Cell, Vol. 56, 867-878,
March
10, 1989. Copyright
0 1989 by Cell Press
Submolecular Domains of Bovine Brain Kinesin
Identified by Electron Microscopy and
Monoclonal Antibody Decoration
Nobutaka Hirokawa,’ K. Kevin Pfister,t
Hiroshi Yorifuji,’
Mark C. Wagner,t
Scott T. Brady,t and George S. Bloomt
Department of Anatomy and Cell Biology
School of Medicine
University of Tokyo
Hongo, Tokyo 113
Japan
TDepartment of Cell Biology and Anatomy
University of Texas
Southwestern Medical Center
Dallas, Texas 75235
l
Summary
Kinesin is a microtubule-activated
ATPase thought to
transport
membrane-bounded
organelles
along MTs.
To illuminate the structural basis for this function, EM
was used to locate submolecular
domains on bovine
brain kinesin. Rotary shadowed kinesin appeared rodshaped and ~60 nm long. One end of each molecule
contained
a pair of *lo x 9 nm globular domains,
while the opposite end was fan-shaped.
Monoclonal
antibodies
against the ~124 kd heavy chains of kinesin decorated
the globular
structures,
while those
specific for the ~64 kd light chains labeled the fanshaped end. Quick-freeze, deep-etch EM was used to
analyze MTs polymerized
from tubulin
and crosslinked to latex microspheres
by kinesin. Microspheres
frequently
attached to MTs by arm-like structures,
2530 nm long. The MT attachment
sites often ap
paared as one or two ~10 nm globular bulges. Morphologically similar cross-links
were observed by qulckfreeze, deep-etch EM between organelles
and MTs in
the neuronal cytoskeleton
in vlvo. These collective observations suggest that bovine brain kinesin binds to
MTs by globular
domains
that contain
the heavy
chains, and that the attachment
sites for organelles
are at the opposite, fan-shaped end of kinesin, where
the light chains are located.
Introduction
The nerve cell axon has proven to be an invaluable model
system for studying mechanisms of intracellular organelle
transport. There are bidirectional
movements of numerous classes of membrane-bounded
organelles in the axon
(Nakai, 1955; Tytell et al., 1961; Allen et al., 1982), and several theories have been proposed to explain the underlying molecular mechanisms (reviewed by Grafstein and
Forman, 1980; Hammerschlag and Brady, 1988). Electron
microscopic studies of the neuronal cytoskeleleton
in
vivo have suggested that microtubules (MTs) and cross
bridges between MTs and membranous organelles form
the structural basis for this class of motility (Smith, 1971;
Hirokawa, 1982; Miller and Lasek, 1985; Hirokawa and
Yorifuji, 1986). Indeed, real-time observations of organelle
movements in isolated axoplasm by video-enhanced
light
microscopy have demonstrated that bidirectional organelle translocation occurs along filaments that correspond
to MTs (Brady et al., 1982,1985; Schnapp et al., 1985; Vale
et al., 1985a).
The observation that a nonhydrolyzable
ATP analog, 5’adenylylimidodiphosphate
(AMP-PNP), halts such movements and causes immotile organelles to bind stably to
MTs in isolated axoplasm (Lasek and Brady, 1985) led to
the discovery in neural tissue of a new protein whose binding to MTs is also stimulated by AMP-PNP (Brady, 1985;
Vale at al., 1985b). This protein, kinesin, fulfills several of
the properties expected of a motor molecule responsible
for the transport of membrane-bounded
organelles along
MTs. For example, in cultured cells, kinesin is localized on
vesicle-like structures that are codistributed
with MTs
throughout the cytoplasm (Pfister et al., 1989). In addition,
kinesin isolated from a variety of sources can bind to MTs,
hydrolyze ATP in a manner stimulated by MTs, and, in the
course of doing so, generate plus-end directed forces
along those MTs (Brady, 1985; Vale et al., 1985h 1985c;
Scholey et al., 1985; Kuznetsov and Gelfand, 1986; Cohn
et al., 1987; Murofushi et al., 1988; Saxton et al., 1988;
Wagner et al., 1989). To account for these properties, kinesin must contain binding sites for membrane-bounded
organelles, ATP and MTs. In order to gain insight into the
molecular basis of these functional domains, a number of
recent studies have been aimed at dissecting the structure of kinesin.
