Am J Physiol Cell Physiol 297: C397–C406, 2009.
First published June 10, 2009; doi:10.1152/ajpcell.00562.2008.
Dynasore inhibits rapid endocytosis in bovine chromaffin cells
Chia-Chang Tsai,1 Chih-Lung Lin,2 Tzu-Lun Wang,2 Ai-Chuan Chou,2 Min-Yi Chou,2 Chia-Hsueh Lee,2
I-Wei Peng,2 Jia-Hong Liao,2 Yit-Tsong Chen,1,3 and Chien-Yuan Pan2,4
1
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan; 2Institute of Zoology, 3Department
of Chemistry, and 4Department of Life Science, National Taiwan University, Taipei, Taiwan, Republic of China
Submitted 4 November 2008; accepted in final form 29 May 2009
actin; atomic force microscope; calcium current; clathrin-mediated
endocytosis; dynamin; FM4-64
CLATHRIN-MEDIATED ENDOCYTOSIS (CME) is a major pathway for
cells to take up specific membrane fractions and extracellular
fluids (6, 28). The fission of clathrin-coated pits from the
plasma membrane involves a GTP-binding protein, dynamin,
and results in the formation of small clathrin-coated vesicles
(CCV) (25, 30). Clathrin then dissociates from the coated
vesicle by interaction with auxilin and heat shock protein 90,
and the budded vesicle fuses with the early endosome for
degradation in the lysosome or is recycled for a further round
of exocytosis.
CME is also involved in recycling the secretory vesicle after
exocytosis in neurons. Using pH-sensitive green fluorescent
protein-fused synaptophysin, it has been suggested that CME
Address for reprint requests and other correspondence: C.-Y. Pan, Institute
of Zoology, National Taiwan Univ., Rm. 730, Life Science Bldg., 1 Roosevelt
Rd., Sec. 4, Taipei 106, Taiwan (e-mail: [email protected]); Y.-T. Chen,
Dept. of Chemistry, National Taiwan Univ., and Institute of Atomic and
Molecular Sciences, Academia Sinica, P. O. Box 23-166, Taipei 106, Taiwan
(e-mail: [email protected]).
http://www.ajpcell.org
is the major vesicle retrieval mechanism at the hippocampal
axon terminal after physiological stimuli (15). However, the
other retrieving mechanism, which does not involve clathrin, is “kiss-and-run,” in which the vesicle does not fully
collapse onto the membrane but forms a transient fusion
pore and is immediately retrieved (16). Both recycling
mechanisms have been suggested to be involved in many
types of nerve terminals (35).
Multiple forms of endocytosis recorded by capacitance measurement have been described in bovine chromaffin cells (5,
14, 34). One is excess retrieval (rapid endocytosis), with an
initial rate ⬎100 fF/s and usually reaching a capacitance value
lower than the prestimulus level. The other is slow compensatory endocytosis, with an initial rate of ⬃6 fF/s. Under mild
stimulation, kiss-and-run-mediated fast endocytosis dominates
the retrieval process, while, when stimulation is stronger and
generates a high cytosolic Ca2⫹ concentration ([Ca2⫹]i), full
fusion events increase (13).
In this study, we examined whether dynamin-mediated endocytosis plays an important role in retrieving the membrane
after evoked exocytosis in bovine chromaffin cells. Dynasore
was used to inhibit the GTPase activity of dynamin to block the
budding of the endocytotic vesicle from the membrane (22).
The cell surface area was monitored using changes in capacitance (23). Our results showed that dynamin was responsible
for a rapid exponential decay after strong depolarizationevoked exocytosis. The number of invagination pits on the
plasma membrane surface, determined using atomic force
microscopy (AFM), was increased by dynasore treatment.
Dynamin is therefore responsible for rapid endocytosis after
strong stimulation.
MATERIALS AND METHODS
Chemicals. FM4 – 64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide], Dulbecco’s modified Eagle’s medium, and all other reagents for cell culture were from
Invitrogen (Carlsbad, CA). Antibodies were purchased from Abcam
(Cambridge, MA). Fura-2 acetomethoxylmethyl ester (fura-2 AM) was
purchased from TefLabs (Austin, TX). All other chemicals were reagent
grade from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.
Cell preparation. Chromaffin cells were prepared by digesting
bovine adrenal glands, obtained from local slaughterhouses, with
collagenase (0.5 mg/ml) and purification by density gradient centrifugation, as described previously (26). The cells were plated at a
density of 2 ⫻ 105 on one 22-mm or three 10-mm poly-L-lysinecoated coverslips in a 35-mm culture dish and cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum.
The medium was replaced every 2 days. All experiments were carried
out between days 3 and 10 after cell isolation.
Solutions. The normal bath buffer used contained (in mM) 150
NaCl, 5 KCl, 2.2 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose, pH 7.3.
The perforated-patch pipette solution contained (in mM) 135 Csglutamate, 10 Na-HEPES, 9.5 NaCl, 0.5 TEACl, and 0.5 CaCl2, pH
0363-6143/09 $8.00 Copyright © 2009 the American Physiological Society
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Tsai CC, Lin CL, Wang TL, Chou AC, Chou MY, Lee CH, Peng
IW, Liao JH, Chen YT, Pan CY. Dynasore inhibits rapid endocytosis
in bovine chromaffin cells. Am J Physiol Cell Physiol 297: C397–C406,
2009. First published June 10, 2009; doi:10.1152/ajpcell.00562.2008.—
Vesicle recycling is vital for maintaining membrane homeostasis and
neurotransmitter release. Multiple pathways for retrieving vesicles fused
to the plasma membrane have been reported in neuroendocrine cells.
Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To
characterize its effects in modulating vesicle recycling and repetitive
exocytosis, changes in the whole cell membrane capacitance of bovine
chromaffin cells were recorded in the perforated-patch configuration.
Constitutive endocytosis was blocked by dynasore treatment, as
shown by an increase in membrane capacitance. The membrane
capacitance was increased during strong depolarizations and declined
within 30 s to a value lower than the prestimulus level. The amplitude,
but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve
fitted to the decay phase were all ⬃110% of the membrane capacitance before stimulation, regardless of the dynasore concentration
used. Membrane depolarization caused clathrin aggregation and Factin continuity disruption at the cell boundary, whereas dynasore
treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma
membrane determined using atomic force microscopy was increased
and the pore was wider in dynasore-treated cells. Our data indicate
that dynamin-mediated endocytosis is the main pathway responsible
for rapid compensatory endocytosis.
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DYNASORE INHIBITS RAPID ENDOCYTOSIS
AJP-Cell Physiol • VOL
for 2 min, this stimulation being repeated five times. The cells were
then incubated in normal bath buffer for 10 min and fixed in 3.7%
formaldehyde in normal bath buffer for 30 min. For cells pretreated
with dynasore, all of the buffers and the fixing solution contained 100
␮M dynasore.
The fixed cells were washed with phosphate-buffered saline (PBS)
and transferred to the stage of a Nikon microscope (TE2000-U,
Nikon, Tokyo, Japan). Silicon-nitride cantilevers with a normal force
constant of 0.06 N/m (PNP-DB, NanoWorld, Switzerland) were
mounted in a AFM fluid tip-holder and carefully rinsed with PBS
before the tips were immersed in the PBS filling the recording
chamber. AFM experiments were performed using a commercial
AFM apparatus (Bioscope SZ, Digital Instruments) combined with an
inverted optical microscope (TE2000-U). The whole system was
placed on an antivibration table and housed in a sound-proof cage to
eliminate vibration and acoustic noise. A patch (⬃1 ⫻ 1 ␮m2) of
plasma membrane was scanned by AFM in tapping mode with 256
scan lines and 512 line samples at a scanning rate of 0.5 Hz, the
resonant frequency of which was set at 3.1 kHz with an amplitude
setpoint of 0.45 V, close to the target amplitude of 0.5 V. The integral
and proportional gains of the scanning parameters were 0.6 and 1.2,
respectively. An invagination pit was defined by a depth ⬎20 nm and
a width ⬎30 nm.
Fluorescence imaging. Chromaffin cells were repetitively stimulated, then fixed as described in AFM imaging. All subsequent steps
were at room temperature. The fixed cells were permeabilized by
incubation in 0.5% BSA and 0.1% Triton X-100 in PBS (PBT) for 30
min, then washed three times with PBS. To detect clathrin and F-actin,
the cells were incubated for 1 h with primary antibody [mouse
monoclonal anti-clathrin (X22) antibody diluted 1:500 in PBS]. After
3 ⫻ 5-min washes with PBT, the cells were incubated for 1 h with
fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG antibody
diluted 1:1,500 in PBS. After 3 ⫻ 5-min washes with PBT, the cells
were incubated in PBS containing rhodamine-conjugated phalloidin
(Invitrogen) to visualize F-actin. To detect dynamin, rabbit polyclonal
antibody (ab64243, Abcam) at a 1:100 dilution in PBS was used as the
primary antibody, and rhodamine-conjugated goat anti-rabbit IgG
antibody was used as the secondary antibody. The stained cells were
observed on a Leica TCS SP2 confocal microscope with a ⫻100
objective.
For FM4 – 64 labeling of the endocytosed membrane vesicle, the
cells were incubated in hypertonic buffer containing 50 ␮M FM4 – 64
for 2 min to evoke exocytosis, then in normal bath buffer containing
50 ␮M FM4 – 64 for another 30 min. The cells were then incubated in
normal bath buffer without FM4 – 64 for another 15 min and observed
on a Leica confocal microscope with a ⫻100 objective.
Curve fitting. To characterize endocytosis, the capacitance trace
from the 3rd to 20th second was fitted using a combination of a
one-order exponential decay function and a linear declination function:
y ⫽ y 0 ⫹ A 1 ⫻ exp共⫺t/␶兲 ⫺ A2 ⫻ t/100
where y is the normalized capacitance value, y0 is the offset of the
function, t is the time in s, ␶ is the time constant, A1 is the amplitude,
and A2 is the slope of the linear declination. All results are presented
as means ⫾ SE and were analyzed with Student’s t-test.
RESULTS
Dynasore increases the membrane capacitance. Exocytosis
and endocytosis in the plasma membrane are in dynamic
balance, and blocking of either leads to a change in cell surface
area (23). To determine whether dynasore inhibited constitutive endocytosis and affected the cell surface area, cells were
treated with different dynasore concentrations, and the whole
cell membrane capacitance was recorded in the perforated-
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adjusted to 7.3 with CsOH; 0.5% amphotericin B (Calbiochem, La
Jolla, CA) was added to the internal solution before the start of the
experiment. The amphotericin B solution was prepared by sonication,
as described previously (26). The initial pipette resistance was 2.5–3.5
M⍀. After perforation, the access resistance was 8 –20 M⍀. Typically, ⬃60% of the access resistance was compensated by the compensation circuitry of the amplifier. To isolate Ca2⫹ currents, the cell
was incubated in N-methyl-D-glucamine (NMG) solution (in mM: 130
NMG, 2 KCl, 5 CaCl2, 1 MgCl2, 5.6 glucose, and 10 HEPES, pH 7.3)
and was whole cell patched with a Cs⫹-containing pipette solution (in
mM: 130 Cs-aspartate, 20 KCl, 1 MgCl2, 0.1 EGTA, 3 Na2ATP, 0.1
Na2GTP, and 20 HEPES, pH 7.3). To depolarize the cell, a high K⫹
buffer consisting of (in mM) 75 NaCl, 75 KCl, 2.2 CaCl2, 1 MgCl2,
10 HEPES, and 5 glucose, pH 7.3, was used. The hypertonic buffer
contained (in mM) 150 NaCl, 5 KCl, 2.2 CaCl2, 1 MgCl2, 10 HEPES,
5 glucose, and 192 mM sucrose, pH 7.3, and the osmolarity was
adjusted to 510 mosM.
