* Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200; and Section of
Molecular and Cellular Biology, University of California, Davis, California 95616
Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin- dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.
TO dock and fuse with the plasma membrane in response to localized calcium influx, vesicles for Ca2+-regulated exocytosis must first be recruited to the
cell surface from intracellular pools. Although much has
been learned about the molecular mechanisms of the
docking and fusion reactions of regulated exocytosis (Sudhof et al., 1993 The motor protein kinesin is a good candidate for part
of the transport machinery in the pathway of regulated
exocytosis. Kinesin has been demonstrated to move along
microtubule tracks towards the plus end by hydrolyzing
ATP in several in vitro assays (Goldstein, 1993 A kinesin holoenzyme is composed of two identical
heavy chains and two light chains. The kinesin heavy chain
(KHC)1 consists of an amino-terminal globular head domain that is linked to the carboxyl-terminal tail domain
through a stalk region that dimerizes two KHCs to form
the kinesin motor (Fig. 1) (Yang et al., 1989
The actin-based motor myosin is another candidate that
may drive vesicle recruitment in regulated exocytotic
pathways (Fath and Burgess, 1994 Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) has been implicated in the regulation of exocytosis
at an actin binding step (Ceccaldi et al., 1995 Using a confocal microscope, we were able to visualize
and quantify individual exocytotic events in real time by
triggering local bursts of regulated exocytosis in early sea
urchin embryonic cells by wounding the cell membrane
with a laser beam and inducing Ca2+ influx through the
wound (Bi et al., 1995 Materials
For isolation and purification of bacterially expressed KHC fragments,
P11 cellulose phosphate cation exchanger was obtained from Whatman
International Ltd. (Maidstone, Kent, UK). DEAE Bio-Gel A (100-200
mesh) anion exchange resin was obtained from Bio-Rad Laboratories
(Hercules, CA). All other reagents were from Sigma Chemical Co. (St.
Louis, MO) unless otherwise specified. SUK4 and SUK2 monoclonal antibody stocks were purified IgG isolated on protein A columns at 4.5 and 3 mg/ml in PBS. They were mixed with Fura-2 salt and aspartate buffer
(containing 100 mM potassium aspartate, 20 mM Hepes, pH 7.2) to make
the injection solutions that contained 2.7 mg/ml of either antibody.
2, 3-butanedione monoxime (BDM, from Sigma Chemical Co.) was dissolved into natural sea water to make a fresh stock of 0.5 M on the same
day of the experiment. It was then added into sea water to reach required
final concentrations. Washes were performed by replacing the extracellular BDM solution with fresh natural sea water at least three times in 5 min. Both the powder and solutions of BDM were kept in dark to avoid
light inactivation.
CaM kinase peptides CaMK(273-302) and CaMK(284-302) were synthesized using an automated synthesizer (Applied Biosystems, Inc., Foster
City, CA), purified by HPLC, and assessed by sequencing. CaMK(273-302) and CamK(284-302) peptide stocks were 2.5 and 5 mM in aspartate
buffer (containing 100 mM potassium aspartate, 20 mM Hepes, pH 7.2).
They were mixed with Fura-2 potassium salt to make the injection solutions that contained 0.25 and 0.5 mM of either peptide.
Isolation and Purification of Bacterially Expressed
KHC Fragments
Previously, two Escherichia coli bacterial strains were transformed with
T7 vectors containing partial inserts of sea urchin conventional kinesin
heavy chain: one 1.4-kb insert encoded the tail and a portion of the stalk
(amino acids 579-1031) and one 0.8-kb insert encoded a portion of the
stalk only (amino acids 579-858), as described (Skoufias et al., 1994 Cells, Microinjection, and Test of Resealing
Lytechinus pictus sea urchins (from Marinus Inc., Long Beach, CA) were
handled as previously described (Bi et al., 1995 Confocal Microscopy
Confocal microscopy was performed using a confocal microscope (model
MRC-600; Bio-Rad Labs) equipped with a 40× Neofluor objective (NA = 1.3, oil immersion; Carl Zeiss, Inc., Thornwood, NY), a 15 mW argon ion
laser, and a UV epifluorescence system (Carl Zeiss, Inc.). For microscopy,
eggs settled on poly-lysine-coated coverslips were injected within 5 min
after fertilization and were allowed to develop to two-cell stage (~2 h at
16.5°C) before being mounted into a handmade chamber that held ~150
µl of natural sea water containing 100 µM rhodamine dextran (3,000 molecular weight; Molecular Probes). Confocal experiments were performed
at 20°C. Two- to four-cell stage embryos with relatively flat surfaces adhering to the coverslip were selected for experiments. Injected embryos could be easily identified by their Fura-2 fluorescence with UV epifluorescence filters (360 nm excitation and >400 nm emission).
