Cell Motility and the Cytoskeleton 23:19-33 (1992)
Association of Kinesin With Characterized
Membrane-Bounded Organelles
Philip L. Leopold, Alasdair W. McDowall, K. Kevin Pfister, George S. Bloom,
and Scott T. Brady
Department of Cell Biology and Neuroscience (P.L.L., A. W.M., K.K.P., G.S.B.,
S.T.5.) and Howard Hughes Medical Institute (A.W.M.), University of Texas
Southwestern Medical Center, Dallas
The family of molecular motors known as kinesin has been implicated in the
translocation of membrane-bounded organelles along microtubules, but relatively
little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a
variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative
immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These
results demonstrate that the organelle binding capacity of kinesin is selective and
specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable
association with membrane-bounded organelles in the cell. In addition, immunoelectron microscopy of mitochondria revealed a patch-like pattern in the kinesin
distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility. 0 1992 Wiley-Liss, Inc.
Key words: synaptic vesicle, mitochondria, coated vesicle, immunogold electron microscopy, motor
protein
INTRODUCTION
The anatomy of neuronal cells requires the existence of efficient transport mechanisms for the movement of materials from the site of synthesis to sites of
utilization [Lasek and Brady, 19821. One class of transport, fast axonal transport, involves the movement of
proteins in association with membrane-bounded organelles along microtubules. Early studies of fast axonal
transport led to the proposal of several physical and
mechanochemical models which might account for the
force production necessary to move organelles [Lasek,
1980; Weiss, 1982; Ochs, 19821. The finding that 5 ’ adenylylimidodiphosphate (AMP-PNP) inhibited vesicle
movement and left organelles attached to microtubules
[Lasek and Brady, 19851 implied the existence of a
force-generating molecule that could interact with both
organelles and microtubules. Centrifugation of microtubules in the presence of AMP-PNP led to the identification of kinesin [Brady, 1985; Vale et al., 1985; Scholey
et al., 19851, a microtubule-stimulated ATPase [Brady,
0 1992 Wiley-Liss, Inc.
1985; Kuznetsov and Gelfand, 19861 with the ability to
induce gliding of microtubules in vitro [Vale et al., 1985;
Porter et al., 1987; Cohn et al., 1987; Howard et a].,
19891. These properties made kinesin a candidate for an
organelle motor.
Several recent studies have documented the association between kinesin and intracellular membranes.
Immunofluorescent localization of kinesin in cultured
cells and in the squid giant axon revealed punctate patterns which were sensitive to detergent solubilization
[Pfister et al., 1989a; Hollenbeck, 1989; Brady et al.,
Received October 25, 1991; accepted February 27, 1992.
Address reprint requests to Scott T. Brady, Department of Cell Biology and Neuroscience, UT Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75235.
K. Kevin Pfister is now at Department of Anatomy and Cell Biology,
University of Virginia Health Science Center, Charlottesville, VA
22908.
20
Leopold et al.
19901, suggesting association of kinesin with organelle
membranes. Wright et al. [1991] demonstrated that an
anti-kinesin immunofluorescence pattern in sea urchin
blastomeres and coelomocytes was similarly detergent
sensitive. Hollenbeck [1989] performed a series of detergent extractions on cultured cells, leading to the suggestion that both soluble and membrane-associated pools
of kinesin exist. Studies of kinesin accumulating at a
nerve ligation are also consistent with an association of
kinesin and membrane-bounded organelles in vivo [Hirokawa et al., 1991; Dahlstrom et al., 19911. Kinesin
transport occurs in the axon at rates which correspond to
the movement of membrane-bounded organelles (R.G.
Elluru and S.T. Brady, unpublished observations). Finally, perfusion of squid axoplasm with anti-kinesin antibodies slows both anterograde and retrograde organelle
traffic, demonstrating a direct involvement of kinesin in
organelle motility [Brady et al., 19901, and kinesin antibodies disrupt endocytic membrane compartments
[Hollenbeck and Swanson, 19901. However, limits in the
resolution available with these approaches have precluded a determination of the identity of organelles with
which kinesin interacts or the nature of that interaction.
This report identifies associations between kinesin and
specific organelle fractions.
Understanding how the cell organizes organelle
transport will require the identification of interactions
between specific organelles and molecular motors. Currently, three putative membrane-bounded organelle motors have been identified (kinesin, cytoplasmic dynein
[Paschal et al., 1987; Euteneuer et al., 19881, and myosin I [Adams and Pollard, 1986]), but it is not yet
known whether these motors are acting individually or
synergistically.
In order to determine the identity of organelles
which bind kinesin, several fractionations of bovine
brain homogenate were performed. Purified fractions of
synaptic vesicles, mitochondria, coated vesicles, and nuclei were tested for the presence of kinesin by immunoblotting and immunoelectron microscopy with anti-kinesin antibodies. In addition, isolated Golgi membranes
prepared from rat liver were examined. Kinesin copurified with some fractions (synaptic vesicles, mitochondria, and coated vesicles) but was not detectable in other
fractions (Golgi membranes and nuclei). These results
demonstrate that kinesin is capable of stable associations
with organelle membranes and that kinesin exhibits organelle-specific binding.
MATERIALS AND METHODS
Vesicle Preparation
Synaptic vesicles and microsomes were prepared
using modifications of the method of Hell et al. [ 19881.
