Proc. Nati. Acad. Sci. USA
Vol. 87, pp. 1061-1065, February 1990
Neurobiology
A monoclonal antibody against kinesin inhibits both anterograde
and retrograde fast axonal transport in squid axoplasm
(fast axonal transport/microtubules/cytoplasmic dynein)
SCOrr T. BRADY*t*, K. KEVIN PFISTER*, AND GEORGE S. BLOOM*t
*Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9039;
and tMarine Biological Laboratory, Woods Hole, MA 02543
Communicated by Keith R. Porter, June 21, 1989
Since discovery of the mechanochemical ATPase kinesin (1,
2), many different physiological functions have been suggested. While kinesins have been proposed to mediate translocation of membrane-bounded organelles (MBOs), cytoskeletal and chromosome movements, secretion, and reorganization of the endoplasmic reticulum (3), these suggestions
have remained at the level of speculation. The ability of
kinesin to mediate gliding of microtubules (MTs) across a
glass coverslip (2, 4) indicated that kinesin could do work, but
the physiological correlate of this phenomenon was uncertain. The most widely accepted role for kinesin has been as
the motor for fast anterograde axonal transport. However,
despite intensive study in a number of laboratories, the
evidence for involvement of kinesin in fast transport or in
other forms of in vivo motility has remained indirect and
correlative.
Direct evidence for involvement of kinesin in cellular
motility could take several forms. One obvious approach
would be reconstitution of the physiological process with
well-characterized constituents. For MBO translocation
along MTs, suitable purified constituents would consist of
MTs assembled without MT-associated proteins, a wellcharacterized fraction of MBOs, and highly purified kinesin.
Alternatively, direct evidence could be provided by suitable
immunochemical probes that inhibit kinesin function. Appropriate antibodies could interfere with kinesin function by
inhibiting ATPase activity, binding of kinesin to either MTs
or MBOs, or conformational changes associated with translocation. A similar approach utilizing myosin-specific antibodies provided direct evidence for involvement of myosin in
cell morphology, locomotion, and cytokinesis, but not chromosome movement (5-7).
While kinesin has been purified to >90% homogeneity (8)
and shown to be a MT-stimulated ATPase (1, 8, 9), the goal
of recombining kinesin with MBOs remains elusive. Recombination studies have proved to be technically difficult, and
little has been known about interactions of kinesin with
MBOs. Surprisingly, direct evidence that kinesin is associated with MBOs in vivo only recently has been obtained (10).
Initially, evidence for kinesin association with MBOs was
inferred from observations that the nonhydrolyzable analogue of ATP, 5'-adenylyl imidodiphosphate (p[NH]ppA;
also called AMP-PNP), caused both vesicles (11) and kinesin
(1, 2) to bind to MTs. Direct evidence for kinesin association
with MBOs has now been obtained by an immunological
approach using our library of monoclonal antibodies to heavy
and light chains of bovine kinesin (10). Unlike previously
described antibodies to the kinesin heavy chain (12-16),
which provided conflicting images for subcellular localization
of kinesin, immunofluorescence studies with our library of
five monoclonal antibodies established kinesin association
with MBOs. All five antibodies (to heavy and light chains)
produce similar, detergent-sensitive, punctate immunofluorescence patterns identified as MBOs in a variety of vertebrate cell types (10). Using the same antibodies, we have
shown kinesin to copurify with MBOs (unpublished observations).
Isolated axoplasm from squid giant axons has proved to be
a powerful model for study of the molecular mechanisms of
fast axonal transport (17, 18). The absence of a plasma
membrane permits introduction of defined amounts of proteins and antibodies that do not readily enter intact cells or
axons, while retaining the native organization and concentration of cytoplasmic components (18-20). One of our
antibodies to bovine kinesin heavy chain (H2) crossreacted
with squid kinesin heavy chain, raising the possibility that H2
could be used as a probe of both the distribution and
physiological function of kinesin in isolated axoplasm. The
amount of kinesin in axoplasm was determined from quantitative immunoblots with H2. Immunofluorescent studies of
The publication costs of this article were defrayed in part by page charge
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Abbreviations: MBO, membrane-bounded organelle; MT, microtubule; p[NH]ppA (AMP-PNP), 5'-adenylyl imidodiphosphate.
