analysis of GLUT4 recycling in pri-
mary adipose cells in the online supplemental material), is dra-
matically augmented by insulin.
Our kinetic model of GLUT4 recycling (see the online
supplemental material) indicates that insulin augments both the
probability of tethering at specific fusion sites and the subse-
quent priming of the vesicles leading to their fusion to the PM.
In insulin-stimulated cells, the GLUT4 vesicles are expected to
fuse at the first encountered fusion site as typical cargo vesicles
(Schmoranzer and Simon, 2003). The probability of fusion is
much less in nonstimulated cells; thus, mobile GLUT4 vesicles
bypass many fusion sites, remaining on the microtubule net-
work for a long time. Fig. 6 summarizes our model for the insu-
lin stimulation of GLUT4 exocytosis. Upon formation, the
GLUT4 vesicles move on microtubules until they fuse, like
vesicles delivering constitutive post-Golgi cargo (Schmoranzer
and Simon, 2003). The GLUT4 vesicles fuse to the PM at spe-
cial fusion sites, with the probability of tethering and fusion be-
ing regulated by insulin.
Discussion
It has long been known that in primary adipose cells GLUT4 is
predominantly sequestered in specialized vesicles uniformly
scattered over the cytoplasm (Lee et al., 1999; Malide et al.,
2000). Such a uniform distribution was shown to be character-
istic of freshly isolated cells; with increasing time in cul-
ture, GLUT4 vesicles tend to relocate to the perinuclear re-
gion (Malide et al., 1997). However, it has remained unclear
whether these scattered GLUT4 vesicles, especially those situ-
ated relatively close to the PM (Lee et al., 1999; Malide et al.,
2000), represent a static pool, awaiting insulin stimulation to
trigger fusion of the vesicles to the PM, or a dynamic pool con-
stantly exchanging GLUT4. We demonstrate here that in
freshly isolated adipose cells, GLUT4 vesicles located near the
PM are mobile. The vesicles move along microtubules, thus
scanning the extensive microtubule network that densely un-
Figure 6.
GLUT4 vesicles to fusion sites in the PM. In the basal state, most of the
GLUT4 is in vesicles that move on microtubules along the PM passing
available fusion sites. Insulin via intracellular signaling cascade stimulates
tethering of GLUT4 vesicles to the first encountered fusion site; the fusing
vesicles accumulate in clusters in the PM. The release of GLUT4 from
fusing vesicles is restricted (see text) in ways that may be regulated.
Hypothetical mechanism of insulin-stimulated recruitment of

Page 7

INSULIN HALTS AND FUSES VESICLES • LIZUNOV ET AL.487
derlies the entire PM (Malide and Cushman, 1997). Accord-
ingly, the mean velocities of the mobile vesicles correspond
well to those reported for microtubule-based transport. The
first characteristic velocity of
?
reported for GLUT4 vesicles driven by conventional kinesin on
microtubules in 3T3-L1 cells (Semiz et al., 2003). The second
one,
?
2
?
m/s, which represents rapid, long-range movements
of GLUT4 vesicles, falls into the range of velocities character-
istic for fast kinesin or dynein motors (Higuchi and Endow,
2002; Ma and Chisholm, 2002). The combination of fast and
slow movements, alternating with periods of idling, results in a
seemingly random distribution of GLUT4 vesicles over the mi-
crotubule network (Fig. 1, C and D). Hence, white adipose
cells appear to use microtubules extensively to support traffick-
ing of GLUT4 vesicles, which is consistent with the general
idea of the microtubular network supporting transport of post-
Golgi vesicles to the PM (Schmoranzer and Simon, 2003).
Importantly, our data indicate that the network architec-
ture plays an important role in the regulation of GLUT4 trans-
location to the PM (Fletcher et al., 2000; Semiz et al., 2003). In
the basal state, GLUT4 vesicles often move close to the PM
where the microtubules do (Schmoranzer and Simon, 2003).