Fresh bovine brains were obtained from Dallas City
Packing, Inc. (Dallas, TX) and were kept in PBS (20
mM Na phosphate, 150 mM NaCl, pH 7.4) at 4°C until
small pieces of tissue could be frozen by immersion in
liquid nitrogen. Frozen tissue was stored at -80°C until
use (<3 months). Frozen tissue (20-25 g) was pulverized at high speed in a pre-chilled Waring Blender. Powder was resuspended in 80 ml of cold homogenization
buffer (320 mM sucrose, 10 mM HEPES, 1 pg/ml pepstatin, I pg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride [PMSF], pH 7.4). The suspension was
homogenized 40 sec using a Polytron with probe PTA
20s (Brinkman Instruments, Westbury, NY) on medium
setting. The homogenate was centrifuged 15 min, 16,500
rpm in a Sorvall SA600 rotor (39,5OOg,,,) at 4°C. The
pellet (Pl) was discarded while the supernatant (Sl) was
centrifuged 40 min, 32,000 rpm, in a Beckman 4STI
rotor (120,OOOg,,,)
at 4°C. The pellet (P2) was discarded, and 20 ml of supernatant (S2) was layered onto
5 ml of 600 mM sucrose, 10 mM HEPES, pH 7.4. The
step gradient was centrifuged in a Beckman TI60 rotor,
50,000 rpm, for 2 hr (260,000gm,,) at 4°C. Four fractions were collected: supernatant (S3), layer of material
over the cushion (L3), cushion (C3), and pellet (P3). P3
was resuspended in 1 ml of 320 mM sucrose, 10 mM
HEPES, pH 7.4 using 10 passages through a 25 gauge
hypodermic needle. The vesicle suspension was diluted
to 5 ml and centrifuged 10 min, 15,000 rpm, in a SA600
rotor (32,S00gm,,) to clear aggregates. The suspension
was layered onto a controlled-pore glass (CPG) (Sigma,
GG-3000-200) sizing column (45 cm X 2.5 cm) which
was equilibrated with 300 mM glycine, 0.02% sodium
azide, 5 mM HEPES, pH 7.4. Flow rate was set at 0.5
ml/min, and 5 ml fractions were collected. Column fractions were tested for absorbance at 260 nm, 280 nm, and
310 nm (Fig. 1). Peak CPG fractions were either pooled
and concentrated (60TI rotor, 60,000 rpm, 2 hr, 4"C,
360,OOOg,,,) to give two vesicle fractions (V1 and V2)
or were pelleted individually (Beckman 70.1TI or Sorvall T1270, 60,000 rpm, 2 hr, 4"C, 335,000gm,,). Vesicle pellets were resuspended in column buffer at approximately 1 mg/ml.
Mitochondria Preparation
Mitochondria were prepared from bovine brain using a modification of the method of Clark and Nicklas
[ 19701. Bovine brains were obtained and homogenized
as described above in 80 ml of 250 mM sucrose, 10 mM
HEPES, 0.5 mM EDTA, pH 7.4 plus protease inhibitors. For some experiments, brain tissue was not frozen
prior to homogenization which allowed the blender step
to be omitted. All centrifugation was conducted in a
Sorvall SA-600 rotor. The homogenate was centrifuged
for S min, 3,750 rpm (2,00Og,,,) at 4°C to give pellet
Kinesin Organelle Association
u)
c
C
260nm
280nm
A 310nm
0.2
a
0
u
m
C
e
:
: 0.1
Q:
0
0.0
0
10
20
30
40
50
Fraction
Fig. 1. Fractions from the final step in the purification of synaptic
vesicles on a controlled pore glass column. Fractions from gel filtration were checked for absorbance at 260 nm (sequestered nucleotides),
280 nm (protein), and 310 nm (scattered light). Two distinct populations of vesicles eluted as reported by Hell et al. [1988]. Populations
were designated V1 and V2.
(Pl) and supernatant (Sl). S1 was centrifuged for 8 min,
9,300 rpm (12,500gmax)to pellet a crude mitochondria1
fraction (P2). Supernatant (S2) was discarded. P2 was
resuspended in 10 ml of 3% Ficoll (Sigma F 9378), 120
mM mannitol, 30 mM sucrose, 25 pM Kf-EDTA, pH
7.4 and was layered onto 6% Ficoll, 240 mM mannitol,
60 mM sucrose, 50 p M K+-EDTA, pH 7.4. The step
gradient was centrifuged for 30 min, 8,700 rpm
(1 1 ,500gma,) at 4°C. Five fractions were collected: supernatant (S3), layer of material over cushion (L3), cushion (C3), soft pellet (P'3), and hard pellet (P3). P3 was
resuspended in homogenization buffer and pelleted ( 10
min, 9,300 rpm, 4"C, 12,500gmax)to give the final mitochondrial pellet (P4).
Other Organelle Fractions
Nuclei were prepared from bovine brain by the
method of Gerace et al. [ 19781. Purity was confirmed by
positive staining with the nucleic acid specific dye,
DAPI (Sigma #D-1388), at 1 pg/ml for 3 min. Purified
coated vesicles from bovine brain were provided by Drs.
John Peeler and Richard Anderson, UT Southwestern
Medical Center, and were purified by modification of the
method of Nandi et al. [1982; Mahaffey et al., 19891.
Golgi membrane factions were prepared from rat liver by
the method of Leelavathi et al. [1970] as modified by
Bloom and Brashear [1989].
Electron Microscopy
The morphology of vesicle fractions (V1 and V2)
and mitochondria was determined using a standard fixa-
21
tiodembedding protocol. Pellets of organelles were
fixed overnight in 4.0% paraformaldehyde, 0.5% glutaraldehyde, 250 mM PIPES, pH 7.2. After osmification
and alcohol dehydration, pellets were embedded in Epon
and stained with uranyl acetate/lead citrate. Microscopy
on 60 nm sections was performed with a JEOL JEM1200EX electron microscope operating at 80 kV.