1To whom reprint requests should be addressed.
One of our monoclonal antibodies against the
ABSTRACT
heavy chain of bovine kinesin (H2) also recognized the heavy
chain of squid kinesin. The immunofluorescence pattern of H2
in axoplasm was similar to that seen in mammalian cells with
antibodies specific for kinesin light and heavy chains, indicating that squid kinesin is also concentrated on membranebounded organeiles. Although kinesin is assumed to be a motor
for translocation of membrane-bounded organelles in fast
axonal transport, direct evidence has been lacking. Perfusion
of axoplasm with purified H2 at 0.1-0.4 mg/ml resulted in a
profound inhibition of both the rates and number of organelles
moving in anterograde and retrograde directions in the interior
of the axoplasm, and comparable inhibition was noted in
bidirectional movement along individual microtubules at the
periphery. Maximal inhibition developed over 30-60 min.
Perfusion with higher concentrations of H2 (>1 mg of IgG per
ml) were less effective, whereas perfusion with 0.04 mg of H2
per ml resulted in minimal inhibition. Movement of membranebounded organelles after perfusion with comparable levels of
irrelevant mouse IgG (0.04 to >1 mg/ml) were not distinguishable from perfusion with buffer controls. Inhibition offast
axonal transport by an antibody specific for kinesin provides
direct evidence that kinesin is involved in the translocation of
membrane-bounded organelles in axons. Moreover, the inhibition of bidirectional axonal transport by H2 raises the
possibility that kinesin may play some role in both anterograde
and retrograde axonal transport.
1061
1062
Neurobiology: Brady et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
squid axoplasm resulted in punctate patterns homologous
with those seen in mammalian cells. Perfusion of H2 into
axoplasm inhibited the rate and number of particles moving
in fast axonal transport. Surprisingly, both anterograde and
retrograde transport were inhibited to a similar extent, in
contrast with in vitro evidence that kinesin is involved only
in anterograde transport (15). Collectively, these observations represent direct evidence that kinesin is involved in the
translocation of MBOs in neurons.
I-j 2
P
sulfate/polyacrylamide gel electrophoresis (SDS/PAGE)
was run as described (22). Protein was determined by the
Bradford assay (23). Samples for immunofluorescence were
fixed in 5% acetic acid/95% ethanol for 10 min at -200C and
then rinsed in Tris-buffered saline (20 mM Tris HCl, pH
7.4/150 mM NaCl) before incubation with primary antibody.
Rhodamine-labeled goat antimouse IgG (Fisher Scientific)
was the second antibody, and p-phenylenediamine was used
in the mounting medium to reduce photobleaching (24).
Chemicals and reagents were obtained from Sigma unless
specified otherwise.
RESULTS
A library of mouse monoclonal antibodies raised against
bovine brain kinesin (10) were checked for cross-reactivity
with kinesin-containing MT pellets prepared by using
p[NH]ppA and squid optic lobe extracts. One antibody to
bovine kinesin heavy chain, H2, recognized squid kinesin
heavy chain in p[NH]ppA pellets (Fig. 1 Left). To determine
specificity of H2 in squid, immunoblots of unfractionated
axoplasm were screened. The H2 antibody recognized a
single polypeptide in squid axoplasm corresponding to squid
kinesin heavy chain (Fig. 1 Right). H2 had the widest
I 1
I.
METHODS AND MATERIALS
Axoplasm and Video Microscopy. Isolated axoplasm was
extruded from giant axons of the squid Loligo pealed (Marine
Biological Laboratory, Woods Hole, MA). Preparations for
video microscopy were as described (17, 18). Axons used for
video microscopy were 400-600 ,Am in diameter, and extrusions typically yielded =5 1.l of axoplasm. Actual volumes of
axoplasm in each experiment were calculated from axoplasm
sizes, and 4 volumes of antibody were perfused into the
chamber. Video microscopy used a Zeiss Axiomat with a
x 100, 1.3 n.a. objective and differential interference contrast
optics, with the zoom lens set for a screen width of 23.8 ,um.