In particular, determination of the quaternary structure
of bovine brain kinesin has provided a framework for explaining the functions of the protein in terms of its molecular design (Bloom et al., 1988; Kuznetsov et al., 1988). The
native protein contains two heavy chains of 120-124 kd
and two light chains in the size range of 62-64 kd. Further
analysis has indicated that bovine brain kinesin exists in
solution as a highly elongated molecule, with an axial ratio
of 2O:l or greater (Bloom et al., 1988), and that multiple
forms of the heavy and light chains exist (Pfister et al.,
1989; Wagner et al., 1989).
The structural studies just cited constitute a foundation
on which to evaluate functions served by the kinesin
heavy and light chains, and to establish how the holoenzyme is physically constructed from its constituent subunit polypeptides. The heavy chains, for example, evidently contain the binding sites for ATP and MTs (Gilbert
and Sloboda, 1986; Penningroth et al., 1987; Bloom et al.,
1988; lngold et al., 1988; Yang et al., 1988; Kuznetsov et
al., 1989). Scant progress had been made, though, toward
either understanding
the organization of heavy and light
chains within the kinesin molecule, or the functions of the
light chains.
To address these issues, we took advantage of the fact
that the quick-freeze, deep-etch, and low angle, rotary
shadowing methods of electron microscopy (EM) can be
used for analyzing the submolecular
structure and bio-
6
1
23456
1
2345
applied to rat spinal cords, where structures similar in appearance to purified kinesin were observed to cross-link
membrane-bounded
organelles to MTs within the neuronal cytoskeleton. Collectively, these experiments have
revealed the positions of multiple heavy and light chain
domains on native kinesin, and have indicated where
those domains reside in relation to the probable binding
sites for MTs and membrane-bounded
organelles.
Results
Figure 1. Electrophoretic
Analysis
ization of Monoclonal
Anti-Kinesin
of Purified Proteins
by lmmunoblotting
and Character-
(A) Six different proteins were purified and used for experiments
summarized in subsequent
figures. SDS-PAGE
was used for analyzing
representative
samplesof
these proteins: tubulin (lane 1) kinesin (lane
2) and four monoclonal
anti-kinesin
IgGs. The antibodies included Hl
(lane 3) H2 (lane 4) Ll (lane 5) and L2 (lane 6). Each lane contained
3-5 ug total protein, and the gel was stained with Coomassie
blue
R250. Note the multiple isoforms of the ~64 kd light (L) and ~124 kd
heavy (H) chains of kinesin.
(B) Microtubules
were polymerized
from bovine brain cytosol using
taxol and, to promote stable binding of kinesin, AMP-PNP was added.
Following centrifugation
and resuspension,
the microtubules
were
analyzed by SDS-PAGE
(lane 1) and immunoblotting
with Hl (lane 2)
H2 (lane 3) Ll (lane 4) and L2 (lane 5). Note that Hl and H2 recognize
only kinesin heavy chain, while Ll and L2 are light chain-specific.
Further analyses of these antibodies on immunoblots
of purified kinesin
have shown that H2 and Ll react with both major and minor forms of
the heavy and light chains, respectively,
whereas Hl and L2 recognize
only the major isoforms (Pfister et al., 1969). Reactivity with the minor
heavy chain isoform can be seen here faintly in lane 3.