Immunogold labeling of cryosections [Griffiths et
al., 19841 was employed to localize kinesin on organelles. Pellets of organelles were initially fixed by a 15
min rinse in 2% paraformaldehyde/250 mM PIPES and
then loosened and suspended in fresh fixative. After 45
min, pellets were transferred through 20 min changes of
1.0, 1.5, and 2.3 M sucrose/PBS allowing pellets to
clarify in the final wash. Fragments of the pellets were
frozen on aluminum pins in liquid nitrogen and sectioned
at a thickness of 100 nm. Sections were picked up on
grids and blocked with 10% fetal calf serum (Gibco,
Grand Island, NY) in 0.12% glycineiPBS. Sections were
labeled with anti-kinesin heavy and light chain antibodies (H-1, H-2, L-1, and L-2 from Pfister et al. [1989a])
in 5% fetal calf serum/0.12% glycine/PBS. Antibodies
were applied at 15 ng of each Protein A affinity purified
antibody per pl for 30 min. Following 4 X 4 min washes
with 0.12% glycine/PBS, grids were floated 30 min on
rabbit-anti-mouse IgG secondary antibody (Organon
TeknikaKappel, West Chester, PA) in 5% FCS/O. 12%
glycine/PBS. Following 4 X 4 min washes in 0.12%
glycineiPBS, grids were floated for 30 min on Protein A
(Pharmacia LKB Technology, Piscataway, NJ) conjugated to 9 nm gold particles in 5% FCS/O.12% glycine/
PBS. Grids were washed 5 X 4 min in 0.12% glycine/
PBS and 5 X 5 min in glass distilled water before
floating 10 min on 2% methyl cellulose/0.3% uranyl
acetate. As controls, either primary antibody was omitted or sections were labeled with 60 ng/pl of an irrelevant primary antibody (2001 Ab from Tolleshaug et al.
[ 19821). In some experiments, rabbit polyclonal antiserum to mitochondrial antigen p24 was used (a gift from
Dr. R. Bravo, Bristol-Myers Squibb, Princeton, NJ); for
these experiments, secondary antibody incubation was
omitted.
Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) and lmmunoblotting
SDS-PAGE was performed according to the
method of Laemmli [1970] using 7% polyacrylamide
gels. Protein patterns were visualized using a minor
modification of the method of Wray et al. [1981] (J.L.
Cyr, UT Southwestern Medical Center, personal communication). Following a 30 min fixation in 50% methano1/0.037% formaldehyde, gels were treated with
Kodak Rapid Fix (Eastman-Kodak, 146 4106) for at least
15 min. Gels were re-equilibrated with methanol/form-
22
Leopold et al.
aldehyde prior to staining. In addition, all glassware was
pre-washed with Kodak Rapid Fix and rinsed with glass
distilled water prior to use. This modification prevented
precipitation of silver during staining.
Following SDS-PAGE, proteins were transferred
to Immobilon-P PVDF membranes (Millipore, IPVH
000 10) using a Transblot apparatus (Hoeffer Instruments, San Francisco, CA). Immobilon-P membranes
were blotted with anti-kinesin antibodies against the
heavy and light chains (H-1, H-2, L-1, and L-2) [Pfister
et al., 1989a1, or monoclonal mouse IgG antibodies to
p38 (synaptophysin) provided by Dr. Paul Greengard
(Rockefeller University, New York). The blotting
technique of Papasozomenos and Binder [ 19871 was
used. Briefly, membranes were blocked 1 hr with 5%
(w/v) Carnation Instant Milk in borate buffered saline
(BBS: 100 mM boric acid, 25 mM sodium borate, 75
mM sodium chloride, pH 8.2). Primary antibody was
applied in 5% milWBBS overnight followed by three 10
min washes with BBS. Secondary antibody (rabbit
anti-mouse IgG, Jackson Irnmunoresearch Laboratories,
315-005-003) was applied for 2 hr in 5 % milWBBS
followed by three 10 min washes with BBS. '251-Protein
A (Amersham Inc. IM 144) was applied in 5%
milWBBS for 2 hr at a concentration of 0.05 p-Ci/ml.
All solutions with 5% milk were brought to 0.02%
sodium azide and were stored at 4°C for reuse. The
membranes were finally washed twice with BBS and
once with BBS + 0.1% Triton X-100. After dying,
protein patterns were visualized by exposure of Kodak
X-ray film (Eastman-Kodak, XAR-5 Diagnostic X-ray
film). Quantitation of kinesin in experimental lanes was
accomplished by comparison to kinesin standards using
a Multi Prias 1 gamma emission counter (Packard
Instrument Co., Laguna Hills, CA) or by densitomem
in the linear range of detection using an Ultroscan-XL
Laser Densitometer (LKB Instrument Co., Gaithersburg, MD).
Other Procedures
All reagents were from Sigma Chemical Co. ( S t .
Louis, MO) or Polysciences (Warrington, PA) unless
otherwise specified. Protein concentrations were determined by the method of Lowry et al. [ 19511 with BSA as
the standard. For individual gel filtration fractions which
had very low protein concentrations, the method of Bradford [ 19761 was used with reagents from Pierce Chemical Co. (Rockford, IL) and gamma globulin as the standard. Assays of enzymatic markers were performed
according to the methods described by Tolbert [1974]
(cytochrome c oxidase) and Vassault [ 19831 (lactate dehydrogenase). All statistical data is presented as mean 2
standard error of the mean.