During each imaging series, the cell was first wounded by "parking" the
confocal beam at maximum power (neutral density filter setting at ND 0)
focused at a selected position on the cell surface for 10-25 s to trigger local
exocytosis. Neutral density filter setting ND 3 was then used for subsequent imaging to attenuate the beam power and reduce cell damage. The
confocal scanning plane was focused just inside the cell surface to visualize
the formation of bright fluorescent "disks." These disks have been shown
to be exocytotic vesicle pockets filled with extracellular fluorescent dextrans, based on their rapid formation and their sensitivity to proteolytic
neurotoxins that specifically target the exocytosis machinery (Bi et al.,
1995 A Slow Phase of Ca2+-triggered Exocytosis Requires a
Functional Kinesin Motor
We used the laser beam of a confocal microscope to
wound the membranes of embryonic sea urchin cells so
that Ca2+ influx through the wound would trigger a burst
of local exocytosis (Bi et al., 1995
Fig. 3 A shows examples of the quantified time course of
exocytosis after individual membrane wounds. Fig. 3 B averages the results from 17 or more experiments and reveals two distinct phases in the temporal pattern of this
regulated exocytosis: a slow phase that is inhibited by
SUK4 and a fast one not affected by this function-blocking
antibody. Although we can not exclude the possibility that
kinesin may play some role in the organization of intracellular structure, which may in turn affect the temporal pattern of exocytosis, it appears more likely that this slow
phase simply represents a kinesin-mediated step in the
regulated exocytosis pathway, the step of vesicle recruitment on microtubules directed toward the release site of
membrane wounding and Ca2+ influx. Furthermore, since
conventional kinesin is currently the only known KRP that
is recognized by SUK4, we suspect that this most abundant
KRP is responsible for the transport of vesicles in this
ubiquitous regulated exocytosis pathway.
Kinesin Mediates Vesicle Transport for Ca2+-regulated
Exocytosis through Its Cargo-binding Tail Domain
To confirm that the effect of SUK4 was due to inhibition
of kinesin-mediated vesicle transport and to further identify the molecular identity of the responsible KRP isoform,
we generated the stalk-tail and stalk only fragments of
KHC expressed in bacteria (Fig. 1). Here the term "kinesin" refers to the conventional kinesin, as opposed to other
KRPs. In an in vitro assay, this stalk-tail fragment, but not
the stalk fragment, effectively competed with kinesin for
vesicle binding (Skoufias et al., 1994 We injected the cells with the stalk-tail fragment at
~650 nM intracellular concentration. This is 32-fold
greater than the Kd of stalk-tail binding to microsomes
(Skoufias et al., 1994
A Myosin Motor May Mediate Vesicle Transport at a
Step Downstream to Kinesin Transport
Members of the myosin family have also been implicated
in membranous organelle transport in several systems
(Adams and Pollard, 1986
To make sure that the BDM effect was not due to any
inhibition of the vesicle fusion step of exocytosis, we tested
exocytosis of previously docked cortical granules found at
the plasma membrane in unfertilized sea urchin eggs at the
same BDM concentration. Exocytosis of these vesicles was
not affected. (Nine out of nine cells tested showed normal
exocytosis in response to membrane wounding; data not
shown.) This is also consistent with the fact that in embryonic cells, BDM inhibited later events (>5 s after wounding) to a much higher degree than the initial burst (Fig. 5).
The initial burst of exocytosis is most likely from previously docked vesicles. Therefore, we believe that BDM affected the regulated exocytosis at a step upstream of the
final fusion event. Furthermore, this step, possibly a myosin-mediated transport step, appears to be downstream of
the kinesin transport step, because in addition to the slow
phase of exocytosis, BDM also inhibited the fast phase of
exocytosis that was not affected by our blocks of kinesin
function.
To obtain further evidence consistent with an actin-based step in vesicle recruitment, we investigated the possible role of CaM kinase II by injecting cells with
CaMK(273-302), an autoinhibitory peptide from the regulatory domain of CaM kinase. Although CaM kinase II is a
multifunctional enzyme and could have multiple targets,
we examined its specific effect on vesicle recruitment to
sites of Ca2+-regulated exocytosis. CaMK(273-302) had
been previously shown to inhibit a CaM kinase II-specific
Ca2+/calmodulin-dependent phosphorylation in cell extracts of Lytechinus pictus (Baitinger et al., 1990
Three Semistable Vesicle Pools in the Pathway of
Regulated Exocytosis
One interesting feature in the above results is that blocking either motor system could not completely block injury-induced regulated exocytosis. When kinesin transport was
blocked, only the slow phase of exocytosis was inhibited.
The fast phase was virtually intact (Figs. 3 and 4). With
BDM inhibition of putative myosin transport, a significant
portion of the very fast events still persisted (Fig. 5). If the
vesicle recruitment process is a simple chain of constitutive sequential steps and the regulation is only at the final
exocytosis step, a blockade at any point in the chain should
similarly inhibit the whole process. However, the above
experiments showed distinct temporal patterns when different motors were blocked, indicating the existence of
semistable intermediate vesicle pools that may be regulated separately in the membrane transport pathway.