chemical nature of cytoskeletal components both in vivo
and in vitro (Heuser and Salpeter, 1979; Tyler and Branton,
1980; Hirokawa, 1982; Hirokawa et al., 1984; Hirokawa
and Hisanaga, 1987). Using these two approaches, we undertook studies aimed at establishing a more refined and
detailed picture of the structure of kinesin. Bovine brain
kinesin at >90% purity (Wagner et al., 1989) was utilized
for all of the in vitro experiments. First, low angle, rotary
shadowing was used for quantitative morphometric analysis of the kinesin molecule. Next, kinesin that had been
decorated with purified monoclonal antibodies to the kinesin heavy or light chains (Pfister et al., 1989) was rotary
shadowed and observed by EM. Finally, samples of MTs
that were assembled from pure tubulin and cross-linked
by kinesin to latex beads were examined by the quickfreeze, deep-etch method of EM. This method was also
The ultrastructure of bovine brain kinesin was studied by
three approaches. Initially, low angle, rotary shadowed
samples were analyzed by a quantitative morphometric
method. Next, native kinesin that had been decorated with
monoclonal antibodies to kinesin heavy or light chains
(Pfister et al., 1989) was observed after low angle, rotary
shadowing. Finally, complexes of kinesin, microtubules,
and latex microspheres were analyzed by quick-freeze,
deep-etch electron microscopy. These experiments were
performed using only highly purified preparations of kinesin, tubulin, and monoclonal IgG antibodies (Figure 1A).
As demonstrated
by immunoblotting
(Figure IS), two
heavy chain-specific
anti-kinesins
(HI and H2) were
used, as well as two monoclonals directed against the
kinesin light chains (Ll and L2). Each of these antibodies
recognizes a distinct kinesin epitope (Pfister et al., 1989).
Kinesin molecules processed for low angle, rotary
shadowing exhibited a number of consistent morphofogical features, as can be seen in the low magnification field
illustrated in Figure 2. Most of the molecules were approximately rod-shaped, between 50 and 100 nm in length, and
contained structurally
distinct domains at each end.
These features are documented in greater detail in the
higher magnification images shown in Figure 3. One end
of each molecule typically displayed one or two globular
structures, referred to henceforth as “heads:’ The opposite
end, or “tail,” of the molecule was usually occupied by a
fan-like structure. Often, two or more fine filamentous
projections were seen to be part of the tail region, which
had a flatter overall appearance than the heads. Connecting the head and tail regions was a long shaft. While the
shaft on the majority of replicas appeared relatively
straight, it was occasionally seen to be bent at a hinge-like
region located near the middle of the molecule. As shown
in the bottom row of Figure 3, the presence of this hinge
was especially evident in samples that had been dissolved in low salt buffer (0.1 M ammonium acetate; see
also Hisanaga et al., 1989).
The dimensions of morphologically
distinct domains on
kinesin molecules were measured, and the results are
presented quantitatively in Figure 4. The overall length of
the molecule was measured to be 80.4 f 8.1 nm. The long
and short diameters of the head were found to be 10.0 f
1.9 and 8.6 * 2.1 nm, respectively. A width of 14.7 rt 2.6
nm was determined for the tail region. The position of the
hinge occasionally found within the shaft region was also
measured, its location typically having been about 35 nm
from the near edge of the globular heads (data not
shown).
EM Analysis
869
Figure
A typical
of Functional
2. Rotary
Domains
Shadowed
field of kinesin
Kinssin
processed
on Kinesin
Viewed
at Low Magnification
for low angle, rotary
shadowing
Figure 5 summarizes a series of experiments, in which
two monoclonal antibodies to kinesin light chains were
used to determine the positions of the corresponding
epitopes on the native kinesin molecule. The top row demonstrates the appearance of normal mouse IgG, which was
morphologically
identical to all of our monoclonal antikinesins (not shown). The next row shows kinesin that had
been incubated with a sample of the identical IgG preparation. Note that the kinesin molecules shown here are essentially indistinguishable
from those displayed in Figure
3, indicating the absence of binding to kinesin by the irrelevant antibodies. Rows 3-5 in Figure 5 illustrate kinesin
decorated with Ll, while the final four rows show kinesin
labeled with l2. Each of these light chain-specific
antibodies labeled 40% of the kinesin molecules, and the
decorations consistently occurred at the fan-shaped ends
of the molecules. The conclusion about the site of labeling
by Ll and L2 is based on three considerations.