Although the vesicles stop at such sites, they are weakly teth-
ered (Fig. 3) and ultimately escape, only rarely fusing with the
PM. However, upon insulin stimulation, the tethering becomes
much tighter (Fig. 3) and the vesicles accumulate in clusters
(Fig. 4) in very close proximity to the PM (Fig. 5 D). The clus-
tering likely takes place at the point of microtubule attachment
to the PM as GLUT4 vesicles appear to move on the microtu-
bules until they tether and fuse (Schmoranzer and Simon,
2003). Furthermore, clustering may also correspond to the in-
sulin signal initiation sites proposed by Saltiel and Pessin
(2003), to which various effector molecules, including molecu-
lar tethers, could be recruited. Transformations of the actin net-
work in the periphery of adipose cells could also play a role in
localization of vesicle fusion at specific sites (Malide and
Cushman, 1997; Saltiel and Pessin, 2003).
In insulin-stimulated adipose cells, GLUT4 vesicles reach-
ing the PM are quickly recruited and the long-range vesicle
movements are thus halted. Contrary to this finding, in 3T3-L1
cells, insulin stimulates long-range movements of GLUT4 vesi-
cles toward the PM, rather than inhibiting trafficking (Patki et
al., 2001; Semiz et al., 2003). The discrepancy could be due to
different basal distributions of GLUT4. If the distribution is
shifted to the perinuclear region, as is characteristic of 3T3-L1
cells (Patki et al., 2001), then more long-range trafficking would
be expected to bring these vesicles to the PM. If a substantial
amount of GLUT4 vesicles is already near the PM (Slot et al.,
1991; Malide et al., 2000), as in adipose cells in primary culture
(Fig. 1 D), then the GLUT4 vesicles need only move slightly to-
ward the PM to augment the rate of GLUT4 exocytosis.
Our data indicate that adipose cells might also have a
means of regulating GLUT4 release at the level of the individ-
ual GLUT4 vesicle: GLUT4 is not freely released from the fus-
ing vesicles. The release may be constrained by the trapping
of GLUT4 by TUG (tether, containing a UBX domain, for
GLUT4; Bogan et al., 2003) or other factors involved in pack-
0.6
?
m/s corresponds to that
ing vesicles with the transporter might interfere with GLUT4
release. Alternatively, GLUT4 may be constrained by the fu-
sion pore, as seen for lipid dyes in small fusion pores induced
by influenza HA (Zimmerberg et al., 1994; Frolov et al., 2000).
We cannot determine the delay in fusion after tethering, but
this gradual release of GLUT4 obviously must come after a fu-
sion pore forms between the vesicle and the PM. If the fusion
pore were sufficiently large, extracellular glucose could access
the GLUT4 constrained in the fusing vesicles. These clustered
transporters would contribute to the cell’s glucose uptake,
which is consistent with the fact that insulin stimulates glucose
uptake in adipose cells maximally in 5–10 min (Vega and
Kono, 1979; Satoh et al., 1993). Importantly, GLUT4 trapped
in a slowly fusing vesicle cannot be retrieved from the PM via
normal endocytosis. Thus, the constrained fusion may explain
the overshoot of the PM GLUT4 seen after insulin application
(Bogan et al., 2001; Zeigerer et al., 2002). The constrained re-
lease of cargo proteins from fused vesicles, also observed for
VAMP, CDC63, and Dopamine-
general mechanism of regulated redistribution of granule mem-
brane proteins to the PM (Allersma et al., 2004).
In summary, we propose that GLUT4 vesicles follow
common pathways of constitutive exocytosis, exploiting mi-
crotubule tracks on their way to the PM and revealing con-
strained release of membrane cargo. However, the probability
of tethering and fusion of these vesicles to the PM is specifi-
cally sensitive to insulin. Insulin is known to stimulate consti-
tutive exocytosis in general (Bryant et al., 2002), though to a
lesser extent than GLUT4 exocytosis. Molecular mechanisms
providing the specificity of insulin action on the GLUT4 vesi-
cles remain to be established.
?
-hydroxylase, could be the
Materials and methods
Cells
White adipose cells were isolated from the epididymal fat pads of 180–
250-g male rats (CD strain; Charles River Laboratories), and then trans-
fected with 8 mg/ml HA-GLUT4-GFP plasmid, and in some experiments
supplemented with 0.25 mg/ml tubulin-GFP plasmid as described previ-
ously (Malide et al., 1997; Dawson et al., 2001). Tubulin-GFP plasmid
was provided by A. Tsvetkov (University of Illinois College of Medicine,
Chicago, IL). Coverslips coated with poly-
face of the medium containing the cells shortly after the transfection. A por-
tion of floating adipose cells spontaneously adhered to the coverslip after
overnight incubation at 37
?