Quantification of the temporal patterns of exocytosis
under different conditions (Figs. 3-5) allows us to mathematically separate these vesicle pools. As shown in Figure
7 A, an "Immediate" pool of vesicles could be defined as
those that can exocytose in the presence of BDM because
they respond to Ca2+ influx most rapidly (within a few seconds) and do not seem to depend on either myosin or
kinesin transport mechanisms. By subtracting the myosin-
independent (BDM-insensitive) component from the kinesin-independent exocytosis (average of SUK4 and stalk-tail
data), a "Fast" pool could be defined as vesicles whose
exocytosis are myosin-dependent but kinesin independent.
Exocytosis of vesicles from this fast pool usually took place
within 10 s after Ca2+ influx. Finally, a "Slow" pool could
be isolated by subtracting the kinesin-independent exocytosis data (summation of immediate and the fast pools)
from the average of control experiments including the
control, SUK2, and Stalk data from Figs. 3-5. Apparently,
vesicles in this slow pool require normal levels of kinesin
and myosin transport for their final exocytosis. These vesicles take a much longer time to exocytose (peaking at 30 s)
than those in the first two pools, presumably because of
the delay in long-distance transport by the kinesin-microtubule system, as will be discussed later.
Motor-driven Vesicle Recruitment Is Essential for Cell
Membrane Resealing
Although the main objective of this study was to directly
observe motor-driven vesicle recruitment steps in a regulated pathway in vivo, we also investigated the role of
these steps in cell membrane resealing (Steinhardt et al.,
1994 Table I.
Effect of KHC fragments on Cell Membrane Resealing
in Activated Eggs of L. pictus
Table II.
Effect of BDM on Cell Membrane Resealing in
Activated Eggs of L. pictus
; Bennett and Scheller, 1994
; De Camilli,
1995
), the few studies of the vesicle recruitment process
have focused on membrane recycled from endocytosis
(Betz and Bewick, 1992
; Ryan et al., 1993
; Betz and Wu,
1995
), and as yet there has been little direct in vivo observation of the roles of motor proteins in recruiting vesicles
to exocytotic sites (Scholey, 1996
; Vallee and Sheetz,
1996
).
; Bloom
and Endow, 1995
). It has also been shown to associate
with vesicle and organelle membranes in different cell
types (Bloom and Endow, 1995
). Several antikinesin antibodies were able to inhibit fast axonal transport (Vale et
al., 1985b
; Brady et al., 1990
), the centrifugal migration of
pigment granules (Rodionov et al., 1991
), and the formation of tubular lysosomal structure (Hollenbeck and Swanson, 1990
). Mutations in Drosophila kinesin impaired the
transport of membrane proteins to their appropriate cellular locations (Saxton et al., 1991
; Gho et al., 1992
). Based on these findings, it has been widely predicted that this
motor protein will be shown to play an essential role in
transporting vesicles to sites of Ca2+-regulated exocytosis.
). The KHC
head domain is highly conserved among different kinesin-related proteins (KRPs) and has been shown to be responsible for ATP hydrolysis and force generation (Yang et al.,
1990
). The tail domain is more variable and is thought to
be important for kinesin cargo binding (Hirokawa et al.,
1989
; Bloom and Endow, 1995
). This was further supported by in vitro observations that the bacterially expressed stalk-tail fragment, but not the stalk fragment of
sea urchin KHC, was able to bind microsomal membranes isolated from sea urchin eggs in a saturable manner and
compete with native kinesin for membrane binding (Skoufias et al., 1994
). However, an in vivo demonstration has
been difficult because the in vivo function of sea urchin kinesin was not known.
Fig. 1.
Primary sequence of KHC. Arrows indicate approximate sites for antibody recognition. Numbers refer to KHC
amino acid sequence number starting from the amino terminus.
The "Stalk-Tail" and "Stalk" are the two KHC fragments used in
this experiment.
[View Larger Version of this Image (25K GIF file)]
; Hasson and Mooseker,
1995
; Langford, 1995
). Evidence that some of the myosin
isoforms may power membrane transport came from several systems including yeast (Johnston et al., 1991
; Drubin
and Nelson, 1996
), algae (Adams and Pollard, 1986
; Grolig et al., 1988
), squid axoplasm (Kuznetsov et al., 1992
), and
polarized epithelial cells (Fath et al., 1994
). There was also
evidence for the presence of both microtubule- and actin-based motors on the same membranous organelles (Fath
et al., 1994
). However, with one exception, there has been
no in vivo demonstration for the role of myosin in regulated exocytosis, and the exact interrelationship between
the microtubule-based and actin-based systems has yet to
be elucidated (Langford, 1995
). The one exception is a
study in which a smooth muscle antimyosin II antibody
microinjected into presynaptic neurons inhibited synaptic
transmission (Mochida et al., 1994
). Transmission was also
inhibited in a dose-dependent manner by two inhibitors of
myosin light chain kinase. The identity of the neuronal
myosin subtype affected in this study remains an open
question since other studies have failed to find myosin II
presynaptically (Miller et al., 1992
).