First, the
fan-shaped tails were not visible on kinesin molecules that
were decorated by antibodies. Next, one end of each kinesin molecule had the characteristic
appearance
and
dimensions of the globular heads. Finally, the opposite
ends contained globular decorations that were in the size
range of mouse IgG, larger than the globular heads of
kinesin, and smaller than the sum of globular kinesin
heads plus IgG. The size of the antibody decorations was
usually consistent with the presence of one or two IgG
is shown
here. The bar in the lower
right corner
represents
100 nm
molecules. Presumably, this reflected the binding of antibody to one or both of the light chain subunits associated
with each molecule of bovine brain kinesin (Bloom et al.,
1988; Kuznetsov et al., 1988).
Analogous data were obtained using heavy chain-specific anti-kinesins, as shown in Figure 6. This gallery of
micrographs compares kinesin that was incubated with
normal mouse IgG (row 1) and kinesin that was labeled
with the Hl (rows 2-5) or H2 antibodies (rows 6-8). Again,
each of the anti-kinesins decorated about half of the kinesin molecules, but the ultrastructure of these immunocomplexes was very distinct from those formed in the
presence of light chain-specific
monoclonals (compare
Figures 5 and 6). The fan-shaped tails of kinesin could be
clearly identified on molecules decorated by Hl or H2, but
the globular head regions appeared unusually large.
These images are consistent with the heavy chain epitopes recognized by Hl and H2 as being located on or
near the globular heads of kinesin. As was found for the
light chain-specific
antibodies, the decorations formed on
each kinesin molecule by Hl or H2 were in the size range
of one or two IgG molecules. This is not surprising, considering that two heavy chains are present in the bovine
brain kinesin molecule (Bloom et al., 1988; Kuznetsov et
al., 1988) and that the extent of antibody binding could be
variable.
To determine which portion of the kinesin molecule is
Cell
870
Figure 3. High Magnification
View of Rotary Shadowed Kinesin
Several, representative
molecules are shown here. One or two globular domains are visible at the right end of each mcslecule, while less well-defil ned.
but generally fan-shaped,
elaborations
can be seen at the opposite ends. The bar in the lower right panel equals ,100 nm.
responsible for binding MTs, purified tubulin was assembled with taxol, and mixed with AMP-PNP, kinesin, and 50
nm carboxylated, latex microspheres. These suspensions
were then centrifuged, resuspended in small volumes and
analyzed by the quick-freeze, deep-etch method of EM. As
can be seen in Figure 7, microspheres often appeared to
be cross-linked to MTs by 25-30 nm long, rod-like molecules, which frequently contained one or two conspicuous
bulges where they contacted MTs. The diameter of each
bulge was 40 nm, and neither cross bridges between
MTs and microspheres,
nor MT-bound bulges, were obsewed when kinesin was omitted from the samples (data
not shown). These findings, combined with those derived
from the antibody decoration experiments (see Figures 5
and 6), strongly suggest that the globular heads, where
two heavy chain epitopes are located, comprise the MTbinding sites on kinesin.
Spinal cords were also processed by the quick-freeze,
deep-etch method and observations were made of their
neurites (Figure 6) where large numbers of membraneboundeded organelles are transported rapidly and continuously along MTs. The purpose here was to compare
the morphologies
of cross-links between MTs and organelles in vivo with the cross-links formed by kinesin between MTs and synthetic microspheres in vitro. Some of
the neuronal cross bridges were strikingly similar in appearance to those produced by kinesin molecules in the
reconstituted system (compare Figures 7 and 8). Although
we cannot at present be certain that any of the neuronal
cross-links actually represent kinesin, it is significant that
some of them, like those composed of kinesin in vitro,
were -25 nm long and contained a pair of globular bulges
at their points of contact with MTs. Other approaches,
such as immuno-EM of kinesin in vivo, will ultimately be
required to determine whether such cross-links are, indeed, kinesin.