Electrophysiological measurements. Cells were transferred to a
recording chamber mounted on the stage of an inverted microscope
and bathed in normal bath buffer at room temperature. Patch pipettes
were pulled from thin-wall capillaries containing a filament (catalog
no. 617000, A-M Systems) using a two-stage microelectrode puller
(P-97, Sutter) and were fire polished with a microforge (MF-830,
Narishige, Tokyo, Japan). Electrodes were coated with Sylgard (catalog no. 184, Silicone Elastomer Kit, Dow Corning, Midland, MI) to
reduce nonspecific noise. For perforated whole cell patch studies, the
electrode was first dipped in a pipette solution without amphotericin
B, then backfilled with pipette solution containing amphotericin B.
Recording was not started until the series resistance was lower than 20
M⍀. Ionic currents, membrane capacitance, and action potentials
were measured from patched cells using an EPC10 patch-clamp
amplifier (HEKA, Lambrecht, Germany) controlled by Pulse software
(HEKA).
To evoke exocytosis, cells were voltage clamped at ⫺70 mV and
depolarized with a train of 10 depolarizations to ⫹10 mV for 100 ms,
with a gap of 100 ms between the start of two consecutive depolarizations. The charge recorded between the 10th and 100th ms of
depolarization was integrated as the amount of the inward Ca2⫹ ions.
A 10-ms sinewave with a frequency of 1 kHz and amplitude of 20 mV
was applied before the start of each depolarization to monitor the
membrane capacitance. After the end of this train of depolarizations,
the same sinewave was applied continuously, and the capacitance
measured was averaged every 100 ms. The membrane capacitance
was obtained using a Lock-in amplifier using “sine ⫹ dc” mode in the
Pulse software program.
To measure the Ca2⫹ current, the cell was incubated in NMG bath
buffer containing 10 mM CaCl2 and whole cell voltage clamped at
⫺70 mV. The cell was depolarized to various potentials for 100 ms
once every 15 s. The maximal inward currents during the depolarizations were normalized to the cell surface area represented by the
capacitance compensation to represent the Ca2⫹ currents for the
current-voltage relationship.
[Ca2⫹]i imaging. To measure [Ca2⫹]i, the cells were incubated in
normal bath buffer containing 5 ␮M fura-2 AM for 1 h at 37°C,
washed three times with bath buffer, and used for the measurements.
To depolarize the cells, high K⫹ buffer loaded in a micropipette was
pressure puffed onto a target cell for 3 s, and the change in fura-2
fluorescence was recorded as described previously (7). The [Ca2⫹]i
before stimulation was averaged as the basal [Ca2⫹]i and the maximal
[Ca2⫹]i after stimulation was recorded as the peak response. A
calcium calibration buffer kit (Molecular Probes, Carlsbad, CA) was
used to transform the fura-2 fluorescence ratio into a Ca2⫹ concentration using the protocol given by the manufacturer.
AFM imaging. To prepare the cells for AFM scanning, chromaffin
cells on coverslips were first incubated in normal bath buffer for 30
min in the presence or absence of 100 ␮M of dynasore, then the buffer
was changed to high K⫹ buffer for 1 min, then to normal bath buffer
DYNASORE INHIBITS RAPID ENDOCYTOSIS
Fig. 1. Membrane capacitance after dynasore treatment. Chromaffin cells were
pretreated for 30 min with the indicated concentration of dynasore, then
perforated patched in whole cell mode. The slow capacitance compensation
used to minimize the capacitance transient was used to represent the membrane
capacitance. The sample number for 0, 12.5, 25, 50, 75, and 100 ␮M dynasore
was 6, 4, 6, 6, 6, and 3, respectively. The data are means ⫾ SE. **P ⬍ 0.01
by Student’s t-test when compared with cells without dynasore treatment.
AJP-Cell Physiol • VOL
the prestimulus level. In contrast, in cells treated with 12.5 or
25 ␮M dynasore, exocytosis increased slightly at each stimulation and rapid endocytosis was inhibited. In cells pretreated
with 50 or 75 ␮M dynasore, the capacitance increase evoked
by depolarization was significantly increased and endocytosis
was markedly inhibited. The averaged results showed that the
increase in capacitance evoked by the first stimulation was
significantly increased from 0.28 ⫾ 0.06 to 0.64 ⫾ 0.08 pF by
75 ␮M dynasore. After the third stimulation, exocytosis was
significantly increased in cells treated with dynasore, regardless of the concentration used. These results suggest that
blocking dynamin GTPase activity depletes the machinery for
endocytosis and augments the capacitance increase caused by
repetitive stimulation.