). CaM kinase
II injected presynaptically in squid giant synapse was
found to facilitate transmitter release (Llinas et al., 1991
).
Additionally, extracellular application of synthetic peptide
inhibitors of CaM kinase II preferentially suppressed the
phosphorylation of synapsin I at the CaM kinase II-specific site and decreased excitatory synaptic responses elicited in the CA1 region of hippocampal slices (Waxham et
al., 1993
). Recently, Ceccaldi showed that the presence of
dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin, and the effect is abolished upon its phosphorylation by CaM kinase II (Ceccaldi et al., 1995
).
Therefore, CaM kinase II is postulated to play an essential
role in recruitment of vesicles since its action would be required to free vesicles from the actin-bound pool.
; Terasaki, 1995
). This system allowed us to directly investigate the in vivo roles of protein
motors in the recruitment of vesicles to exocytotic sites and address several related issues. By analyzing the temporal patterns of exocytosis when the functions of kinesin,
myosin, and CaM kinase were inhibited, we concluded
that exocytotic vesicles were recruited by a two-step transport mechanism mediated by kinesin and myosin sequentially. Our results also revealed distinct intermediate vesicle pools along the transport pathway. In addition, our
results demonstrated the in vivo cargo binding function of
the KHC tail domain.
Materials and Methods
). Isolation of the KHC fragments was performed as previously described
(Skoufias et al., 1994
) with the following modifications. Frozen transformed cell stocks were grown up in Luria Broth containing 50 µg/ml each of ampicillin and chloramphenicol. Bacteria were induced, harvested, washed as before, and then lysed by sonication followed by three rounds
of freezing and thawing in lysis buffer as described (Skoufias et al., 1994
).
Stalk and stalk-tail fragments were isolated by ion exchange chromatography in 20 mM Tris, pH 8, 6 M urea, 1 mM DTT and eluted as before, except DEAE Bio-Gel A was substituted for DE52 DEAE. Column elutates were dialyzed sequentially with 1 liter of 3 and 1.5 M urea in 20 mM
Tris, pH 8, 1 mM EGTA, 1 mM EDTA, 150 mM NaCl, 1 mM MgSO4, 2 mM DTT, plus protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, 20 µg/ml
benzamidine, and 1 mg/ml TAME). Final purification was performed by
sedimentation through a 1-15% sucrose density gradient in the same
buffer with 1.5 M urea. Peak gradient samples were pooled and dialyzed
sequentially against 1 liter each of 1.5, 0.75, and 0 M urea in aspartate
buffer that contained 100 mM L-aspartic acid, 20 mM Hepes, pH 7.2, plus protease inhibitors. Final concentrations of KHC stalk fragment was 2.7 mg/ml = 80 µM; stalk-tail fragment was 0.75 mg/ml = 14.5 µM.
). Microinjections and resealing tests were performed as described (Steinhardt et al., 1994
; Bi et al.,
1995
). Briefly, eggs and sperm were obtained by injection of 0.55 M KCl
into the sea urchin interperitoneal cavity. Sperm was collected dry and
stored at 4°C for up to 1 wk. Eggs were collected in natural sea water, dejellied by passing through 80-µm Nitex mesh (Tetko Inc., Depew, NY),
washed three times in natural sea water, and were allowed to settle onto
glass coverslips coated with 10 mg/ml poly-DL-lysine (Sigma Chemical
Co.). Kinesin reagents or buffer were pressure-injected into unfertilized
or newly fertilized sea urchin eggs. All injection solutions contained 2.5 mM Fura-2 salt (Molecular Probes, Eugene, OR) as a fluorescent marker
for injected cells. Injection volume was about 5% of cell volume. For tests
of resealing, injected cells were wounded in natural sea water with a glass
micropipette that penetrated the plasma membrane and was withdrawn
immediately after wounding. The size of this wounding is similar to that
produced by a typical microinjection. Resealing was monitored by photometric measurement of Fura-2 emission fluorescence at 510 nm after alternate excitation with 357- and 385-nm ultraviolet light (Steinhardt et al.,
1994
) and subsequently confirmed visually.
). Cells were scanned continuously (1 frame/s), and image frames
were collected and stored when new exocytotic events occurred or after a
period of time (~20 s) without new events. A semiautomatic macro program was written to perform these operations. Images were processed by
NIH Image (Bethesda, MD) and Adobe Photoshop (San Jose, CA).
Quantification was performed manually during playback of recorded image series by direct counting the number of new exocytotic events in each
frame.