RESULTS
Characterization of Vesicle Fractions From
Bovine Brain
Brain vesicle fractions were prepared by an adaptation of the method of Hell et al. [1988]. This method,
originally applied to rat brain, was designed as a rapid
preparation of highly purified synaptic vesicles. Hell et
al. [I9881 reported that two populations of vesicles were
obtained in a final controlled pore glass sizing column
step; the first population of vesicles to pass through the
column was heterogenous and was enriched for microsomes while the second fraction contained purified
synaptic vesicles. Our preparation employs bovine brain
rather than rat brain and modifies the homogenization
procedures. Despite these changes, the elution profile of
the CPG column is comparable to that reported by Hell et
al. [1988] (Fig. 1). Two vesicle peaks (V1 and V2) can
be clearly distinguished based on the absorbance of light
by proteins and sequestered nucleoside triphosphates
(280 and 260 nm), as well as by light scattering (310
nm) .
The contents of the V1 and V2 vesicle fractions
were characterized using a variety of experimental approaches. In each case, the characteristics of vesicle fractions from bovine brain were consistent with published
results [Hell et al., 19881. Electron microscopy of ultrathin sections after Epon embedding showed that V1
contained vesicular structures ranging from 30 nm to 500
nm in diameter (Fig. 2a). The mean diameter of vesicle
profiles was 104 2 3.4 nm (N = 146). V1 vesicles
displayed spherical as well as irregular shapes. V2
contained smaller vesicles with a mean diameter of vesicle profiles of 46 2 1.3 nm (N = 311) (Fig. 2b). The
V2 fraction represented a more homogenous population
of organelles. A small, spherical morphology is typical
of presynaptic vesicles [reviewed by Klein et al., 19821
and anterogradely transported vesicles [Tsuluta and
Ishikawa, 1980; Fahim et al., 1985; Miller and Lasek,
19851. Cytoskeletal structures were not observed in
membrane-bounded organelle fractions.
Hell et al. [1988] conducted an extensive analysis
of the vesicle fractions using a series of marker enzymes
and immunochemical markers. Their analysis demonstrated that V1 was enriched for microsomes, while V2
was enriched for synaptic vesicles. In contrast, plasma
membrane and mitochondria1 markers were eliminated
from these fractions. In order to confirm that the vesicle
fractions derived from bovine brain were comparable to
those reported by Hell et al. [l988], markers for mitochondrial inner membrane (cytochrome c oxidase) and
cytosolic proteins (lactate dehydrogenase) were assayed.
These markers were reduced more than 28-fold in the V1
and V2 fractions (Table I). The V2 fraction was further
Kinesin Organelle Association
23
Fig. 2. Ultra-thin sections of pelleted vesicles from fractions V l (a) and V2 (b). Fraction V1 contained
both large, irregularly shaped membranes as well as small, round organelles. Fraction V2 contained
predominantly small, round vesicle profiles with a diameter of 30 to 50 nm. Bar = 500 nm.
TABLE I. Enzymatic Analysis of V1 and V2 Fractions
~~
Lactate dehvdrogenase
la
,024
ND~
Homogenate
V1'
V2'
H S1 P1 S2 P2 S3 L3 C3 P3 V1 V2
Cvtochrome C oxidase
Ib
,035
,025
aHomogenate activity for LDH was 208 pmol min-' mg-'.
bHomogenate activity for cytochrome c oxidase was 117 pmol min-'
mg-'.
'Activity for V1 and V2 presented relative to homogenate activity
of 1.
dNot detectable.
characterized by the presence of p38 (synaptophysin), an
integral membrane protein found on synaptic vesicles
[Wiedenmann and Franke, 1985; Jahn et al., 19851. Immunoblots of steps during the preparation of the vesicles
demonstrated that p38 was enriched in V2 relative to the
initial homogenate (Fig. 3 ) . The p38 antigen was also
present in the V1 fraction but was not enriched to the
same degree.
Fig. 3. Enrichment of p38 (synaptophysin) in fraction V2. The integral membrane protein, p38, serves as a marker for synaptic vesicles.
p38 was identified by immunoblotting of fractions from vesicle preparations and was enriched in fraction V2 relative to the initial homogenate (H). Fractions included low speed supernatant (Sl) and pellet
(Pl), high speed supernatant (S2) and pellet (P2), sucrose cushion
supernatant (S3), layer between the supernatant and cushion (L3), 600
mm sucrose cushion (C3), and pellet (P3), and the final controlledpore glass purified vesicle fractions (V1 and V2). Lanes contain equal
loads of protein.
nor depleted in the vesicle fractions. In contrast, immunoblotting with antibodies against tubulin and MAP 1B
Association of Kinesin With Vesicles
showed that these two proteins were significantly dePurified vesicles were assayed for the presence of pleted while p38 was enriched in the vesicle fractions
kinesin by immunoblotting. Kinesin was detected using (data not shown; Fig. 3 ) . Light chains of kinesin were
mouse monoclonal antibodies directed against the 124 not detected in organelle fractions. Pfister et al. [1989a]
kD heavy chain [Pfister et al., 1989al. Kinesin was characterized a series of light chain antibodies to kinesin
found in both the V1 and V2 fractions following exten- using blots containing microgram quantities of kinesin
sive purification including separation of vesicles by size per lane. We have recently determined that light chains
on controlled-pore glass columns (Fig. 4). Kinesin rep- have a lower affinity for blotting membranes than heavy
resented 0.14 0.06% of the total protein in V1 (N = chains (D.S. Stenoien and S.T. Brady, unpublished ob9) and 0.11 rt 0.04% of total protein in V2 (N = 9). servations). With kinesin present at less than 0.14% of
Kinesin made up 0.16 k 0.04% of total protein in brain total protein in purified organelles, the light chain prohomogenate indicating that kinesin was neither enriched teins may be obscured by the large number of proteins
*
24
Leopold et al.