The contribution of the exponential component is decreased
by dynasore. To characterize the contribution of the dynasoresensitive mechanism to the evoked endocytosis, the capacitance trace was first normalized to the prestimulation level,
then the trace between the 3rd and 20th second was fitted by a
combination of a one-order exponential decay and a linear
declination. Since there was no significant difference between
the effects of each of the three repetitive stimulations, the
results were pooled to calculate the averages. The results
(Table 1) showed that the time constant for the exponential
decay were unaffected by dynasore. In addition, the intercept
of the fitted curve at time 0 was ⬃110%, regardless of the
dynasore concentration used. However, the amplitude of the
exponential decay was significantly decreased from 9.9 ⫾
0.9% in control cells to 6.4 ⫾ 1.5 and 2.9 ⫾ 0.8% by 50 and
75 ␮M dynasore, respectively. The slope of the linear declination was significantly increased from 2.1 ⫾ 0.1% per 100 s
in control cells to over 3.5% per 100 s in dynasore-treated cells.
These results indicate that dynasore does not affect the total
depolarization-evoked exocytosis and the exponential decay
time constant, but decreases the amplitude of the exponential
decay in retrieving the membrane.
To determine whether the inhibitory effects of dynasore
were reversible, cells were pretreated with 100 ␮M dynasore
for 30 min, then were moved to normal bath buffer for another
30 min before recording. The capacitance trace for a representative cell (bottom trace, Fig. 3A) shows that the decay phase
after the first train of depolarization was similar to that in
control cells, but the second and third decay phases were
inhibited. The averaged increases in capacitance from the first
to the third stimulation were 0.24 ⫾ 0.03, 0.25 ⫾ 0.03, and
0.21 ⫾ 0.03 pF, respectively, which were comparable to those
in control cells. However, after washout of 100 ␮M dynasore,
the contribution of the exponential phase (A1) at the third
stimulation (5.5 ⫾ 1.4%) was significantly smaller than that at
the first stimulation (7.5 ⫾ 1.1%) (n ⫽ 8, Student’s paired
t-test, P ⬍ 0.05). These results show that the inhibitory effect
of dynasore on endocytosis is reversible.
Dynasore inhibits the Ca2⫹ currents. Ca2⫹ is the pivotal ion
involved in triggering exocytosis and endocytosis (4, 41). The
total Ca2⫹ influx during the first train of depolarizations shown
in Fig. 3 demonstrates a trend to a decline as the concentration
of dynasore was increased. The results showed that the total
charge influx was decreased by 75 ␮M dynasore from 1.59 ⫾
0.95 to 0.63 ⫾ 0.20 pQ/pF, but the difference was not significant due to the large variance in the control group. Since the
above experiment was carried out in Na⫹-containing bath
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patch configuration. The plasma membrane can be looked on
as a capacitor, and its capacitance value can be used to estimate
the cell surface area, so the slow capacitance compensation
values measured at the amplifier were used to represent the cell
surface area. The averaged results (Fig. 1) showed that the
capacitance compensation was 7.3 ⫾ 0.3 pF in control cells
and showed a significant increase to 9.4 ⫾ 0.4 or 11.7 ⫾ 1.5 pF
in cells pretreated with 75 or 100 ␮M dynasore, respectively.
These results show that dynasore increases the cell membrane
capacitance.
Slow endocytosis is inhibited by dynasore. To determine
whether endocytosis after evoked exocytosis was blocked by
dynasore, perforated-patched cells in whole cell mode were
depolarized 10 times to ⫹10 mV for 100 ms at an interval of
100 ms. When exocytosis is evoked by membrane depolarization, the fusion of the secretory vesicles to the plasma membrane increases the surface area, which is reflected in an
increase in the membrane capacitance; conversely, endocytosis
decreases the surface area and membrane capacitance (23).
Figure 2A shows that, in control cells, the membrane capacitance increased immediately during the depolarizations, then
decreased rapidly and dropped to a value below the prestimulus
level. The decrease in capacitance slowed as the concentration
of dynasore increased. The normalized averaged capacitance
traces are shown in Fig. 2B. As the dynasore concentration
increased, the maximal elevation of the capacitance increased
and the exponential decay became less obvious, showing that
dynasore affects the evoked capacitance change in a dosedependent manner.
Dynasore enhances the increase in the capacitance caused
by repetitive stimulations. To determine whether blocking
endocytosis interfered with repetitive exocytosis, cells were
stimulated using three strong depolarization trains at an interval of 120 s. The representative results shown in Fig. 3A
demonstrate that the capacitance of a control cell after the first
stimulation decreased to less than the prestimulus level, then
gradually increased. The second and third depolarization trains
evoked a similar exocytosis to the first, but endocytosis after
the second and third stimulations did not decrease to less than
C399
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DYNASORE INHIBITS RAPID ENDOCYTOSIS
Fig. 2. Dynasore inhibits rapid endocytosis. The cell was perforated patched and voltage clamped in whole cell mode at a holding potential of ⫺70 mV.
Exocytosis was evoked by a train of 10 depolarizations to ⫹10 mV for 100 ms with an interval of 100 ms, and capacitance was monitored. A: representative
capacitance traces from a control cell without dynasore treatment and cells pretreated with the indicated concentrations of dynasore. The numbers to the left of
each trace are the capacitance in pF recorded before the depolarization train, which is indicated by the black line under each trace. B: normalized averaged
capacitance traces. The capacitance values were normalized to the prestimulus level and averaged. The sample number for 0, 12.5, 25, 50, and 75 ␮M dynasore
was 8, 6, 8, 8, and 8, respectively.
dynasore. The averaged maximum Ca2⫹ response was significantly decreased from 1.49 ⫾ 0.27 ␮M in control cells to
0.65 ⫾ 0.07 and 0.43 ⫾ 0.03 ␮M in cells treated with 75 and
100 ␮M dynasore, respectively (Fig. 5B). The basal [Ca2⫹]i
was also significantly decreased by 100 ␮M of dynasore from
27.8 ⫾ 4.0 nM to 8.3 ⫾ 4.2 nM. These results indicate that
high concentrations of dynasore inhibit the Ca2⫹ response in
chromaffin cells and also decrease the resting [Ca2⫹]i.