EM Analysis
of Functional
Domains
on Kinesin
871
total length
-
mean
a.4 f I.1 ma
long diameter
n=l34
r
of head
mean 10.0f 1.9 nm
n=77
A
diameter of head
1 short mean
8.6k2.1nm
n= 96
tail
mean 14.7k 2.6
n= 104
DIh
9.5 10.7 12.0 13.2 14.4
c
Figure
4. Morphometric
Analysis
The total length, tail width,
ure 3. The size distribution
1,
nm
!M 231
nm
of Kinesin
and long and short
for each parameter
diameters of the heads
is summarized
here.
Discussion
The observations reported here have made it possible to
construct a rational model that describes the functional
properties of bovine brain kinesin in terms of submolecular structure (Figure 9). The overall morphology of the protein was demonstrated to be rod-like, but each end of the
molecule was characterized
by its own distinct type of
structural elaboration. A pair of globular heads located at
were measured
for numerous
kinesin
molecules,
like those
shown
in Fig-
one end were shown to function as attachment sites for
MTs and to contain two distinct antigenic sites of the kinesin heavy chain. The fan-shaped tail at the opposite end
of kinesin was found to contain two light chain epitopes,
and the available evidence strongly suggests that this tail
serves as the binding site for the membrane-bounded
organelle.
A hinged region was occasionally observed approximately halfway between the head and tail. The presence
(rows 3-5). and L2 (rows S-S)
e row 2 with Figure 3), but that
ht panel represents 100 nm.
EM Analysis
673
Figure
of Functional
6. Monoclonal
Domains
Antibodies
on Kinesin
to Kinesin
Heavy
Chains
Decorate
the Globular
Head
Regions
Samples of kinesin that had been incubated with normal mouse IgG (top row), Hl (rows 2-5),
rotary shadowing.
Note that normal mouse IgG failed to decorate kinesin, but that the anti-heavy
heads of the molecules.
The bar in the lower right panel represents
166 nm.
of this hinge might explain why the total length of kinesin
was measured to be ~80 nm, but the protein appeared to
be only about one-third as long when cross-linking MTs to
latex microspheres. As postulated in Figure 9A, perhaps
a portion of the shaft of kinesin, extending from the hinge
to the fan-shaped
tail, accomodates
the membranebounded organelle or microsphere. Consistent with this
of Kinesin
and H2 (rows 6-8) were processed
for low angle,
chain antibodies,
Hl and H2, labeled the globular
idea is the observation of similarly sized cross bridges between MTs and vesicles in vivo (Hirokawa, 1982; Miller and
Lasek, 1985; Hirokawa and Yorifuji, 1988). It is also tempting to speculate that the hinge marks a point at which kinesin can bend, and that such a conformational
change is
related to ATP hydrolysis and organelle motility along MTs,
or the regulation of those processes.
Cell
874
Figure
7. The Globular
Heads
of Kinesin
Contain
the Microtubule
Binding
Sites
Fifty nanometer carboxylated,
latex microspheres
were cross-linked
by kinesin to microtubules
made from pure tubul lin and taxol, and the sa lmples
were analyzed by the quick-freeze,
deep-etch method of EM. Three pairs of stereo images are shown here. Note that several kinesin cross b ridges
contain globular structures
at their attachment sites for microtubules
(see arrows). The size and appearance
of these structures
are consiste, at with
the globular heads seen in rotary shadowed
samples of kinesin (see Figure 3). The bar in the lower left panel eqi Jals 100 nm.