The video processing system was a Photonics Microscopy
C-1966 and Chalnicon camera. Velocity was measured with
a Photonics Microscopy C2117 videomanipulator by matching the speed of cursors moving across the screen to the
particle movements. All procedures were conducted at room
temperature unless indicated otherwise.
Immunological Studies. A full characterization of the antibodies has been published (10, 21). H2 is an IgG2b monoclonal antibody that recognizes the full range of bovine
kinesin heavy chain isoforms. Homogeneous IgG2b was
purified from ascites fluid by using (i) protein A-Superose
chromatography (Pharmacia) (10) or (it) the MASS protein
A-based filter system (Nygene; Yonkers, NY). Before perfusion, purified IgG was dialyzed against buffer X2 [10 mM
Hepes, pH 7.2/150 mM potassium aspartate/5 mM MgCl2/1
mM EGTA]. Immunoblots were visualized by peroxidaselabeled goat anti-mouse IgG (Cappel Laboratories) second
antibody with 4-chloro-1-naphthol as the chromogen (10).
Some immunoblots were scanned with an LKB laser scanning densitometer to quantitate the kinesin. A serial dilution
of purified kinesin was run on the same gel. All lanes were
coincubated with primary and then secondary antibodies and
were batch-developed to visualize kinesin. Sodium dodecyl
AV\
-.m,
.ii :
:.
T
....
FIG. 1. Recognition of squid kinesin heavy chain by an antibody
raised against bovine kinesin heavy chain. (Left) Lane P shows
polypeptide composition of a 1 mM p[NH]ppA pellet obtained by
centrifugation of a squid optic lobe extract and taxol-stabilized MTs
in SDS/PAGE. The Coomassie blue pattern indicates the presence
of squid kinesin heavy chain (120 kDa). In immunoblots of a similar
gel lane (H2), a single band corresponding in molecular mass to squid
kinesin heavy chain was recognized in the squid p[NH]ppA pellet by
H2, a monoclonal antibody against bovine kinesin heavy chain. The
positions of tubulin (T) and a series of molecular mass standards (205,
116, 97, 66, 45, and 29 kDa) are indicated. (Right) To confirm
specificity of H2 in squid, axoplasmic protein was immunoblotted.
Lane AX shows the Coomassie blue-stained pattern for 16 ,ug of
axoplasmic protein, and lane H2 is the immunoblot of a lane
containing 80 ,g of axoplasmic protein probed with H2 antibody. A
single band corresponding in molecular mass to squid kinesin heavy
chain is seen in the immunoblot of axoplasm. Similar immunoblots
were scanned by laser densitometry to determine the concentration
of kinesin in squid axoplasm.
cross-reactivity with kinesins from different sources and
appeared to recognize the full complement of isoforms for
bovine kinesin heavy chain (8, 10).
The concentration of kinesin in squid axoplasm was determined by laser densitometry of quantitative immunoblots.
Defined amounts of axoplasm were probed with H2 on blots
that contained dilutions of bovine kinesin covering the linear
range for densitometry. Quantitative immunoblots gave a
kinesin concentration in squid axoplasm of =0.5 uM. By
comparison, tubulin concentration in axoplasm is -4 /.M
(25), giving a molar ratio of 1:8 for kinesin to tubulin in squid
axoplasm.
Immunocytochemical localization of kinesin in squid axoplasm by indirect immunofluorescence with H2 (Fig. 2) gave
a pattern similar to that seen in mammalian cells with our
antibodies specific for kinesin light and heavy chains. The
kinesin-positive structures form a characteristically punctate
pattern (Fig. 2A). No specific staining was seen either with
control IgG (Fig. 2B) or with H3, an antibody that does not
recognize squid kinesin in immunoblots (not shown). Immunoreactive structures often formed linear arrays similar to
Neurobiology: Brady et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
1063
FIG. 2. Subcellular location of kinesin in squid axoplasm by indirect immunofluorescence with H2. (A) A punctate pattern of fluorescence
characteristically obtained with H2 in isolated axoplasm, comparable in appearance to the pattern seen in mammalian tissue culture cells
(10). Punctate structures often appear in linear arrays that are thought to represent MBOs attached to MTs. (B) A similar region of isolated
axoplasm, processed and photographed as in A, with purified control IgG as the primary antibody. No specific staining of axoplasm was obtained
either with control IgG or with H3, a monoclonal antibody to bovine kinesin that did not cross-react with squid kinesin.