Results
). By setting the confocal
scanning plane right underneath the cell surface, individual exocytotic events could be visualized as bright disks in
the confocal images (Bi et al., 1995
; Terasaki, 1995
). In
Fig. 2, each newly emerged bright disk indicated by an arrow represents a single newly exocytosed vesicle. Under
normal conditions, the exocytotic burst usually lasts for about a minute and ceases after the wound reseals, Ca2+
influx stops, and intracellular free Ca2+ levels return to
normal (Fig. 2 A). To investigate the possible transport
role of the motor protein kinesin (Scholey et al., 1985
; Vale et al., 1985a
) in this exocytotic pathway, we injected
cells with SUK4, a monoclonal antibody targeting the motor domain of KHC (Fig. 1) (Wright et al., 1991
). This antibody had been shown to block the kinesin-mediated microtubule motility in vitro (Ingold et al., 1988
) and to
inhibit exocytosis-dependent membrane resealing (Steinhardt et al., 1994
). Surprisingly, the early exocytotic events
were virtually unaffected by the antibody injection (5 to
10 s in Fig. 2 B). However, the later exocytotic events were inhibited (Fig. 2 B, 26 s and later). As a control, some cells were injected with another antikinesin antibody, SUK2,
that targets the kinesin stalk domain (Fig. 1) (Wright et al.,
1991
) and does not interfere with the motor function (Ingold et al., 1988
) or membrane resealing (Steinhardt et al.,
1994
) at similar intracellular concentrations. These cells
exhibited a normal pattern of exocytosis in response to
Ca2+ influx through the laser wound (Fig. 2 C).
Fig. 2.
Effect of reagents targeted against motor proteins on Ca2+-regulated exocytosis induced by laser wounding. The pseudocolor
pictures are confocal fluorescence images of extracellular rhodamine dextran showing the exocytotic pockets into confocal focal plane
just under the plasma membrane (Bi et al., 1995). Exocytotic events are visualized as the appearance of bright disks (0.5-1 µm diameter) against the dark intracellular background indicated by arrows. Blue arrows indicate early events, while green arrows indicate later
events. (A) The exocytotic pattern of a sea urchin embryonic cell in natural sea water. In other series, the embryos were previously injected with (B) SUK4, (C) SUK2 antikinesin, (D) stalk-tail fragment, and (E) stalk fragment of KHC and were kept in natural sea water. In series F, the embryo was treated with 50 mM BDM. In series G, the embryo was injected with CaMK(273-302), an autoinhibitory peptide from the regulatory domain of CaM kinase type II. The large central stain in G is dye entering the unhealed wound. The first
frame in each series was collected before wounding. Time 0 is defined as the moment immediately after wounding. The unit for time labels is seconds. Arrows indicate new exocytotic events that occurred since the previous frame. Bar, 5 µm.
[View Larger Version of this Image (112K GIF file)]
Fig. 3.
Specific inhibition of a slow phase of Ca2+-regulated
exocytosis by function-blocking antikinesin antibody SUK4. A
quantifies cumulative number of exocytotic events in individual
cells from typical experiments. The control cell (Ctrl) was not injected with any reagent. B summarizes the average number of
exocytotic events that occurred within different time ranges from
n experiments. n = 37 for Ctrl, 17 for SUK4, and 23 for SUK2. A
biphasic temporal pattern of exocytosis is seen by comparing the
SUK4 injection data with the Ctrl or SUK2 injection data. The
slow phase (after 16 s), but not the fast phase (0-15 s) of exocytosis
is inhibited by SUK4 antikinesin. Error bars are standard errors.
[View Larger Version of this Image (37K GIF file)]
). If the KHC tail domain is responsible for vesicular cargo binding in vivo, as
has been postulated, introduction of the stalk-tail fragment, but not the stalk alone into the cell, should arrest kinesin-mediated transport by competition for the binding
of cargo receptors. If kinesin, but not other KRPs, does
mediate vesicle recruitment for regulated exocytosis, this
arrest of transport should alter the temporal pattern of
exocytosis, as did the SUK4 antibody.
). These cells indeed showed a block
in the slow phase of exocytosis similar to that seen in cells
injected with SUK4 (Figs. 2 D and 4). In contrast, stalk
fragment injection (intracellular concentration ~2.4 µM)
had no effect on exocytosis (Fig. 2 E and 4). Because the
tail domain is less conserved among different KRPs, the
stalk-tail competition for putative vesicle receptors should
be very specific. It is therefore highly likely that conventional kinesin is the protein motor that drives vesicle recruitment in Ca2+-regulated exocytosis. In addition, this
result also provides direct in vivo evidence for the postulated vesicular cargo binding function of the kinesin tail
domain.
Fig. 4.
Specific inhibition of the slow phase of Ca2+-regulated
exocytosis by KHC stalk-tail fragment. (A) Typical examples of
the cumulative number of exocytotic events in individual cells.
(B) The average number of exocytotic events that occurred
within different time ranges from n experiments. n = 37 for Ctrl,
27 for Stalk-tail, and 33 for Stalk.