EM Analysis
875
Figure
of Functional
8. Structures
Domains
on Kinesin
with the Morphology
of Kinesin
Appear
to Cross-Link
Membrane-Bounded
Organelles
to Microtubules
in the Neurite
A rat spinal cord was processed
for quick-freeze,
deep-etch EM, and neurite regions rich in membrane-bounded
organelles and microtubules
were
examined. Numerous,
rod-shaped
structures
were observed to span the distance between organelles and microtubules,
and many of these apparent
cross bridges had globular bulges where they seemed to contact microtubules
(see arrows). The dimensions
and appearance
of these presumptive
cross bridges were reminiscent
of kinesin invovled in cross-linking
synthetic
microspheres
to microtubules
(compare this micrograph
with Figure
7). The bar in the lower right indicates 100 nm.
A number of structural and functional features of kinesin
invite a comparison with myosin (see Figure 9C). Both proteins are long, rod-like molecules that contain heavy and
light chain subunits, and a pair of globular heads, where
polymers of cytoskeletal proteins can attach. These polymers stimulate a Mg*+-ATPase activity associated with
kinesin and myosin, and are mechanochemical
partners
of the enzymes. It is likely that the principal form of work
performed intracellularly by kinesin is to move membranebounded organelles. Although myosin functions mainly
as a contractile protein, it too appears able to be an organelle transport motor in a few cell types (Adams and
Pollard, 1986; Warrick and Spudich, 1987). Contrasting
these similarities, though, are numerous profound differences between kinesin and myosin. First, the mechanochemical partner of kinesin is the MT, while that of myosin
is F-actin. Second, the order of the various steps in the
mechanochemical
cycle of kinesin apparently differs from
that used by myosin (Lasek and Brady, 1985; Hackney,
1988). Third, most forms of myosin are nearly twice as
long as bovine brain kinesin. Next, the light chains of kinesin appear to be located at the tail end of the molecule
(see Figure 5), whereas the myosin light chains are part
of the globular heads (Flicker et al., 1983; Yamamoto et al.,
1985). Finally, there has been no indication yet that kinesin and myosin are related immunologically
or by primary
structure.
The contrasts between kinesin and dynein, the two MTbased mechanochemical
ATPases, are equally pronounced. Dyneins are complex, multimeric proteins found
in flagellar axonemes, where they are known to cause sliding between adjacent MTs and in the cytoplasm, where
they have been proposed to be responsible for moving
membrane-bounded
organelles
along MTs (Johnson,
1985; Lye et al., 1987; Paschal et al., 1987; Paschal and
Vallee, 1987; Euteneuer et al., 1988; Gibbons, 1988). The
cytoplasmic and axonemal dyneins consist of either two
or three globular domains of lo-15 nm diameter each,
Cell
876
A
0
I
-
Heavy Chain
Light Chain
124 KD
64 KD
= 10 nm
-
IOnm
-;f
IOna
Figure 9. Models for the Substrudure
of Bovine Brain Kinesin
(A) Based on the data summarized
in Figures l-8, bovine brain kinesin
appears to contain a pair of globular heads that bind microtubules
and
a fan-shaped tail involved in binding to membrane-bounded
organelles
or synthetic microspheres.
A hinged region was found near the center
of the molecule, and the tailward side of the hinge may rest against the
surface of the organelle or microsphere.
This could explain why cross
bridges between microtubules,
and either organelles or microspheres,
are -25 nm, while the overall length of kinesin is ~80 nm.
(8) Shown here is a scale model for the structure of kinesin that takes
into account the diameters
of the two globular head domains, the
length of the central shaft region, the position of the hinge within the
shaft (arrow), the width of the tail, and the apparent locations of each
of the two heavy and light chains. The degrees to which the heavy and
light chains extend within the shaft represent estimates, but all other
details are based on the date shown in Figures 1-i’.
(C)The morphological
similarities between kinesin and myosin are superficial. Both kinesin and myosin are long, rod-shaped
molecules
with a pair of globular head domains, and heavy and light chain
subunit polypeptides.
As can be seen, though, kinesin contains a fanshaped tail, which myosin lacks; the light chains of kinesin, but not myosin, are found at the tail end of the molecule; and kinesin is substantially shorter than most myosins.
joined together by separate, thin strands connected at a
common base (Johnson and Wall, 1983; Witman et al.,
1983; Goodenough and Heuser, 1984; Vallee et al., 1988).