was
in tissue culture cells. Double-label studies indiare aligned along MTs (10). We
assume, therefore, that kinesin-positive structures in axoplasm are MBOs aligned along MTs. As in vertebrate cells,
immunocytochemical studies of squid axoplasm indicated
that kinesin is associated with MBOs.
The effects of H2 on fast axonal transport were evaluated
by perfusion of axoplasm. Concentrations of H2 between 0.1
and 0.6 mg/ml resulted in a profound inhibition in the rates
and number of MBOs moving in both anterograde and
retrograde directions (see Table 1 and Fig. 3). Inhibition of
bidirectional transport was comparable at the axoplasm periphery along individual MTs and in the interior of the
axoplasm. In both regions, inhibition by H2 had a characteristic time course (Fig. 3). An initial =25% reduction in
velocity was seen in the first 10 min, followed by a second 30to 60-min phase of increasing inhibition. At 60 min and
optimal antibody concentrations, velocities were typically
reduced by 60-70%o. The extent and timing of inhibition were
similar but not identical in the axoplasm interior and on
isolated peripheral MTs; reductions in velocity became apparent somewhat later in the interior rather than on the
periphery, presumably due to the time required for H2
diffusion into axoplasm (18, 20). Inhibition was maximal by
30-60 min throughout the preparation. Optimal inhibition by
H2 occurred at molar ratios of antibody/kinesin of -1-5,
assuming 0.5 ,uM kinesin. Perfusion with higher H2 concentrations (7-10 ,uM IgG) were less effective at reducing rates,
while perfusion with H2 < 0.3 ,uM was ineffective. No effect
on fast axonal transport was seen for similar concentrations
of control IgG (Table 1 and Fig. 3).
The biochemical basis of H2 inhibition was evaluated by in
vitro assay systems for MT-stimulated ATPase of kinesin and
nucleotide-sensitive binding of kinesin to MTs. Molar ratios
of H2 to kinesin from 1 to 5 did not inhibit the MT-stimulated
ATPase (M. Wagner, S.T.B., K.K.P., and G.S.B., unpublished data), although higher molar ratios (60- to 110-fold
excess of antibody) partially inhibited ATPase activity. Similarly, kinesin-H2 complexes bound to MTs in the presence
of p[NH]ppA, and H2-kinesin complexes could be used to
decorate MTs in vitro (data not shown). These results are
consistent with video microscopy in which MBOs slowed or
stopped but were not displaced from MTs. The stoichiometry
of H2 to kinesin required for effective inhibition and the time
course of inhibition suggest that H2-crosslinked adjacent
those
seen
cate that linear arrays
kinesins on the MBO surface, analogous to immunoprecipitation, were responsible for fast axonal transport inhibition.
DISCUSSION
Discovery of the kinesins, a family of mechanochemical
ATPases distinct from myosin and dynein, represented a
beginning for understanding molecular mechanisms of fast
Table 1. Effects of antibody perfusion on velocity
Transport velocity,
,um/sec
Velocity
RetroExp., on fibrils,* AnteroAntibody
gradet
gradet
no.
/Lm/sec
(mg/ml)
11 0.74 ± 0.23 0.78 ± 0.16 0.78 ± 0.16
H2 (0.1-0.6)
(n =6)
(n =6)
(n =35)
0.9
1.19
2 1.28 ± 0.04
H2 (>1)
(n= 1)
(n =1)
(n =3)
3 1.56 ± 0.06 1.31 ± 0.05 1.16 ± 0.09
H2 (0.04)
(n= 3)
(n =3)
(n =8)
1.19
1.78
6 1.89 ± 0.01
IgG (0.1-1.0)
(n 1)
(n =1)
(n= 12)
2.21 ± 0.02 1.79 ± 0.10 1.09 ± 0.05
Control axoplasm
(n 16)
(n= 16)
(n =9)
(- IgG, + 2 mM
ATP) (10-30 min)
Velocity was measured by matching the rate of calibrated cursor
movements to the velocity of multiple particles for each determination. Measurements of velocity on MTs represent values for bidirectional movement on individual MTs at the axoplasm periphery.