9-00
I
I
6.00
-
-E
3.00
z
2.00
2
.-u
E
v
.-c
1 .oo
3.00
v)
(u
c
E
0.00
~
0.00
22 24 26 28 30 32 34 36
Fraction
Fig. 5 . Quantitation of total protein and kinesin in fractions from the
controlled-pore glass column. Kinesin content of individual column
fractions was determined by densitometry of immunoblots. The distribution of kinesin and the protein concentrations of column fractions
show that kinesin co-elutes with organelles, and reflect the fact that
the kinesin concentration in V1 was slightly greater than that of V2.
Data are from a representative experiment.
Fig. 4. Silver-stained gel (A) and immunoblot (B) showing protein
profiles and kinesin content of each of the steps in the vesicle preparation. The heavy chain (124 kd) of kinesin was detected by immunoblotting in each step of the preparation (see Fig. 3 for abbreviations). Lanes contain equal loads of protein. The only immunoreactive
band observed was 124 kD.
migrating at the same position as the light chains, which
compounds the difficulty in obtaining efficient light
chain binding to blotting membranes. As a result, detection of kinesin light chains using the '251-Protein A immunoblotting method employed in these experiments is
not feasible.
Once purified vesicle fractions were obtained, kinesin remained associated through further pelleting and
resuspension of vesicles or following several days of
incubation at 4°C (data not shown). Examination of individual fractions from gel filtration demonstrated that
the peak of kinesin fractionation directly coincided with
the V1 and V2 protein peaks (Fig. 5). The elution volume of pure kinesin on the controlled-pore glass (CPG)
column cannot be evaluated due to the unusually high
affinity of kinesin for glass surfaces. However, several
facts argue that kinesin could not elute with vesicles
unless a specific association between organelles and kinesin existed. According to the distributer (Sigma Chemical Co., St. Louis, MO), the CPG-3000 matrix used in
these experiments fractionates particles with molecular
weights ranging from 1,200 kD to 2,700,000 kD. Since
the molecular weight of synaptic vesicles exceeds this
range (MW = 176,000,000kD [Wagner et al., 19781)
and kinesin's molecular weight falls short of the range
(MW = 379 kD [Bloom et al., 1988]), the CPG column
should efficiently separate the two populations. Finally,
kinesin has a Stokes radius of 9.64 nm while the average
radii of organelles in the V1 and V2 fractions was 104
nm and 46 nm, respectively. As a result, the 300 nm pore
size of the CPG-3000 matrix should efficiently separate
the soluble kinesin from organelles.
The association of kinesin with V1 and V2 vesicles
was observed directly using immunogold staining. Cryosections of both V I and V2 fractions exhibited gold
labeling adjacent to membranous structures (Fig. 6a,c).
The gold particles often occurred in clumps with an average of 5.1 t- 0.17 (N = 207 clusters) and 4.8 0.15
(N = 260 clusters) gold particles per cluster on V1 and
V2 vesicles, respectively. Gold particles were rare or
absent from regions of the sections which did not clearly
contain membranous structures, When an irrelevant primary antibody was substituted for anti-kinesin antibodies
(Fig. 6b,d) or when primary antibody was not included
+_
Kinesin Organelle Association
(data not shown), a very low background was present
and clumps of gold were not observed.
Characterization of Mitochondria
Characterization by electron microscopy demonstrated that the mitochondria were obtained intact and at
high purity. Thin sections of mitochondrial pellets revealed that the organelles exhibited the characteristic
double membrane structure (Fig. 7). Isolated mitochondria appeared in both condensed and orthodox structures
[Hackenbrock, 19681 indicating that the mitochondria
were isolated in a range of metabolic states. In no case
were cytoskeletal structures observed in the mitochondrial fractions. A mitochondrial marker enzyme, cytochrome c oxidase, was enriched nearly 18-fold while a
marker for cytosolic proteins, lactate dehydrogenase,
was decreased by 11-fold (Table 11).
Association of Kinesin With Mitochondria
The association of kinesin with mitochondria was
investigated using immunoblots and immunogold staining of cryosections. As in the vesicle fractions, kinesin
heavy chain was detected in each fraction of the mitochondria preparations including the most highly purified
fraction, P4 (Fig. 8). Kinesin accounted for 0.02
0.01% of the protein in the P4 fraction (N = 3).
Cryosections of mitochondrial (P4) pellets exhibited specific staining when treated with anti-kinesin antibodies that were visualized with 9 nm gold particles
conjugated to Protein A . The gold staining appeared in
large clusters containing an average of 10.4 0.54 particles per cluster (N = 428 clusters) (Fig. 9a). When an
irrelevant primary antibody was substituted (Fig. 9b) or
when the primary antibody was omitted (data not
shown), no specific staining appeared. A polyclonal antiserum to mitochondrial antigen p24, a protein associated with the mitochondrial inner membrane [MoseLarsen et al., 19821, gave a distinctly different pattern of
staining (Fig. 9c). The p24 was uniformly distributed
and did not exhibit clusters.
Mitochondria were prepared from both fresh and
frozen bovine brain. Mitochondria prepared from fresh
tissue exhibited better preservation of cristae structure.