C with 5% CO
(fraction V; Intergen) as described in Malide et al. (1997). Cells were stim-
ulated with 67 nM (10 mU/ml) insulin added to the extracellular medium.
L
-lysine were placed on the sur-
2
in DME containing 3.5% BSA
Confocal microscopy and TIRFM
A coverslip with adhered cells was placed into Delta-TPG dish (Bioptechs)
in Krebs-Ringer bicarbonate Hepes buffer, pH 7.4, containing 200 nM
adenosine and 5% BSA (Al-Hasani et al., 1998), supplemented with 2%
Ficoll to avoid cell damaging. The cells were slightly squashed between
the coverslip and the bottom of the dish (Fig. 1, A and B). All experiments
were performed at 37
?
C maintained by Delta T4 temperature controller
(Bioptechs). Confocal microscopy was performed on a microscope (model
LSM510; Carl Zeiss MicroImaging, Inc.) using a 100
sion objective. Stacks of confocal images were collected from each cell
and three-dimensional reconstructions were performed by Imaris software
(Bitplane). The Prism-less TIRFM setup (Schmoranzer et al., 2000) was
based on a microscope (model IX-70; Olympus) equipped with a 60
1.45 NA objective (Olympus), argon laser (488 nm; Spectra Physics),
and intensified CCD camera (VE1000SIT; MTI). Images were digitized by
acquisition board (Flashbus; Integral Technologies) controlled by Meta-
Morph (Universal Imaging Corp.). Time-lapse images were acquired at
?
1.4 NA oil-immer-
?

Page 8

JCB • VOLUME 169 • NUMBER 3 • 2005488
high rates (10–30 frames/s) to monitor vesicle trafficking and fusion and
at slow rates (
?
0.5 frames/s) to monitor changes of total fluorescence
from an area of the PM. For the slow acquisition, a mechanical shutter
(UniBlitz D122; Vincent Associates) synchronized with the MetaMorph
software was used to block laser illumination between frames. Penetration
depth
d of the evanescent field was measured to be 180
calibration procedure with 40-nm fluorescent beads attached to the piezo-
driven micropipette.
?
20 nm by a
Image analysis
Stacks of time-lapse images were processed using projection algorithms
implemented in ImageJ 1.32 (National Institutes of Health). Mean projec-
tion image of a stack was subtracted from its max projection image to ob-
tain the traces of all moving vesicles. The number of traces (length
within a square region of interest (ROI) of 100
The Gaussian fit of radial pixel intensity profile showed that for the moving
vesicles 90% of fluorescence was confined within a circular vesicle ROI of
3 pixels. Thus, bright spots were counted as vesicles if (a) the pixel inten-
sity profile had a local maximum and (b) the average intensity in the vesi-
cle ROI positioned on the maximum was 20% higher than the background
fluorescence. The thickness of the layer where we could detect single vesi-
cles was ?400 nm. To measure intensity of the vesicle fluorescence, im-
ages were first background subtracted using a rolling-ball algorithm (Im-
ageJ) with a 10-pixel radius; and then the fluorescence intensity was
calculated as a sum of all pixels values in the vesicle ROI.
The degree of vesicle clustering after application of insulin was esti-
mated by pair-distance correlation analysis. Coordinates of the peak in-
tensity of the vesicle ROI were taken as vesicle coordinates and trans-
ferred in ASCII format to Maple (Waterloo Maple Inc.). Custom written
macros counted the number of vesicles within a certain distance r (running
from 0.5 to 30 ?m) from each vesicle and built the cumulative frequency
distribution of all vesicle-vesicle distances:
?
2
?
m)
?m2 was counted visually.
where n is the total number of vesicles, A is the region area, and Ir is a
counter variable that is set to one if the distance between vesicles i and j
dij ? r, otherwise Ir ? 0. For the analysis, circular regions of 30-?m-radius
were selected from TIRF images of the cells. Because the cumulative func-
tions were built for the same boundary conditions and were normalized
over the number of vesicles, it allowed us to average it between different
cells. To compare the vesicle distribution with a random spatial distribu-
tion, the random point patterns were generated by computer simulation.