Anterograde and retrograde moving particles on MTs are combined,
because no reliable markers for MT polarity in the periphery are
available. When bidirectional transport was seen on a single MT,
velocities in both directions were similarly affected. All rates were
obtained 40-60 min after perfusion with the antibody solution.
*Measurements of velocities on fibrils include data from individual
MBOs moving in either direction on MTs in randomly selected
fields of view. Velocities indicated were measured for a number of
MBOs moving along a MT by two or more individuals (n = number
of microtubules for which MBO movement was determined).
tVelocities for anterograde and retrograde transport represent average velocities for the population of vesicles moving in each
direction in randomly selected fields of view in the axoplasm
interior. Each velocity measurement (n = number of velocity
measurements) is an average of three to five independent measurements by two or more individuals. This method permits velocity
measurements for large numbers of MBOs.
1064
Neurobiology: Brady et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
1.500
rb1.000
0. 500
0.000
0
10
1
20
1.
1
30
40
50
60
Min after Perfusion
FIG. 3. Time course for inhibition: inhibition of fast axonal
on individual MTs in the axoplasm periphery following
perfusion with H2. Results of three experiments (o, *, and A) are
shown. Aliquots of H2 ranging in concentration from 0.1 to 0.6
mg/ml in buffer X2 were perfused into axoplasm at a H2/kinesin
molar ratio of 1-5. Velocities typically were reduced to 75% of
control values within the first 10 min. Over the course of 60 min,
velocities continued to decline until they were 30-40% of control
values. By contrast, velocities at 60 min after perfusion with control
IgG (X) were not significantly different from buffer controls. The
time course and stoichiometry of H2 inhibition of fast "xonal
transport suggest that H2 crosslinks kinesin molecules on the surface
of MBOs. Both anterograde and retrograde transport of organelles
were affected similarly (see Table 1).
transport
axonal transport. Despite a substantial amount of correlative
evidence, defining the role of kinesin in intracellular motility
proved elusive. Moreover, the discovery of cytoplasmic
dyneins (26-29) complicated interpretation of pharmacological assays and in vitro studies implicating kinesin as the
motor in fast axonal transport. To understand the physiological roles of kinesin, direct evidence for kinesin involvement
in axonal transport or other physiological prqcesses is
needed. Such direct evidence has two components- (i) kinesin
must have an appropriate subcellular location and (it) molecular (biochemical, pharmacological, or immunochemical)
probes specific for kinesin must inhibit the physiological
activity. Antibodies specific for kinesin polypeptides have
the potential to satisfy both criteria.
Our library of monoclonal antibodies (10) constitutes an
important set of tools for exploring the role of kinesin in many
forms'of cell motility. The specificity of these antibodies,
each recognizing a unique epitope, has been demonstrated on
both purified kinesin and cellular extracts. Ultrastructural
localization of epitopes for these antibodies on kinesin documented specificity and provided details on the molecular
architecture of kinesin (21). Immunocytochemical studies
with' the antibodies showed kinesin localized on MBOs in
both neuronal and nonneuronal cells in culture (10). Kinesin
was notably absent from cytoskeletal structures, suggesting
that its primary association is with MBOs. The size, distribution, and number of kinesin-positive structures in squid
axoplasm (see Fig. 2) and other cell types are consistent with
kinesin being localized on transported MBOs. Thus, kinesin
has the appropriate cellular and subcellular location to be a
motor for fast axonal transport.
Once reactivity of H2 with squid kinesin was demonstrated, theeffects of H2 on fast axonal transport in axoplasm
could be evaluated. Perfusion of suitable concentrations of
H2 into axoplasm inhibited both anterograde and retrograde
axonal transport to a similar extent with a similar time course.