However, the two methods yielded mitochondria which
had similar amounts of associated kinesin (as determined
by immunoblotting), and the kinesin was present in clusters on the surface of the organelles (as determined by
immunoelectron microscopy).
*
*
Other Organelle Fractions
Several additional organelle fractions were assayed
for the presence of kinesin. Both nuclei and coated vesicles were purified from bovine brain. Isolated Golgi
membranes were obtained from rat liver. Coated vesicles
25
were found to contain a substantial amount of kinesin
(Fig. 10). Immunoblotting of SDS-PAGE samples containing equal protein loads did not reveal the presence of
kinesin in nuclear or Golgi membrane fractions (Fig.
10). These fractions were not assayed by immunoelectron microscopy.
DISCUSSION
Kinesin Associates With Purified Organelles
Structural and biochemical studies have provided a
detailed picture of three characteristics of kinesin: the
ability to bind microtubules, to hydrolyze ATP, and to
induce microtubule motility in vitro. These three properties of kinesin support proposals that it is responsible
for organelle translocation along microtubules in cells.
However, a fourth characteristic is predicted by this
model. Kinesin should associate with organelles that
move in the fast component of axonal transport and exhibit similar forms of motility in other cell types. We
designed a set of experiments to test whether kinesin
satisfies this requirement for organelle association. Three
criteria were utilized to determine whether kinesin associated with organelle fractions: 1) kinesin should be detectable by immunochemical methods in highly purified
fractions of organelles, with purity of organelle fractions
established by electron microscopy, enrichment of proteins indicative of the particular organelle fraction, and
depletion of proteins characteristic of other cellular fractions; 2) kinesin should localize to organelle membranes
in purified fractions; and 3) the stoichiometry of kinesin
binding should reflect the physiology of the neuron. Enrichment of kinesin in organelle fractions was not included as one of the criteria due to the fact that both
soluble and membrane-bound pools of kinesin may exist
[Brady, 1985; Vale et al., 1985; Hollenbeck, 19891.
Moreover, the membrane-bound pool should be distributed among a wide variety of organelles; therefore, the
amount of kinesin in any one organelle fraction is likely
to represent only a small proportion of total cellular kinesin. Compatible with this prediction, kinesin was detected in every fraction of both the synaptic vesicle and
mitochondria preparations (Figs. 4, S), although other
subcellular fractions, including nuclei and Golgi membranes, lacked kinesin.
The two independent approaches described in this
paper demonstrate that kinesin has the ability to bind
stably to a variety of organelles which are moved in fast
axonal transport and that the association is organelle specific. Immunoblotting was used to detect kinesin in association with various subcellular fractions. Purified synaptic vesicles, mitochondria, and coated vesicle fractions
contain kinesin, while no kinesin was detected in nuclear
or Golgi membrane fractions. Immunoelectron micros-
Fig. 6.
Kinesin Organelle Association
27
TABLE 11. Enzymatic Analysis of Mitochondria1 Fraction
Homogenate
P4 (mitochondriaY
Lactate dehydrogenase
Cytochrome C oxidase
1"
.088
17.8
lb
aHomogenate activity for LDH was 277 Fmol min-' mg-'.
bHomogenate activity for cytochrome c oxidase was 113 Fmol min-'
mg-'.
'Activity for P4 presented relative to homogenate activity of 1.
Fig. 7. Ultrathin sections of pelleted mitochondria. Purified mitochondria exhibited both the orthodox (Or) and condensed (Cn) conformations. In some cross sections, the matrices of orthodox mitochondria were swollen to the point that no intermembrane space was
visible. Bar = 500 nm.
copy demonstrated that kinesin in purified organelle fractions was directly associated with membrane surfaces.
Purity of synaptic vesicles was shown by electron microscopy, enrichment of a synaptic vesicle specific protein (p38), and depletion of enzymatic markers specific
for mitochondria and cytosolic fractions. Purity of mitochondrial fractions was demonstrated by electron microscopy, enrichment of cytochrome c oxidase, and depletion of an enzymatic marker for cytosolic proteins.
Fig. 6. Kinesin found in association with membranes in cryosections
of vesicles. Cryosections of vesicle pellets were treated with antibodies to kinesin and then labeled using 9 nm gold particles conjugated to
Protein A. Clusters of gold particles were evident in areas of sections
which contained membranous organelles in both V1 (a) and V2 (c)
vesicles. In contrast, cryosections of V1 and V2 vesicles treated with
an irrelevant primary antibody did not label with gold-conjugated Protein A (b,d). Bar = 200 nm.
Several additional pieces of evidence support the conclusion that kinesin was specifically associated with synaptic vesicles (V2) and a microsomal fraction (Vl). Kinesin coeluted with vesicle fractions on a CPG sizing
column (Fig. 5 ) indicating a tight association between
kinesin and the vesicle fractions. The amount of kinesin
associated with the V1 and V2 fractions remained constant during extended incubations of several days. Specific association of kinesin with mitochondria is supported by the observation that kinesin has a non-random
distribution on mitochondria (Fig. 9). Furthermore, the
lack of kinesin in nuclear and Golgi membrane fractions
argues against adventitious kinesin binding in vesicle and
mitochondria1 fractions (Fig. 10). Therefore, the kinesin
found in organelle fractions satisfied the first two criteria
for specific association set forth above.