Dynasore affects the distribution of clathrin. To characterize
whether the internalization of CCVs was blocked by dynasore,
the distribution of clathrin, F-actin, and dynamin in the cell was
examined using specific antibodies (Fig. 6). In control cells,
clathrin was mainly distributed in the cytosol and concentrated
at an area near the nucleus, while F-actin formed an intact ring
along the cell plasmalemma. The merged result showed that
little clathrin was colocalized with F-actin (data not shown).
After either high K⫹ stimulation or dynasore treatment, part of
the clathrin accumulated at the plasma membrane and formed
some high-density spots. In contrast, the continuity of the
F-actin ring was disrupted by high K⫹ stimulation, but not by
dynasore. In cells pretreated with dynasore and stimulated with
high K⫹ solution, the F-actin ring was disrupted and part of the
Fig. 3. Dynasore enhances repetitively evoked capacitance changes. A: representative capacitance recordings from cells treated with the indicated concentrations
of dynasore. Exocytosis was evoked with a train of 10 depolarizations (arrows) to ⫹10 mV from a ⫺70-mV holding potential, each lasting 100 ms with an
interval of 100 ms. Stimulations were separated by a 2-min interval. The numbers to the left of each trace indicate the capacitance in pF recorded before the first
stimulation. Cells were treated with different concentrations of dynasore for 30 min before recording. In the washout experiment (indicated as “after 100 ␮M
washout”), cells were pretreated with 100 ␮M dynasore for 30 min, then moved to normal bath buffer for another 30 min before recording. B: averaged peak
capacitance changes at each stimulation. The increase in capacitance was calculated from the difference between the maximal capacitance after the stimulation
train and the prestimulus level. The data are means ⫾ SE. *P ⬍ 0.05 and **P ⬍ 0.01 when compared with control cells by Student’s t-test. The sample number
was 6, 4, 7, 7, and 7 for 0, 12.5, 25, 50, and 75 ␮M dynasore treatment, respectively, and 8 for the washout experiment.
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buffer, to examine the effects of dynasore on Ca2⫹ currents and
to avoid Na⫹ current contamination, cells were pretreated with
50 or 100 ␮M dynasore and whole cell patched in Na⫹-free
NMG buffer to isolate the Ca2⫹ currents. The representative
current traces shown in Fig. 4A demonstrate that there was not
much difference in the shape of the evoked currents, but that
the amplitude was decreased by dynasore. The averaged current-voltage relationship (Fig. 4B) shows that the peak current
at ⫹30 mV was significantly decreased from ⫺23.2 ⫾ 2.0 in
control cells to ⫺14.3 ⫾ 1.6 and ⫺6.2 ⫾ 0.8 pA/pF by 50 and
100 ␮M dynasore, respectively. Thus, dynasore inhibits the
Ca2⫹ currents.
Dynasore inhibits the averaged increase in the [Ca2⫹]i. To
investigate whether dynasore affects depolarization-evoked
Ca2⫹ responses, cells were stimulated by high K⫹ solution and
the change in the [Ca2⫹]i was monitored by fura-2 fluorescence. Figure 5A shows representative [Ca2⫹]i responses from
cells treated with different concentrations of dynasore. In
control cells or cells pretreated with dynasore concentrations
lower than 50 ␮M, the [Ca2⫹]i was immediately increased by
high K⫹ depolarization and reached ␮M concentrations, but
the effect was much less in cells pretreated with 75 or 100 ␮M
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DYNASORE INHIBITS RAPID ENDOCYTOSIS
Table 1. Curve-fitting parameters of the decay phase
Dynasore
Control
12.5 ␮M
25 ␮M
50 ␮M
75 ␮M
y0
A1
␶, s
A2
99.1⫾0.4
9.9⫾0.9
3.4⫾0.3
2.1⫾0.1
98.1⫾1.0
12.7⫾1.0
3.6⫾0.3
2.9⫾0.5
99.0⫾2.4
12.1⫾2.8
4.0⫾0.3
3.5⫾0.7*
106.3⫾1.3*
6.4⫾1.5*
4.2⫾0.4
3.8⫾0.7*
106.4⫾0.9*
2.9⫾0.8*
3.7⫾0.6
3.3⫾0.7*
Values are means ⫾ SE. The normalized capacitance between the 3rd and 20th second after the start of the depolarization train was fitted by y ⫽ y0 ⫹ A1 ⫻
exp(⫺t/␶) ⫺ A2 ⫻ t/100, where y is the normalized capacitance, y0 is the offset of the function, A1 is the amplitude, t is the time in seconds, ␶ is the time constant,
and A2 is the slope. The sample number for 0, 12.5, 25, 50, and 75 ␮M dynasore was 6, 4, 7, 7, and 7, respectively. *P ⬍ 0.05 when compared with control
cells by Student’s t-test.
0.3 ⫾ 0.1 per ␮m2 in cells stimulated with K⫹ (n ⫽ 45) and
was significantly increased by dynasore pretreatment to 1.1 ⫾
0.1 per ␮m2 (n ⫽ 42). Furthermore, the averaged width and
depth of the invaginations were significantly increased by
dynasore pretreatment from 136.9 ⫾ 14.2 and 59.6 ⫾ 7.9 (n ⫽
15) to 172.9 ⫾ 9.2 and 89.6 ⫾ 7.7 (n ⫽ 46) nm, respectively
(Fig. 7C). These results indicate that dynasore treatment increases the number of invaginations on the membrane surface
which may represent the endocytotic pores.