Thus, studies with H2 satisfy both criteria for direct evidence
that kinesin is involved in translocation of MBOs along MTs.
Comparisons between the axoplasm periphery, where transport of MBOs can be followedvalong individual MTs, and the
axoplasm interior, where axoplasmic organization was retained, were particularly instructive. The extent of inhibition
was similar in both regions, although initial declines in
velocity occurred later in the interior than the periphery. By
30-60 min, transport was inhibited throughout the preparation, so inhibition was not limited by restricted access of
antibody to the interior. Furthermore, while inhibition in the
interior might have been due to nonspecific antibody
crosslinks restricting passage of MBOs through the axoplasmic matrix, reduced velocities on isolated MTs at the periphery effectively rule out this possibility. Invariably, both
anterograde and retrograde transport were inhibited to a
similar extent on individual MTs at the periphery and in the
interior (see below). Inhibition of bidirectional transport does
not appear to require axoplasmic components other than
kinesin, MTs, and MBOs.
Although the percent reduction in velocity after H2 treatment is greater for anterograde than retrograde-moving particles (see Table 1), final velocities at maximum inhibition are
indistinguishable. Since anterograde and retrograde velocities on individual MTs in the periphery were comparable,
retrograde transport rates appear limited by steric constraints
imposed by the axoplasmic matrix on the larger, retrogrademoving MBOs (17, 18). This interpretation is consistent with
observations that differences between anterograde and retrograde velocities in the interior became less apparent after
inhibition by H2 and that transport rates for both directions
were comparable on isolated MTs in both H2 and control IgG
treatments. While modest differences in effect cannot be
excluded, available facts suggest that H2 actions on anterograde and retrograde transport were comparable.
Previous studies with other antibodies to kinesin (12, 14,
15) had not inhibited either gliding of MTs or MBO transport
along MTs. However, several monoclonal antibodies to sea
urchin kinesin were recently described that inhibit MT gliding
(30, 31). However, none of these crossreact with preparations
suitable for analyzing MBO translocation along MTs. Antibodies to other motors and structural proteins have produced
variable results. Antibodies that interfere with myosin activity inhibit cell locomotion (5, 32) and cytokinesis (6, 7) but not
translocation of MBOs in most animal cell types. With one
exception, antibodies against tubulin have not interfered with
MBO transport. Interestingly, an affinity-purified tubulin
antibody that inhibited MBO transport reduced bidirectional
transport (19). No reports have appeared to date of antibodies
against cytoplasmic dyneins suitable for immunocytochemical or functional studies. Thus, little is known at
present about the subcellular distribution of cytoplasmic
dyneins, and hypotheses regarding the precise roles they play
in motility in vivo remain dependent on correlative in vitro
studies. Perhaps the best evidence to date for dynein-based
organelle transport is in Reticulomyxa (28, 33), which has a
dynein-like ATPase but apparently lacks a kinesin homologue. In this system, the dynein-like ATPase may mediate
bidirectional MBO movements (33).
Several mechanisms of action might be invoked to explain
H2 inhibition of fast axonal transport: (i) H2 could inhibit
MT-stimulated ATPase, (it) H2 might interfere with kinesin
binding to MTs, or (iii) H2 could sterically interfere with
mechanochemical movements by crosslinking adjacent kinesins. Since addition of H2 at antibody/kinesin molar ratios of
1-5 had no detectable inhibition on the MT-stimulated ATPase
of bovine kinesin and kinesin/H2 complexes still exhibit
nucleotide-sensitive binding to MTs, the first two mechanisms
are unlikely. The third suggestion is, however, consistent with
available experimental evidence. Both the time course for
reducing MBO velocities and the incomplete blockade of
transport are compatible with a process analogous to kinesin
Neurobiology: Brady et al.
immunoprecipitation. This mechanism is plausible, since kinesin contains two heavy chains (22) and two binding sites for
H2. Thus, the effect of H2 on fast axonal transport may result
from steric constraints on conformational changes in kinesin
associated with mechanochemical activity.