[View Larger Version of this Image (37K GIF file)]
; Grolig et al., 1988
; Kuznetsov
et al., 1992
). Does it play any role in the regulated exocytotic pathway? If yes, what is the relationship between the
kinesin-microtubule and actomyosin transport systems?
To address these questions, we treated the cells with
BDM, a known reversible inhibitor of myosin ATPase that
has been shown to block myosin-dependent cell spreading
in epithelial cells (Cramer and Mitchison, 1995
) and retrograde F-actin flow in neuronal growth cones (Lin et al.,
1996
) but has not been found to affect kinesin ATPase activity (Cramer and Mitchison, 1995
). BDM is at present
the only potent general inhibitor of myosin function available. In the absence of knowledge of specific myosin isoforms and in the absence of more specifically targeted reagents, BDM is as close as one can get now to implicating
myosin in a functional assay. BDM is fully reversible in
functional studies of myosin. Exocytosis was reversibly inhibited by 50 mM BDM (Figs. 2 F and 5). In contrast to the
inhibition by SUK4 and KHC stalk-tail, BDM inhibited
exocytotic events from both the slow and the fast phase
(Fig. 5). This is particularly obvious when comparing the exocytosis under these conditions within the 6-15-s range
(Fig. 3-5). The onset of BDM effect on exocytosis pattern
took about 10 min. After that, the exocytosis pattern was
stable for at least 1 h, until BDM was washed away. Exocytosis recovered to its normal pattern within 15 min after
BDM was washed away (Fig. 5).
Fig. 5.
Reversible inhibition of both the slow and the fast
phases of exocytosis by BDM. The cells were in 50 mM BDM for
10-60 min. For "Wash" experiments, cells were in 50 mM BDM
for at least 45 min and were then transferred to BDM-free sea
water for at least 15 min before imaging. (A) Quantified examples of individual experiments under different conditions. (B)
Average number of exocytotic events that occurred within different time ranges from n experiments. n = 37 for Ctrl, 17 for BDM,
and 8 for Wash.
[View Larger Version of this Image (36K GIF file)]
) and to
inhibit exocytosis-dependent membrane resealing (Steinhardt et al., 1994
). Figs. 3 G and 6 A show typical examples
of cumulative exocytotic events. Fig. 6 B averages the results from 18 or more experiments and reveals that both
fast and slow vesicle exocytosis have been inhibited by
CaMK(273-302). CaMK(273-302) did not block exocytosis
completely, which is consistent with its action on CaM kinase II in vitro (Baitinger et al., 1990
). To demonstrate
that the inhibition was not on the fusion of vesicles, we
tested the effect of CaMK(273-302) on laser wound-triggered exocytosis of previously docked cortical granules
found at the plasma membrane in unfertilized sea urchin eggs. Exocytosis of these vesicles was not affected (four of
four cells tested). As a control, some cells were injected
with the shorter peptide, CaMK(284-302), which does not
inhibit Ca2+/calmodulin-dependent phosphorylation in
vitro (Baitinger et al., 1990
) or membrane resealing (Steinhardt et al., 1994
). Those cells exocytosed normally in response to Ca2+ influx through the laser wound (Fig. 6). It
appears that CaMK(273-302) and BDM have similar effects on the recruitment of vesicles, a result consistent with
a block of the late step of the vesicle transportation process from the actin matrix to the plasma membrane.
Fig. 6.
Inhibition of both the slow and the fast phases of exocytosis by CaMK(273-302), an autoinhibitory peptide from the regulatory domain of CaM kinase type II. Control peptide
CaMK(284-302) did not inhibit exocytosis. (A) Typical examples
of cumulative number of exocytotic events in individual cells. (B)
The average number of exocytotic events that occurred within
different time ranges from n experiments. n = 18 for Ctrl, 39 for
CaMK(273-302), and 22 for CaMK(284-302).
[View Larger Version of this Image (33K GIF file)]
; Bi et al., 1995
; Miyake and McNeil, 1995
). After injection into the cell, the stalk-tail fragment of KHC inhibited membrane resealing in sea urchin embryonic cells or
activated eggs (Table I) when tested by mechanical
wounding with a glass needle, as did the SUK4 antibody
(Steinhardt et al., 1994
). Stalk injection had no significant
effect on resealing (Table I). As expected, BDM treatment also inhibited membrane resealing in activated eggs
or embryonic cells but not in unfertilized eggs, which all
have thousands of cortical vesicles already docked at their
plasma membrane (Table II). We also found that resealing in activated eggs and embryonic cells was inhibited by cytoskeleton-disrupting drugs. (Nine out of nine cells tested
failed to reseal after 0.5 h in 25-30 µM nocodozole, which
disrupts microtubules; 10 out of 10 cells failed to reseal after 0.5 h in 10-20 µM cytochalasin D or B, which both disrupt actin filaments.) This is consistent with the function of
microtubules and actin filaments as tracks for appropriate
protein motors. Therefore, both kinesin-microtubule and
actomyosin transport systems appeared to be not only important for the ubiquitous process of Ca2+-regulated exocytosis but were also essential for normal cell membrane
resealing.