The stoichiometry of kinesin binding to synaptic
vesicles in the V2 fraction may be calculated using the
percentage of total vesicle protein represented by kinesin
determined in this report in combination with physical
parameters of synaptic vesicles published by Wagner et
al. [1978]. The ratio calculated in this way was 1 kinesin
molecule: 16 vesicles. This calculated ratio was supported by immunoelectron microscopic images showing
that colloidal gold particles do not appear on every vesicle. While this stoichiometry may be lower than one
would expect a priori, the value becomes a reasonable
estimate when one considers the cellular physiology of
synaptic vesicles in the neuron. Many synaptic vesicles
are derived from a pool of vesicles stored in the presynaptic terminal prior to fusion at the active zone. Such
vesicles may not have kinesin bound to their surfaces
since they have reached their destination and are not
expected to undergo further anterograde axonal transport. In addition, some kinesin which is initially bound
to vesicles undergoing axonal transport may be stripped
off by the buffer conditions and physical manipulations
encountered during homogenization and preparation of
vesicles. The relative contribution of each explanation
remains to be determined. As a result of these variables,
the amount of kinesin found associated with synaptic
vesicles and other organelles in this study is not likely to
reflect maximal binding of kinesin or levels of binding
found on actively translocated organelles in situ. The
28
Leopold et al.
A.
H
11E
97
66
45
29
S1
P1 S2
P2 S3
L3 C3
P 3 P3
P4
tions as thin as 100 nm reveal clusters of kinesin molecules on mitochondria. Estimates of mitochondrial
kinesin based on such images suggest a value for the
number of kinesins on an intact mitochondrion comparable to that obtained by calculations from quantitative
immunoblots.
The presence of kinesin on some classes of organelles and absence from others indicates specificity in
kinesin-membrane interactions. This result raises questions about the molecular basis of such specificity. The
possible mechanisms for confemng specificity to kinesin
organelle interactions include the presence of organelle
specific receptors and/or organelle specific isoforms of
kinesin. The possibility that different forms of kinesin
may interact with different organelles is particularly attractive. Multiple isoforms of bovine kinesin heavy and
light chains have been reported [Wagner et al., 1989; Cyr
et al., 19911. These isoforms may arise from posttranslational modifications of kinesin [Murphy et al., 19891
(Elluru, Bloom, and Brady, unpublished results) or from
genetic diversity [Cyr et al., 19911. Preliminary evidence
indicates that kinesin isoforms are differentially distributed in purified organelle fractions (Leopold, McDowall,
and Brady , unpublished observations).
Implications of Kinesin Binding
The association of kinesin with synaptic vesicles
and mitochondria from bovine brain strongly supports
the proposal that kinesin mediates fast axonal transport.
124 KD
The microtubule dependence of fast transport is a common characteristic of intracellular motility in many cell
types [reviewed by Schliwa, 1984; Kelly, 1985; Vale,
Fig. 8. Silver-stained gel (A) and immunoblot (B) showing protein
19871. While many properties of organelle transport in
profiles and kinesin content of each fraction of the mitochondria prepaxons
are distinct from motility in other cells, the basic
aration. Kinesin could be detected in each fraction of the mitochondria
machinery
may be the same.
preparation including homogenate (H), low speed supernatant (S 1 )
Several pieces of evidence suggested that kinesin is
and pellet (Pl), high speed supernatant (S2) and pellet (P2), 3% Ficoll
supernatant (S3), layer between supernatant and cushion (L3), 6% bound to axonally transported vesicles. Kinesin antibodFicoll cushion (C3), soft pellet (P’3), pellet (P3), and washed pellet ies inhibit organelle motility in squid axoplasm [Brady et
(P4). Lanes contained equal loads of protein. The only immunoreactive band observed was 124 kD. The immunoblot shown in B was al., 19901, and kinesin is found near membrane-bounded
exposed on the radioactive blot for a longer period of time than the organelles in mammalian nerve [Hirokawa et al., 19911.
immunoblot shown in Figure 4B and has resulted in a stronger signal In addition, the pharmacology of organelle transport in
in the early steps of the fractionation.
axons [Forman et al., 1984; Brady et al., 1985; Pfister et
al., 1989b; Leopold et al., 19901 is similar to the pharmacology of ATPase activity and force generation of
amount of kinesin obtained in these studies is more likely kinesin in vitro [Porter et al., 1987; Cohn et al., 1987,
to represent minimum levels of kinesin in purified or- 1989; Pfister et al., 1989b; Wagner et al., 19891. With
ganelle fractions, and the amount of kinesin per or- this demonstration that kinesin copurifies with transganelle in vivo may well be greater.
ported organelles, the role of kinesin in fast axonal transUsing data provided by Srere [1985] to approxi- port is further substantiated. There remains a possibility
mate mitochondrial protein content and dimensions in that other organelle motors act synergistically with kinecombination with the concentration of kinesin in the sin in the control of axonal transport.
A consideration of the molecular dimensions of kimitochondrial fraction as determined in this study, the
number of kinesin molecules per mitochondrion was es- nesin and of a synaptic vesicle provides some indication
timated to be approximately 200. Mitochondria1 cryosec- about the number of kinesins that could be associated
B.
Kinesin Organelle Association
29
Fig. 9. Kinesin found in association with mitochondrial membranes in
cryosections of mitochondria. Cryosections of mitochondrial pellets
were treated with antibodies to kinesin (H-1, H-2, L-I, and L-2) and
labeled with 9 nm gold particles conjugated to Protein A. Clusters of
gold particles were present in association with the membranous organelles in the sections (a). Clusters contained more gold particles, on
average, than those clusters found on vesicle sections (Fig. 6). Gold
particles were not uniformly distributed over the surface of mitochondria giving the sections a patch-like appearance. In contrast, cryosections treated with an irrelevant primary antibody did not exhibit gold
particle staining (b). A mitochondrial inner membrane marker, p24,
did not exhibit a clustered pattern of staining (c). Bar = 200 nm.
with a single vesicle. The kinesin holoenzyme is 80 nm
in length [Hirokawa et al., 19891 of which 20-30 nm is
thought to project away from the membrane surface [Hirokawa et al., 1989; Miller and Lasek, 19851. As a result, the surface of a 30-50 nm diameter synaptic vesicle
is unlikely to have room for more than 1-5 kinesin molecules and the probability that a kinesin will interact with
other motors on a vesicle surface is increased [Brady,
19911.