Inhibition of bidirectional transport by H2 is not consistent
with previous suggestions that kinesin was involved only in
anterograde transport (15). Earlier suggestions were based on
the fact that MT gliding mediated by kinesin was unidirectional (4, 15). Subsequently, cytoplasmic dynein was found to
produce MT gliding in the opposite direction, leading to
proposals that cytoplasmic dynein was the motor for retrograde transport (27). A number of biochemical and pharmacological differences have been noted between gliding due to
kinesin (4, 15) and that due to cytoplasmic dynein (27),
including rates of gliding, sensitivity to N-ethylmaleimide,
nucleotide specificity, and vanadate sensitivity. However,
studies of fast axonal transport do not exhibit these same
differences between anterograde and retrograde movements.
Pharmacological studies of fast transport in axoplasm using
a variety of agents expected to differentiate between kinesinmediated and dynein-mediated transport, including p[NH]ppA (11), erythro-9-[3-(2-hydroxynonyl)]adenine (18), Nethylmaleimide (34), and vanadate (unpublished observations)
failed to differentiate clearly between anterograde and retrograde transport in axoplasm. The effects of these agents on
MBO transport in axoplasm differ quantitatively and qualitatively from MT gliding because of either kinesin or dynein.
Finally, electron microscopic morphometric analyses of cross
bridges between MBOs and MTs indicated that cross bridges
on anterograde- and retrograde-moving MBOs were indistinguishable (35). Although the hypothesis that kinesin moves
MBOs anterogradely and dynein moves them retrogradely
remains attractive, other models are also consistent with
available evidence. A number of questions must be answered
before a specific model can be considered proven.
To determine which motors are involved in anterograde
and retrograde axonal transport, the identity of kinesinpositive MBOs and the presence of other putative motors
must be established. Preliminary experiments aimed at determining whether kinesin is associated with anterogradeand retrograde-moving MBOs suggest that kinesin may be
present on MBOs moving in both directions. Similar studies
must establish whether cytoplasmic dynein is also associated
with MBOs and determine the identity of these MBOs. Until
immunological or pharmacological reagents with suitable
properties can be obtained, the question of the roles played
by kinesin and by cytoplasmic dynein in fast axonal transport
will remain uncertain.
In summary, the role of kinesin in the intracellular transport of MBOs has been explored by using H2, an antibody
specific for kinesin heavy chain. Inhibition of fast axonal
transport by H2, when combined with localization of kinesin
on MBOs, provides direct evidence that kinesin is involved
in axonal transport of MBOs. The fact that bidirectional
transport is inhibited by H2 raises the possibility that kinesin
may play a role in both anterograde and retrograde axonal
transport.
Note. Video sequences illustrating the effects of control
IgG and H2 antibody on transport of membrane-bounded
organelles are to be included in the second video supplement
of "Cell Motility and the Cytoskeleton," scheduled for
publication in 1989.
This work was supported in part by grants from the National
Institutes of Health (NS 23320 to S.T.B. and NS23868 to S.T.B. and
Proc. NatL. Acad. Sci. USA 87 (1990)
1065
G.S.B.), from the Welch Foundation (1-1077 to G.S.B. and S.T.B.),
from the National Science Foundation Biological Instrumentation
Program (DMB-8701164 to G.S.B. and S.T.B.), and by a National
Institutes of Health Postdoctoral Fellowship Award (GM 10143 to
K.K.P.).
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Brady, S. T. (1985) Nature (London) 317, 73-75.
Vale, R., Reese, T. & Sheetz, M. (1985) Cell 42, 39-50.
Vale, R. (1987) Annu. Rev. Cell Biol. 3, 347-378.
Porter, M. E., Scholey, J. M., Stemple, D. L., Vigers,
G. P. A., Vale, R. D., Sheetz, M. P. & McIntosh, J. R. (1987)
J. Biol. Chem. 262, 2794-2802.
Honer, B., Citi, S., Kendrick-Jones, J. & Jockusch, B. M.
(1988) J. Cell Biol. 107, 2181-2189.
Kiehart, D. P., Mabuchi, I. & Inoue, S. (1982) J. Cell Biol. 94,
165-178.