Effect of dynasore on the accumulation of FM4 – 64 at the
cell boundary. To determine whether membrane retrieval was
blocked by dynasore, FM4 – 64 was used to stain the plasma
membrane. FM4 – 64 is nonfluorescent in the aqueous medium,
but it becomes intensely fluorescent when inserted in the outer
leaflet of the surface membrane and can thus be used to track
the fate of the internalized membrane (39). Cells were left
untreated or were pretreated with 100 ␮M dynasore, then
challenged with hypertonic buffer for 2 min to evoke exocytosis in the presence of FM4 – 64. Fig. 8 shows that, at 15 min
after wash-off of excess FM4 – 64, the FM4 – 64-stained membrane was evenly distributed inside the control cell, while, in
dynasore-treated cells, the fluorescence was mostly concentrated at the cell surface. The integrated intensity profile
showed that FM4 – 64 was significantly concentrated along the
membrane boundary. These results show that dynasore treat-
Fig. 4. Dynasore inhibits voltage-gated Ca2⫹
channel activity. Ca2⫹ currents (ICa) in chromaffin
cells pretreated with different concentrations of
dynasore were recorded in the whole cell patch
configuration. Cells were incubated in N-methylD-glucamine bath buffer, and the patch pipette
solution was Cs⫹-containing buffer. The cells
were voltage clamped at ⫺70 mV and depolarized
to different potentials for 100 ms with an interval
of 15 s to evoke voltage-gated Ca2⫹ currents.
A: representative current traces from cells treated
with the indicated concentrations of dynasore.
B: averaged current-voltage curve from cells
treated with different concentrations of dynasore.
The data are means ⫾ SE, and the sample number
for 0, 50, and 100 ␮M dynasore was 17, 12, and 5,
respectively. **P ⬍ 0.01 and ***P ⬍ 0.001 by
Student’s t-test when compared with the control
group.
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clathrin accumulated along the cell boundary. In control cells,
dynamin was localized at the cell boundary, and treatment with
dynasore and/or high K⫹ had little effect. These results show
that dynasore modulates the distribution of clathrin but has no
effect on the integrity of the F-actin ring in the subplasmalemma region.
Effect of dynasore on the invaginations on the plasma
membrane detected by AFM. Dynamin is responsible for the
budding of CCVs from the plasma membrane, and inhibition of
its activity results in invaginations at the membrane surface
(22). To characterize the properties of the endocytotic pore, a
1.2 ⫻ 1.2 ␮m membrane patch from a fixed cell was scanned
by AFM (8). An invagination pit is defined by both a depth
⬎20 nm and a width ⬎30 nm. Almost no invaginations were
detected in untreated cells, as reported previously (38). After
high K⫹ stimulation and rest for 10 min, some invaginations
were observed in the membrane patch (data not shown). To
study the effect of dynasore on the invagination pits, the cells
was pretreated with 100 ␮M dynasore for 30 min, then stimulated with high K⫹ buffer in the presence of dynasore. Fig. 7A
shows an invagination pit and its corresponding three-dimensional image (right) from a dynasore treated and high K⫹
stimulated cell. The cross section (Fig. 7B) of this pit shows
that its depth and width were about 250 and 375 nm, respectively. The averaged number of invaginations detected was
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DYNASORE INHIBITS RAPID ENDOCYTOSIS
ment prevents the FM4 – 64-stained plasma membrane from
being endocytosed.
DISCUSSION
Dynamin is involved in several mechanisms responsible for
membrane retrieval from the surface after evoked exocytosis
(35). In this study, we showed that blocking dynamin activity
using dynasore increased the membrane capacitance and inhibited evoked rapid endocytosis. Bright spots of clathrin labeling
were seen in the subplasmalemma region of dynasore-treated
cells, indicating that clathrin coats were formed, but the clathrin-coated invaginations did not pinch off from the plasma
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Fig. 5. Cytosolic Ca2⫹ concentration ([Ca2⫹]i) increase induced by high K⫹
depolarization. Single chromaffin cells were stimulated with high K⫹ buffer
(containing 75 mM KCl) for 3 s as indicated by the arrow, and the change in
[Ca2⫹]i was monitored by the change in the fura-2 fluorescence ratio.
A: representative [Ca2⫹]i responses from single chromaffin cells pretreated
with the indicated concentrations of dynasore. B and C: averaged peak after
stimulation and basal [Ca2⫹]i before stimulation. The data are means ⫾ SE for
n ⫽ 11, 9, 14, 11, and 10 for 0, 10, 50, 75, and 100 ␮M dynasore, respectively.
*P ⬍ 0.05 by Student’s t-test when compared with control cells.
membrane. This interfered with the endocytotic recycling machinery and increased the capacitance jump levels during
repetitive exocytosis. AFM scanning of the membrane surface
showed that dynasore caused in an increase in the number of
invagination pits. This report therefore suggests that rapid
endocytosis decay occur via a dynamin-mediated pathway.
Endocytosis is blocked by dynasore. Cells undergo constitutive exocytosis and endocytosis as a way of communicating
with the extracellular environment (6). CME has been reported
to be responsible for most of the constitutive endocytosis and
requires dynamin for the budding stage. Clathrin distribution in
chromaffin cells has been shown to be punctate in the cytosol
and concentrated at a juxtanuclear region, possibly the Golgi body
(37), as also observed in our control cells (Fig. 6). The dynasoreinduced appearance of bright clathrin spots at the subplasmalemma region is support for the inhibition of the budding off of
CCVs. Our results therefore indicate that dynasore treatment
prevents the budding off of CCVs and causes clathrin accumulation in the subplasmalemma region. This may explain
why the cell surface capacitance is increased by dynasore
treatment, as constitutive endocytosis is inhibited. However,
since the change in membrane capacitance reflects the sum of
exocytosis and endocytosis, we cannot exclude the possibility
that part of the increase in membrane capacitance is contributed by constitutive exocytosis.