The overall length of the dynein complex is 35-50 nm,
considerably shorter than kinesin. In summary, kinesin,
dynein, and myosin may be regarded as proteins that
share some gross structural and functional features essential for performing work, but appear to be unrelated to
one another in molecular terms.
A previous report on the ultrastructure of porcine brain
kinesin indicated that the protein is a long, rod-shaped
molecule with morphologically
distinct ends, but the accompanying model is at odds with our data for the equivalent bovine brain enzyme (Amos, 1987). The porcine protein was described as being ~100 nm long and containing
a small, bifurcated fork-like structure at one end of the
molecule. This fork, along with a substantial length of the
shaft region, was hypothesized to be involved in MT binding. In contrast, bovine brain kinesin was found to be only
~80 nm long (see Figures 2-4). In addition, we never observed a fork-like region, but consistently noted the presence of a pair of globular domains at one end of each kinesin molecule (see Figures 2 and 3). As shown in Figure
7, these globular heads alone apparently correspond to
the MT binding sites. Finally, at the opposite end of the
molecule, porcine brain kinesin was envisioned as containing an elaborate, multibranched
structure that attaches the molecule to the organelle, whereas our data do
not support such a detailed model (compare Figure 9 here
with Figure 6 in Amos, 1987).
Besides characterizing
the overall morphology of bovine brain kinesin, the present study also supplies the first
evidence for how both subunit polypeptides of kinesin are
organized in the holoenzyme, and relates those findings
to the physical locations of functional domains. Here, we
have shown that a pair of heavy chain epitopes and the
MT binding sites are on the globular heads of bovine brain
kinesin, while two light chain domains are located on the
fan-shaped tail at the opposite end of the molecule.
Moreover, our data stongly imply that the binding sites
for membrane-bounded
organelles are in the tail region,
and that heavy and light chains are involved in the complementary functions of MT and organelle binding, respectively. The overall morphology of kinesin from other
sources that have been examined very recently is consistent with that of the bovine brain enzyme (Hisanaga et al.,
1989; Scholey et al., 1989). Furthermore, molecular biological and immuno-EM studies concurrent with those described here have also indicated that the globular heads
of kinesin contain the MT-binding sites (Scholey et al.,
1989; Yang et al., 1989, see accompanying paper). It is anticipated that these advances in illuminating the structure
of kinesin will promote an improved understanding of the
functional, biochemical, enzymatic, and molecular biological properties of the protein.
Experimentel
Procedures
Puriticatlon
of Kineeln, tibulin,
and Monoclonal
Antlbodlee
Kinesin was purified from bovine brain as described in detail previously
(Wagner et al., 1989). The purity of the final product ranged from
EM Analysis
677
of Functional
Domains
on Kinesin
90%-95%,
as judged by quantitative densitometry
of SDS-polyacrylamide gels stained with Coomassie
brilliant blue R250. Tubulin was purified from porcine or bovine brain using Phosphocellulose
(Whatman;
Clifton, NJ) or DEAE-Sepahdex
chromatography
(Pharmacia;
Piscataway, NJ), as described earlier (Weingarten
et al., 1975; Hirokawa et al.,
1986; Bloom et al., 1988). Four extensively
characterized
monoclonal
antibodies to kinesin (Pfister et al., 1989) were employed for the present study. Two of these, Hi and H2, recognize the major kinesin heavy
chain, while the other antibodies, Ll and L2, react with the major light
chain. H2 and Ll are also immunoreactive
with minor forms of the
heavy and light chains, respectively.
Each of the antibodies was purified out of ascites fluid as a homogeneous
IgG subisotype
by Protein
A-Superose
(Pharmacia)
or Protein A-Affi-Gel (BieRad;
Richmond,
CA) chromatography
(Pfister et al., 1989). Prior to use, all purified proteins were dialyzed into PEM buffer (0.1 M PIPES (pH 8.81, 1 mM EGTA,
1 mM MgQ).