30 simulations were performed using a region with the same boundary
conditions and with the number of random points equal to the average
amount of vesicles in the ROI defined on cells. The SD and confidence in-
tervals were approximated from simulations of complete random spatial
distributions.
Online supplemental material
Online supplemental material describes in detail the imaging of the micro-
tubular network in adipose cells, immunofluorescence of GLUT4 exposure
on cell surface, and FRAP analysis of GLUT4 mobility in the PM. Videos 1
and 2 show traffic of GLUT4 vesicles in adipose cells in the basal state,
and Video 3 demonstrates the movement of the vesicles on fluorescent mi-
crotubule tracks. Video 4 shows the traffic reduced by insulin stimulation.
Video 5 shows a docking-fusion event and release of GLUT4 to the PM.
Fig. S1 shows the TIRFM image of the PM in the TIRF zone, the microtubu-
lar network, and a histogram of vesicle velocities. Fig. S2 shows colocal-
ization of immunofluorescence to surface GLUT4 in the PM after applica-
tion of insulin and in control experiments with wortmannin. Fig. S3 shows
the analysis of the GLUT4 diffusion, whereas Fig. S4 and Table S1 de-
scribe the kinetic model of GLUT4 recycling in primary adipose cells. On-
line supplemental material is available at http://www.jcb.org/cgi/content/
full/jcb.200412069/DC1.
The authors would like to thank Mary Jane Zarnowski for assistance in isola-
tion and transfection of rat adipose cells.
Submitted: 10 December 2004
Accepted: 17 March 2005
References
Al-Hasani, H., C.S. Hinck, and S.W. Cushman. 1998. Endocytosis of the glu-
K r ( )
n2
–AIr
ji
≠
n
∑
i
n
∑
dij
(),=
cose transporter GLUT4 is mediated by the GTPase dynamin. J. Biol.
Chem. 273:17504–17510.
Allersma, M.W., L. Wang, D. Axelrod, and R.W. Holz. 2004. Visualization of
regulated exocytosis with a granule-membrane probe using total internal
reflection microscopy. Mol. Biol. Cell. 15:4658–4668.
Bogan, J.S., A.E. McKee, and H.E. Lodish. 2001. Insulin-responsive compart-
ments containing GLUT4 in 3T3-L1 and CHO cells: regulation by amino
acid concentrations. Mol. Cell. Biol. 21:4785–4806.
Bogan, J.S., N. Hendon, A.E. McKee, T.S. Tsao, and H.F. Lodish. 2003. Func-
tional cloning of TUG as a regulator of GLUT4 glucose transporter traf-
ficking. Nature. 425:727–733.
Bryant, N.J., R. Govers, and D.E. James. 2002. Regulated transport of the glu-
cose transporter GLUT4. Nat. Rev. Mol. Cell Biol. 3:267–277.
Cushman, S.W. 1970. Structure–function relationships in the adipose cell. I. Ul-
trastructure of the isolated adipose cell. J. Cell Biol. 46:326–341.
Cushman, S.W., and L.J. Wardzala. 1980. Potential mechanism of insulin action
on glucose transport in the isolated rat adipose cell. Apparent transloca-
tion of intracellular transport systems to the plasma membrane. J. Biol.
Chem. 255:4758–4762.
Dawson, K., A. Aviles-Hernandez, S.W. Cushman, and D. Malide. 2001. Insu-
lin-regulated trafficking of dual-labeled glucose transporter 4 in primary
rat adipose cells. Biochem. Biophys. Res. Commun. 287:445–454.
Fletcher, L.M., G.I. Welsh, P.B. Oatey, and J.M. Tavare. 2000. Role for the mi-
crotubule cytoskeleton in GLUT4 vesicle trafficking and in the regula-
tion of insulin-stimulated glucose uptake. Biochem. J. 352:267–276.