In addition, the accumulation of FM4 – 64 at the cell boundary in dynasore-treated cells is further evidence that endocytosis was blocked. The last step of endocytosis requires the
GTPase activity of dynamin to pinch off the vesicle. Since
FM4 – 64 can be washed off the cell surface, its retention at the
cell boundary shows that the membrane was endocytosed but
did not leave the cell surface. These results also suggest that
the necks of the invaginations are constricted by nonfunctional
dynamin, thus limiting the diffusion of FM4 – 64 out of the
endocytosed vesicles.
Although dynasore prevented the pinching off of CCVs, it
did not affect the continuity of the actin filament network
immediately below the plasma membrane; in contrast, the
continuity of the filament was disrupted by high K⫹ stimulation. It has been suggested that disassembly of the actin
filament meshwork allows the movement of secretory vesicles
to the exocytotic site (12, 36). It is not clear whether vesicle
endocytosis requires the disassembly of actin filaments, but
this process may not be required before the budding stage.
Dynasore blocks recycling of the endocytotic machinery.
Since a change in membrane capacitance reflects both exocytotic and endocytotic processes, it is difficult to interpret the
results if exocytosis is mixed with the endocytosis (1, 18).
Using 75 ␮M dynasore, the evoked capacitance jump was
significantly increased at each of the three stimulations,
whereas, at lower dynasore concentrations, the capacitance
increase was only significantly enhanced at the third stimulation (Fig. 3). If endocytosis occurs after the evoked exocytosis,
there should be no difference in the exocytosis. Similarly, the
intercepts of the fitted curves at time 0 were all ⬃110% (Table
1) of the prestimulation membrane capacitance, regardless of
the dynasore concentration used, suggesting that there was no
significant difference in the stimulation-evoked exocytosis.
Although the capacitance trace did not start declining until the
end of the depolarizations in most cells recorded, some cells
did show a significant decrease in capacitance during the train
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DYNASORE INHIBITS RAPID ENDOCYTOSIS
of depolarizations (data not shown). This hints at the occurrence of endocytosis during stimulation and hinders the interpretation of the increase in capacitance.
Blocking of endocytosis in nerve terminals by dynasore
decreases the postsynaptic response during repetitive stimulation (22, 24). In contrast, at dynasore concentrations lower than
25 ␮M, the level of exocytosis evoked at the first stimulation
was about the same as that in control cells and exocytosis was
significantly increased at the second and third stimulations
(Fig. 3). Taking this result together with the accumulation of
clathrin and FM4 – 64 in the subplasmalemma region caused by
dynasore treatment, it is possible that the endocytosis machin-
Fig. 7. Invaginations observed by atomic force microscopy (AFM). Chromaffin cells were stimulated
4 times by alternate incubation in high K⫹ buffer
and normal bath buffer for 1 and 2 min, respectively,
then were rested for 10 min and fixed for AFM
scanning. In the case of cells pretreated with dynasore, 100 ␮M dynasore was added 30 min before the
start of K⫹ stimulation and was present in all buffers
used. The membrane patches scanned were selected
randomly, and each cell was scanned only once.
A: AFM image of a cell pretreated with dynasore,
then stimulated with K⫹; the corresponding threedimensional image is shown on the right. B: cross
section of the white line shown in A. C: averaged
width and depth of the invaginations observed in 45
cells after K⫹ stimulation (open columns, n ⫽ 15) or
pretreated with dynasore, then K⫹ stimulated (filled
columns, n ⫽ 47) cells. *P ⬍ 0.05 and **P ⬍ 0.01
when compared with the K⫹ group by Student’s
t-test.
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Fig. 6. Clathrin distribution in chromaffin
cells. Chromaffin cells were stimulated 4
times by incubation in high K⫹ buffer (high
K⫹, columns 2 and 4) for 1 min, then in
control bath buffer for another 2 min. The
cells remained in the control bath buffer for
10 min, then were fixed in 3.7% formaldehyde for staining. For dynasore treatment
(dynasore, columns 3 and 4), the cells were
either incubated in control bath buffer containing 100 ␮M dynasore for 30 min, then
fixed for staining (dynasore alone) or preincubated with dynasore for 30 min, then incubated in high K⫹ buffer as above, with 100
␮M dynasore in all buffers used. A: costaining of clathrin and F-actin. Images of endogenous clathrin were obtained by confocal
microscopy after double-labeling with anticlathrin antibody (clathrin, row 2) and rhodamine-conjugated phalloidin (F-actin, row
1) to visualize F-actin. The arrows indicate
some clathrin aggregation sites. B: localization of dynamin. Monoclonal antibody was
used to label the endogenous dynamin.
Scale bar, 5 ␮m.
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DYNASORE INHIBITS RAPID ENDOCYTOSIS
Fig. 8. Distribution of FM4 – 64 in chromaffin cells.
Chromaffin cells were incubated in normal bath
buffer for 30 min, then in hypertonic buffer for 2
min in the presence of 50 ␮M FM4 – 64. The cells
were then moved to bath buffer containing FM4 – 64
for 30 min, then incubated in bath buffer without
FM4 – 64 for another 30 min. FM4 – 64 fluorescence
images were obtained using a confocal microscope.