SDS-PAGE
and immunoblotting
were performed
as
described
(Pfister et al., 1989).
Low Angle,
Rotary
Shadowing
EM
Ammonium
acetate and glycerol were added to the protein solutions
to final concentrations
of 0.5 M (occasionally
0.1 M) and 50% (by volume), respectively.
For antibody decoration experiments,
this step was
completed after mixtures of kinesin at -50 pglml and a P-fold molar
excess of antibody had incubated together for 1 hr at room temperature. Protein solutions
supplemented
with ammonium
acetate and
glycerol were sprayed onto freshly cleaved mica flakes, which were
subsequently
dried under vacuum as described
previously
(Tyler and
Branton, 1980; Hirokawa, 1986). Rotary shadowing with platinum was
peformed at an angle of 8O using a Balzers (Hudson,
NH) model 301
freeze fracture apparatus,
and the replicas were processed
further as
described
previously
(Hirokawa
et al., 1988).
The dimensions
of various domains on kinesin molecules were determined by a quantitative
morphometric
method. Micrographs
were
printed at a magnification
of 140,000x.
Then, length measurements
were made by using a magnifying
glass to examine profiles of kinesin,
along side of which a ruler was placed. All specimens
were observed
and photographed
on a JEOL 2000 EX electron microscope
at 100 kV.
Quick-Freeze,
Deep-Etch
EM
One hundred micrograms
each of kinesin and tubulin were incubated
for 15 min at 3PC in the presence
of 1 mM 5’-adenylylimidodiphosphate (AMP-PNP),
1 mM GTP, 20 PM taxol (gift from Matthew Suffness,
NIH), and 0.8% (by volume) Fluoresbrite
(50 nm) carboxylated
microspheres
(Polysciences;
Warrington,
PA). The suspension
was then
pelleted in a Beckman (Palo Alto, CA) model TL 100 tabletop ultracentrifuge using a TLA 100.2 rotor spun at 55,000 rpm for 30 min at 30°C.
The resulting pellets were resuspended
into small volumes of PEM
containing
1 mM each GTP and AMP-PNP, and 20 PM taxol. Finally,
droplets of these samples were adsorbed onto fragmented
mica chips,
and the specimens
were rapidly frozen, deeply etched, and replicated
(Heuser, 1983; Hirokawa et al., 1985, 1988a). Stereo micrographs
were
taken at -c 10“ on a side-entry goniometer
stage. Rat spinal cords were
dissected,
and thin slices of tissue were processed
for quick-freeze,
deep-etch
EM as described
previously
(Hirokawa et al., 1985, 1988).
Observations,
photography,
and morphometric
measurements
were
as described
for rotary shadowed
samples.
Acknowledgments
This work was supported
by a Special Grant in Aid for Scientific Research,
number 82065007, from the Japan Ministry
of Education,
Science and Culture (N. H.); a grant from the Muscular Dystrophy Association
of America
(N. H.); National
Institutes
of Health grant
NS23888 (S. T. B. and G. S. 6.); NIH Postdoctoral
Fellowship Award
GM10143 (K. K. P); grant number DMB-8701164
from the National
Science Foundation Biological Instrumentation
Program (G. S. B. and
S. T. B.); and Welch Foundation grant l-1077 (G. S. B. and S. T. B.). We
would like to thank Matthew Suffness of the National Cancer Institute
for supplying us with taxol, Y. Kawasaki for her secretarial
assistance,
Y. Fukuda for his photographic
expertise, David L. Stenoien for providing technical assistance,
and Mary Seither for artistic contributions.
The costs of publication of this article were defrayed in part by the
payment
of page charges.
This article must therefore
be hereby
marked “advertisemenf”
in accordance
solely to indicate this fact.
Received
December
28, 1988; revised
Adams, R. J.. and Pollard,
lated from Acanthamoeba
322, 754-756.
with
18 USC.
January
Section
1734
17, 1989.
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