Translocation of mitochondria is a well-recognized
30
Leopold et al.
A.
CV
G
N
116
97
66
45
B.
124 KD
Fig. 10. Association of kinesin with other organelle fractions. The
kinesin content of coated vesicles (CV), isolated Golgi membranes
(C), and nuclei (N) were checked using immunoblots. Panel A shows
a silver stained gel of each fraction. Panel B shows the corresponding
immunoblot with antibodies to the heavy chain of kinesin. Kinesin
was detected only in coated vesicles and not in Golgi membranes or
nuclei. Kinesin appears as a doublet at 124 kD in the coated vesicle
lane. The presence of a 124 kD doublet is well established [see, for
example, Wagner et al., 19891. The doublet resolved on this gel due
to fact that the gel was allowed to run for a longer period of time than
the gels displayed in Figures 4 and 8 (note position of molecular
weight standards). Arrow marks clathrin which was enriched in the
coated vesicle fraction. Lanes contained equal loads of protein.
phenomenon with an extensive history of study [reviewed by Newcomer, 1940; Murray and Kopech, 1953;
and Novikoff, 19611. Two models which have recently
provided access to the study of mitochondrial motility
are neurons [Willard et al., 1974; Lorenz and Willard,
1978; Allen et al., 1982; Brady et al., 1982, 1985; Martz
et al., 19841 and primary cultures of endothelial cells
from heart tissue [Bereiter-Hahn and Morawe, 1972;
Bereiter-Hahn, 1976, 1978; Bereiter-Hahn and Voth,
19831. Martz et al. [1984] conducted a characterization
of mitochondrial motility in extruded axoplasm from
squid giant axon. Besides linear translocations, two other
properties of mitochondrial motility were noted. Mitochondria appear to branch, producing tri-radial structures. Branching is more common in preparations containing non-parallel microtubules, suggesting that the
branching results from multiple focal sites of contact
between mitochondria and microtubules. In addition, mitochondria occasionally undergo elastic recoil suggesting
that one site of attachment can prevent mitochondrial
movement mediated by a second site of attachment.
Combining these results with ultrastructural observations
of crossbridges between mitochondria and microtubules
[Ellisman and Porter, 1980; Tsukita et al., 1982; Raine et
al., 19711, Martz et al. [1984] proposed that binding
sites on the surface of mitochondria were localized to
focal sites or clusters. The data presented here provide
direct evidence for this model. Anti-kinesin immunogold
staining of mitochondria showed that kinesin was restricted to clusters on the surface of mitochondria.
A second set of observations indicates that mitochondrial motility is more sensitive to the energetic state
of squid axoplasm than the motility of other organelles
[Brady et al., 1982, 19851. Bereiter-Hahn and Voth
[ 19831 have found a relationship between the metabolic
state of mitochondria and mitochondrial motility by correlative phase contrast microscopy and transmission
electron microscopy. The authors report that mitochondria in the condensed state, where the matrix appears to
have increased electron density and decreased volume,
are non-motile. The transition from condensed to orthodox appears to be a graded phenomenon with motility
increasing as the orthodox shape is attained. The authors
also reported that the ultrastructural state and motility of
mitochondria could be influenced by microinjection of
ADP, ATP, or metabolic inhibitors. The possibility of a
connection between the metabolic state of the cytoplasm
and clustering of motors is under investigation.
An analysis of kinesin-organelle interactions relies
on knowledge of the identity of organelles which associate with kinesin and a means of describing that association. This report verifies that kinesin associates with
organelles that undergo fast axonal transport which fulfills a prediction of the current model for organelle mo-
Kinesin Organelle Association
tility. More importantly, this report identifies three organelle fractions which associate with kinesin and
thereby provides a basis for quantitative and comparative
analyses of kinesin interactions with organelles.
ACKNOWLEDGMENTS
The authors would like to thank Drs. John S. Peeler
and Richard G.W. Anderson, UT Southwestern, for providing coated vesicles; and Dr. Ulrich Seydel, Scripps
Clinic, and Dr. Ricardo Azpiroz and Ravindhra G. Elluru, UT Southwestern, for helpful discussions and technical advice during the preparation of this paper. We
would also like to thank Jason Rios for help with preparation of figures and Cheryl Hartfield for technical assistance. Initial experiments leading to this work were
performed by Lili Yamasaki. Antibodies to p38 were
provided by Dr. Paul Greengard, Rockefeller University;
antibodies to p24 were provided by Dr. Rodrigo Bravo,
Bristol-Meyers Squibb, Princeton, NJ; and, 2001 antibodies were provided by Drs. Michael s. Brown and
Joseph L. Goldstein, UT Southwestern. This work was
supported by National Institutes of Health (NIH) grants
NS 23320 (S.T.B .) and NS 23868 (S.T.B. and G.S.B.),
NIH Training Fellowship GM 08203 (P.L.L.), and by
Welch Foundation Grant 1-1077 (G.S.B. and S.T.B.).
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