During the past few years, a great deal has been learned
about vesicle docking and fusion mechanisms (Sudhof et
al., 1993; Bennett and Scheller, 1994
; De Camilli, 1995
) in
Ca2+-regulated exocytosis. However, much less is known
about the recruitment of vesicles in the regulated exocytotic pathway (Scholey, 1996
; Vallee and Sheetz, 1996
). In
these experiments, the distinct temporal patterns of inhibition of exocytosis by reagents targeting two different
motor systems are consistent with two sequential motor-driven recruitment steps for vesicle exocytosis
a kinesin-mediated transport step followed by an actomyosin-based
step. Fig. 7 B summarizes the essential features of this
transport/exocytosis pathway. This picture is also consistent with the fact that astral microtubules extend from the
cell center to the cell periphery, while short actin filaments
form networks near the cell membrane (Hollenbeck and
Cande, 1985
), and with the coexistence of kinesin-microtubule and actomyosin motor systems on the same organelles (Fath et al., 1994
; Morris and Hollenbeck, 1995
).
Similar mechanisms have been postulated in other systems
based on elegant in vitro observations (Fath et al., 1994
;
Allan, 1995
; Langford, 1995
). Our results, however, provide in vivo evidence for this postulated two-step pathway
in general vesicular trafficking. Furthermore, our results also indicate that the recruitment of an average vesicle
may need about 20 s of kinesin transport and about 5-10 s
of myosin transport. Translating into spatial measures,
these numbers correspond to about 20 µm of microtubule
track and about 5-10 µm of actin filament track, assuming
the average motor speeds are both around 1 µm/s (Vale et
al., 1985b
; Porter et al., 1987
; Langford et al., 1994
; Bearer
et al., 1996
). Considering that the cell size is ~40 µm in diameter for a four-cell stage embryo, there is enough time to recruit vesicles from any intracellular location to the
cell surface.
Conventional kinesin is the most abundant member in
the large family of KRPs. In sea urchin embryonic cells,
there is at least 10-fold more kinesin than the next most
abundant KRP, kinesin-2. (Scholey, J., unpublished observation). Conventional kinesin associates with the vesicles
in the mitotic spindle asters (Wright et al., 1991) but is apparently not essential for mitosis (Wright et al., 1993
). Because SUK4 is specific for kinesin and does not cross-react with any other known KRPs, and more importantly, because the KHC tail domain is variable among different
KRPs, the inhibition of exocytosis by SUK4 and KHC
stalk-tail domain strongly suggests that the conventional
kinesin, rather than any other KRPs, is the protein motor
for vesicle transport in regulated exocytosis. In addition,
these results clearly demonstrate that the variable tail domain is responsible for in vivo vesicle cargo binding, as has
been suggested based on in vitro observations (Skoufias et
al., 1994
; Bloom and Endow, 1995
; Vallee and Sheetz,
1996
). Kinectin, an integral membrane protein of the endoplasmic reticulum, has been identified as a membrane
anchor protein for kinesin in the chick brain (Kumar et al.,
1995
). A similar protein may be responsible for the KHC
tail binding in our system.
The myosin motor protein family consists of many structural and functional distinct isoforms (Hasson and Mooseker, 1995). Which isoforms may be involved in vesicular
transport is still an open question (Hasson and Mooseker,
1995
). BDM inhibits many myosin isoforms and could also
have other effects. Possible complications from BDM effects on membrane conductance do not play a role in our
preparation since Ca2+ influx is directly from a membrane
wound. By having the advantage of direct visualization of
regulated exocytotic events, our system provides a relatively simple in vivo assay that may help more explicitly
identify the myosins responsible for vesicle recruitment when more specific reagents become available.
The inhibition of CaM kinase II has a similar effect on
exocytosis as BDM, and this encourages us to believe both
are affecting an actin-based step in vesicle delivery. However, the inhibitory effects we observed on exocytosis,
while consistent with interruption of vesicle transport at
the myosin-actin-dependent step, cannot be conclusive.