Frolov, V.A., M.S. Cho, P. Bronk, T.S. Reese, and J. Zimmerberg. 2000. Multi-
ple local contact sites are induced by GPI-linked influenza hemaggluti-
nin during hemifusion and flickering pore formation. Traffic. 1:622–630.
Grusovin, J., and S.L. Macaulay. 2003. Snares for GLUT4—mechanisms direct-
ing vesicular trafficking of GLUT4. Front. Biosci. 8:d620–d641.
Hashiramoto, M., and D.E. James. 2000. Characterization of insulin-responsive
GLUT4 storage vesicles isolated from 3T3-L1 adipocytes. Mol. Cell.
Biol. 20:416–427.
Higuchi, H., and S.A. Endow. 2002. Directionality and processivity of molecu-
lar motors. Curr. Opin. Cell Biol. 14:50–57.
Holman, G.D., L. Lo Leggio, and S.W. Cushman. 1994. Insulin-stimulated
GLUT4 glucose transporter recycling. A problem in membrane protein
subcellular trafficking through multiple pools. J. Biol. Chem. 269:
17516–17524.
Inoue, M., L. Chang, J. Hwang, S.H. Chiang, and A.R. Saltiel. 2003. The exo-
cyst complex is required for targeting of Glut4 to the plasma membrane
by insulin. Nature. 422:629–633.
Karylowski, O., A. Zeigerer, A. Cohen, and T.E. McGraw. 2004. GLUT4 is re-
tained by an intracellular cycle of vesicle formation and fusion with en-
dosomes. Mol. Biol. Cell. 15:870–882.
Lampson, M.A., J. Schmoranzer, A. Zeigerer, S.M. Simon, and T.E. McGraw.
2001. Insulin-regulated release from the endosomal recycling compart-
ment is regulated by budding of specialized vesicles. Mol. Biol. Cell. 12:
3489–3501.
Lee, W., J. Ryu, R.P. Souto, P.F. Pilch, and C.Y. Jung. 1999. Separation and
partial characterization of three distinct intracellular GLUT4 compart-
ments in rat adipocytes. Subcellular fractionation without homogeniza-
tion. J. Biol. Chem. 274:37755–37762.
Li, C.H., L. Bai, D.D. Li, S. Xia, and T. Xu. 2004. Dynamic tracking and mobil-
ity analysis of single GLUT4 storage vesicle in live 3T3-L1 cells. Cell
Res. 14:480–486.
Loerke, D., W. Stuhmer, and M. Oheim. 2002. Quantifying axial secretory-
granule motion with variable-angle evanescent-field excitation. J. Neu-
rosci. Methods. 119:65–73.
Ma, S., and R.L. Chisholm. 2002. Cytoplasmic dynein-associated structures
move bidirectionally in vivo. J. Cell Sci. 115:1453–1460.
Malide, D., and S.W. Cushman. 1997. Morphological effects of wortmannin on
the endosomal system and GLUT4-containing compartments in rat adi-
pose cells. J. Cell Sci. 110:2795–2806.
Malide, D., N.K. Dwyer, E.J. Blanchette-Mackie, and S.W. Cushman. 1997. Im-
munocytochemical evidence that GLUT4 resides in a specialized trans-
location post-endosomal VAMP2-positive compartment in rat adipose
cells in the absence of insulin. J. Histochem. Cytochem. 45:1083–1096.
Malide, D., G. Ramm, S.W. Cushman, and J.W. Slot. 2000. Immunoelectron
microscopic evidence that GLUT4 translocation explains the stimulation
of glucose transport in isolated rat white adipose cells. J. Cell Sci. 113:
4203–4210.
Martin, S., J. Tellam, C. Livingstone, J.W. Slot, G.W. Gould, and D.E. James.
1996. The glucose transporter (GLUT-4) and vesicle-associated mem-
brane protein-2 (VAMP-2) are segregated from recycling endosomes in
insulin-sensitive cells. J. Cell Biol. 134:625–635.
Oatey, P.B., D.H. Van Weering, S.P. Dobson, G.W. Gould, and J.M. Tavare.

Page 9

INSULIN HALTS AND FUSES VESICLES • LIZUNOV ET AL. 489
1997. GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized
with green-fluorescent protein. Biochem. J. 327:637–642.