Mabuchi, I. & Okuno, M. (1977) J. Cell Biol. 74, 251-263.
Wagner, M. C., Pfister, K. K., Bloom, G. S. & Brady, S. T.
(1989) Cell Motil. Cytoskel. 12, 195-215.
Kutznetsov, S. & Gelfand, V. (1986) Proc. Natl. Acad. Sci.
USA 83, 8530-8534.
Pfister, K. K., Wagner, M. C., Stenoien, D. S., Brady, S. T. &
Bloom, G. S. (1989) J. Cell Biol. 108, 1453-1463.
Lasek, R. J. & Brady, S. T. (1985) Nature (London) 316,
645-647.
12. Neighbors, B. W., Williams, R. C. & McIntosh, J. R. (1988)J.
Cell Biol. 106, 1193-1204.
13. Leslie, R. J., Hird, R. B., Wilson, L., McIntosh, J. R. &
Scholey, J. M. (1987) Proc. Natl. Acad. Sci. USA 84, 27712775.
14. Scholey, J. M., Porter, M. E.,.Grissom, P. M. & McIntosh,
J. R. (1985) Nature (London) 318, 483-486.
15. Vale, R. D., Schnapp, B. J., Mitchison, T., Steuer, E., Reese,
T. S. & Sheetz, M. P. (1985) Cell 43, 623-632.
16. Murofushi, J., Ikai, A., Okuhara, K., Kotani, S., Aizawa, H.,
Kumakura, K. & Sakai, H. (1988) J. Biol. Chem. 263, 1274412750.
17. Brady, S. T., Lasek, R. J. & Allen, R. D. (1982) Science 218,
1129-1131.
18. Brady, S. T., Lasek, R. J. & Allen, R. D. (1985) Cell Motil. 5,
81-101.
19. Johnston, K. T., Brady, S. T., van der Kooy, D. & Connolly,
J. A. (1987) Cell Motil. Cytoskel. 8, 155-164.
20. Brady, S. T., Lasek, R. D., Allen, R. D., Yin, H. & Stossel, T.
(1984) Nature (London) 310, 56-58.
21. Hirokawa, N., Pfister, K. K., Yorifuji, H., Wagner, M. C.,
Brady, S. T. & Bloom, G. S. (1989) Cell 56, 867-878.
22. Bloom, G. S., Wagner, M. C., Pfister, K. K. & Brady, S. T.
(1988) Biochemistry 27, 3409-3416.
23. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.
24. Johnson, G. D. & Nogueira Araujo, G. (1981) J. Immunol. Res.
43, 349-350.
25. Morris, J. R. & Lasek, R. J. (1984) J. Cell Biol. 98, 2064-2076.
26. Paschal, B., Shpetner, H. & Vallee, R. (1987) J. Cell Biol. 105,
1273-1282.
27. Paschal, B. M. & Vallee, R. B. (1987) Nature (London) 330,
181-183.
28. Euteneuer, U., Koonce, M. P., Pfister, K. K. & Schliwa, M.
(1988) Nature (London) 332, 176-178.
29. Lye, R. J., Porter, M. E., Scholey, J. M. & McIntosh, J. R.
(1987) Cell 51, 309-318.
30. Ingold, A. L., Cohn, S. A. & Scholey, J. A. (1989) J. Cell Biol.
107, 2657-2667.
31. Scholey, J. M., Heuser, J., Yang, J. T. & Goldstein, L. S. B.
(1989) Nature (London) 338, 355-357.
32. Sinard, J. H. & Pollard, T. D. (1989) Cell Motil. Cytoskel. 12,
42-52.
33. Euteneuer, U., Johnson, K., Koonce, M., McDonald, K.,
Tong, J. & Schliwa, M. (1989) in Cell Movement, eds. Warner,
F. & McIntosh, J. R. (Liss, New York), Vol. 2, pp. 155-167.
34. Pfister, K. K., Wagner, M. C., Bloom, G. S. & Brady, S. T.
(1989) Biochemistry 28, 9006-9012.
35. Miller, R. H. & Lasek, R. J. (1985)J. CellBiol. 101, 2181-2193.