CaM kinase II has been shown to be able to phosphorylate a number of targets in docking/fusion: synaptobrevin, synaptotagmin, and rabphilin-3A (Popoli, 1993; Fykse et al.,
1995
; Nielander et al., 1995
; Hirling and Scheller, 1996
;
Popoli et al., 1997
). Any of these proteins might conceivably be a target, and their phosphorylation by CaM kinase
II may yet be shown to play a regulatory role. However, at
the moment, there is no evidence suggesting that these
phosphorylations must precede successful docking and fusion. More telling, in our results when we specifically
tested the block of CaM kinase on previously docked cortical granules, it had no effect on the fusion step of exocytosis. The most abundant synaptic phosphoprotein, synapsin and its homologues, are more likely to be the targets
affected by CaM kinase II in vesicle recruitment for exocytosis. Double knockouts of synapsin I and II exhibited decreased posttetanic potentiation and severe synaptic depression upon repetitive stimulation (Rosahl et al., 1995
). Peptide inhibitors of CaM kinase II preferentially suppressed the phosphorylation of synapsin I at the CaM kinase II-specific site and decreased excitatory synaptic responses elicited in the CA1 region of hippocampal slices
(Waxham et al., 1993
). These inhibitory effects on exocytosis could be explained by deficient recruitment of vesicles to the active zone, in line with the need for phosphorylation of synapsin by CaM kinase II to free vesicles from
actin (Ceccaldi et al., 1995
).
The three vesicle pools identified by the action of blockers of kinesin function and BDM are closely related to the
two-step transport mechanism. As shown in Fig. 7, the immediate pool represents vesicles that can exocytose when
transport mechanisms are blocked. These vesicles must
have passed the recruitment steps and are functionally
docked on the plasma membrane, ready to fuse in response to Ca2+ influx. The fast pool, on the other hand,
should consist of vesicles that have just passed the kinesin
transport step but still require myosin-mediated transport
before docking and fusion. The slow pool is the most upstream in this trafficking pathway. These vesicles must be
first transported via a kinesin-microtubule system and then via an actomyosin system before approaching the
plasma membrane. Recent studies on the nerve termini
have defined vesicle pools using different criteria (Borges
et al., 1995; Pieribone et al., 1995
; Rosahl et al., 1995
;
Stevens and Tsujimoto, 1995
; Rosenmund and Stevens,
1996
). The time constant for replenishment of the readily
releasable pool at nerve termini is also about 10 s (Stevens and Tsujimoto, 1995
). It will be interesting to know how
much of the vesicle recruitment scheme described above is
applicable to neuronal transmission. One study found that
a smooth muscle antimyosin II antibody microinjected
into presynaptic neurons inhibited synaptic transmission;
however, the antibody fraction with this activity is no
longer available (Mochida et al., 1994
).
The fast pool, which we are labeling myosin dependent,
must be relatively stable in the absence of Ca2+ influx because it would otherwise have been depleted when kinesin transport was blocked. Therefore, the myosin-mediated
transport is not constitutively active but is probably turned
up by intracellular Ca2+ elevation. This may be accomplished, at least in part, by the activation of CaM kinase II
since BDM and CaM kinase II autoinhibitory peptide disrupt at the fast pool step. Likewise, the kinesin-mediated transport should also be activated by Ca2+ influx through
the wounding because otherwise the vesicle pools after the
kinesin transport step would have accumulated and would therefore be much larger in the control cases than in the
cases with SUK4 or KHC stalk-tail injection. The nature
and kinetics of the regulation for either transport system
are not clear. However, Ca2+ ions appear to play a key
role in both steps because it is probably the first signal the
cell can receive after membrane wounding. This is also
consistent with the observation of multiple calcium-dependent processes related to secretion in bovine chromaffin
cells (Neher and Zucker, 1993).
Finally, this study adds to our knowledge of the process
that reseals disrupted cell membrane. Recent experiments
have indicated that Ca2+-regulated exocytosis is a ubiquitous process in many cell types (Dan and Poo, 1992; Steinhardt et al., 1994
; Bi et al., 1995
; Girod et al., 1995
; Coorssen et al., 1996
; Rodriguez et al., 1997
) and is essential for
cell membrane resealing (Steinhardt et al., 1994
; Bi et al.,
1995
; Miyake and McNeil, 1995
). Although the detailed
physical picture of the resealing process is not clear, it has
been demonstrated that the addition of membrane lipids proximal to the wound site by vesicle exocytosis is necessary for the final "closure" of wounded membrane (Bi et
al., 1995
). The inhibition of membrane repair by block of
CaM kinase II (Steinhardt et al., 1994
) is now directly correlated with the inhibition of exocytosis. In addition, consistent with our earlier results (Steinhardt et al., 1994
), we
found that cells could not reseal when their kinesin transport was inhibited (Table I), even though the early phases
of exocytosis were intact under the same conditions (Figs. 2-4). It therefore appears that exocytosis must be sustained and prolonged by the active transport of vesicles to
successfully reseal a disrupted membrane and ensure cell
survival.
Received for publication 19 December 1996 and in revised form 26 June 1997.
Address all correspondence to Richard A. Steinhardt, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200. Tel.: (510) 642-3517. Fax: (510) 643-6791. E-mail: rick{at}mendel.berkeley.eduWe thank A. Tieu and D. MacDermed for technical assistance.
This work was supported by the Committee on Research of the University of California, Berkeley, the National Institutes of Health, and the American Cancer Society.
BDM, 2,3-butanedione monoxime; CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; KHC, kinesin heavy chain; KRP, kinesin-related protein.
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