Patki, V., J. Buxton, A. Chawla, L. Lifshitz, K. Fogarty, W. Carrington, R. Tuft,
and S. Corvera. 2001. Insulin action on GLUT4 traffic visualized in sin-
gle 3T3-l1 adipocytes by using ultra-fast microscopy. Mol. Biol. Cell.
12:129–141.
Pessin, J.E., D.C. Thurmond, J.S. Elmendorf, K.J. Coker, and S. Okada. 1999.
Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Loca-
tion! Location! Location! J. Biol. Chem. 274:2593–2596.
Rea, S., and D.E. James. 1997. Moving GLUT4: the biogenesis and trafficking
of GLUT4 storage vesicles. Diabetes. 46:1667–1677.
Saltiel, A.R., and J.E. Pessin. 2003. Insulin signaling in microdomains of the
plasma membrane. Traffic. 4:711–716.
Satoh, S., H. Nishimura, A.E. Clark, I.J. Kozka, S.J. Vannucci, I.A. Simpson,
M.J. Quon, S.W. Cushman, and G.D. Holman. 1993. Use of bismannose
photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking
kinetics in rat adipose cells. Evidence that exocytosis is a critical site of
hormone action. J. Biol. Chem. 268:17820–17829.
Schmoranzer, J., and S.M. Simon. 2003. Role of microtubules in fusion of post-
Golgi vesicles to the plasma membrane. Mol. Biol. Cell. 14:1558–1569.
Schmoranzer, J., M. Goulian, D. Axelrod, and S.M. Simon. 2000. Imaging con-
stitutive exocytosis with total internal reflection fluorescence micros-
copy. J. Cell Biol. 149:23–32.
Semiz, S., J.G. Park, S.M. Nicoloro, P. Furcinitti, C. Zhang, A. Chawla, J.
Leszyk, and M.P. Czech. 2003. Conventional kinesin KIF5B mediates
insulin-stimulated GLUT4 movements on microtubules. EMBO J. 22:
2387–2399.
Slot, J.W., H.J. Geuze, S. Gigengack, G.E. Lienhard, and D.E. James. 1991. Im-
muno-localization of the insulin regulatable glucose transporter in brown
adipose tissue of the rat. J. Cell Biol. 113:123–135.
Suzuki, K., and T. Kono. 1980. Evidence that insulin causes translocation of
glucose transport activity to the plasma membrane from an intracellular
storage site. Proc. Natl. Acad. Sci. USA. 77:2542–2545.
Tengholm, A., and T. Meyer. 2002. A PI3-kinase signaling code for insulin-trig-
gered insertion of glucose transporters into the plasma membrane. Curr.
Biol. 12:1871–1876.
Timmers, K.I., A.E. Clark, M. Omatsu-Kanbe, S.W. Whiteheart, M.K. Bennett,
G.D. Holman, and S.W. Cushman. 1996. Identification of SNAP recep-
tors in rat adipose cell membrane fractions and in SNARE complexes co-
immunoprecipitated with epitope-tagged N-ethylmaleimide-sensitive fu-
sion protein. Biochem. J. 320:429–436.
Vega, F.V., and T. Kono. 1979. Sugar transport in fat cells: effects of mechani-
cal agitation, cell-bound insulin, and temperature. Arch. Biochem. Bio-
phys. 192:120–127.
Watson, R.T., A.H. Khan, M. Furukawa, J.C. Hou, L. Li, M. Kanzaki, S. Okada,
K.V. Kandror, and J.E. Pessin. 2004. Entry of newly synthesized GLUT4
into the insulin-responsive storage compartment is GGA dependent.
EMBO J. 23:2059–2070.
Zeigerer, A., M.A. Lampson, O. Karylowski, D.D. Sabatini, M. Adesnik, M.
Ren, and T.E. McGraw. 2002. GLUT4 retention in adipocytes requires
two intracellular insulin-regulated transport steps. Mol. Biol. Cell. 13:
2421–2435.
Zimmerberg, J., R. Blumenthal, D.P. Sarkar, M. Curran, and S.J. Morris. 1994.
Restricted movement of lipid and aqueous dyes through pores formed by
influenza hemagglutinin during cell fusion. J. Cell Biol. 127:1885–1894.