A: control cell. B: dynasore-treated cell. In the case
of the dynasore-treated cell, all buffers used contained 100 ␮M dynasore. The fluorescence intensity
inside the dashed box was integrated and plotted at
the bottom of each graph.
AJP-Cell Physiol • VOL
slow endocytosis. Clathrin has been shown to be colocalized
with dynamin-1 and dynamin-2 in both resting and stimulated
PC12 cells (31). In addition, RNA interference knockdown of
clathrin inhibits both fast and slow endocytosis at hippocampal
synapses after exocytosis (42). Thus, it is possible that clathrin
is involved in the rapid endocytosis mediated by dynamin in
bovine chromaffin cells, but this remains to be confirmed.
Ca2⫹ influx and exo/endocytosis. Although Ca2⫹ influx
during the repetitive stimulations was not significantly inhibited by dynasore, the averaged increase in the [Ca2⫹]i and
Ca2⫹ currents measured by whole cell patch was significantly
inhibited by high dynasore concentrations (Figs. 4 and 5).
Considering the increase in cell size caused by dynasore
treatment, the same amount of Ca2⫹ influx would result in a
smaller increase in the [Ca2⫹]i; in addition, exocytosis has
been suggested to be mainly determined by the local [Ca2⫹]i at
the microdomain where Ca2⫹ flux into the cell occurs and not
the averaged [Ca2⫹]i increase (10, 27). Thus, although Ca2⫹
influx was inhibited by dynasore at concentrations above 50
␮M, the local [Ca2⫹]i at the exocytotic site was still high
enough to support exocytosis. However, some reports have
suggested that Ca2⫹ may not have an effect on endocytosis (29,
33) or may even play an inhibitory role (9, 40). In addition, the
GTPase activity of dynamin-1 is inhibited by Ca2⫹, with an
IC50 of 30 ␮M (20), so it is possible that dynamin-1 activity is
increased at a low [Ca2⫹]i. Our results showed that dynasore
inhibited the Ca2⫹ currents and lowered the [Ca2⫹]i, which
should increase dynamin activity and endocytosis. However,
endocytosis was not increased, suggesting that the inhibition of
endocytosis by dynasore is not due to the decrease in Ca2⫹
influx. How dynasore inhibits the Ca2⫹ currents requires further investigation.
Dynasore widens the invagination pits. AFM provides threedimensional information of the surface scanned and has been
used to study the exocytotic fusion pores of endocrine cells. In
an untreated cell, the surface is quite flat, with little fluctuation
(8, 38). Invaginations were seen after high K⫹ stimulation, and
their number was increased in cells treated with dynasore, then
stimulated with high K⫹. Recently, Lou et al. (21) reported that
the number of CCVs with a neck observed by electron microscopy after high K⫹ stimulation in the calyx of Held is ⬃0.1 and
⬃1.3 counts/␮m2 in wild-type and dynamin-1 knockout mice,
respectively. These results are close to the number of invaginations we observed, indicating that the increase in number is
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ery is depleted during repetitive stimulation and thus increases
the capacitance level. The contribution of the exponential
component to endocytosis was decreased at the third stimulation after dynasore washout, further support for the idea that
the endocytosis machinery was depleted. The number of secretory vesicles in chromaffin cells is much greater than that in
the axon terminal, so, even if vesicle recycling is blocked, there
are still enough vesicles to replenish the readily releasable pool
and maintain the evoked exocytosis at a similar level (19).
It has been suggested that the effect of dynasore in blocking
endocytosis at the axon terminal is reversible (24). In chromaffin cells after dynasore washout, the evoked increase in the
capacitance was similar to that in control cells and the endocytosis rate at the first stimulation was comparable to that in
control cells (Fig. 3). These results show that the effect of
dynasore on endocytosis in chromaffin cells is reversible.
Dynamin is involved in exponential compensatory retrieval.
The time constant of the exponential decay after evoked
exocytosis was about 3– 4 s, which is comparable to reported
values for rapid endocytosis (3, 14, 34). This time constant was
not affected by dynasore treatment, but the amplitude of this
decay decreased as the dynasore concentration increased. This
suggests that rapid compensatory endocytosis is mediated by a
dynamin-dependent pathway.
In control cells, the excess retrieval after evoked exocytosis
caused the membrane capacitance to drop to a value below the
prestimulus level, then increase gradually to the prestimulus
level within 6 min (Fig. 3) (14, 34). Such an increase in
membrane capacitance was not observed in dynasore-treated
cells, and the mechanism needs to be characterized. However,
this increase may underestimate the decay rate, especially the
linear declination component. This may explain the smaller
slope factor in control cells than in dynasore-treated cells
(Table 1).
It has been reported that perfusion of anti-dynamin-1 or
anti-dynamin-2 antibodies through a patch pipette blocks, respectively, rapid or slow endocytosis in bovine chromaffin
cells and that rapid endocytosis may not be clathrin dependent
(2, 3). When the function of both dynamin-1 and dynamin-2
was inhibited by dynasore, the contribution of the rapid decay,
but not the slow linear declination, to the total evoked endocytosis was greatly suppressed. This discrepancy in the effect
on slow endocytosis may be due to the patch protocol used or
the existence of an unidentified dynamin-independent ultra-
DYNASORE INHIBITS RAPID ENDOCYTOSIS
ACKNOWLEDGMENTS
The authors thank Dr. Chung-Chih Lin for comments on the manuscript and
the technicians in the TechComm Center, School of Life Science, National
Taiwan University, for fluorescence imaging analysis.
GRANTS
This work was supported by grants from the National Science Council
(NSC 96-2311-B-002-008-MY2 & 97-2627-M-002-020), Taiwan, R. O. C.
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