Purification and assay of kinesin from sea urchin eggs and early embryos
DAN BUSTER and JONATHAN M. SCHOLEY
University o f California at Davis, Department o f Zoology and Graduate Groups in Cell and Developmental Biology, and
Biochemistry, Davis, CA 95616, USA
Summary
This paper describes the procedures used to purify
the microtubule motor, kinesin, from mitotic cells,
namely sea urchin eggs and cleavage stage embryos,
and describes methods for assaying its motor ac­
tivity.
Key words: kinesin purification, motility and ATPase, sea
urchins, microtubule-based transport.
Introduction
Intracellular transport systems that move and position
cytoplasmic particles such as organelles, chromosomes
and perhaps macromolecules, play essential roles in the
functioning of eukaryotic cells. Many particles are
thought to be transported along microtubule ‘tracks’ by
enzymes called ‘motors’. One such motor, kinesin, is a
microtubule-activated ATPase that transports particles
towards the plus ends of microtubules (Vale et al. 19856;
Porter et al. 1987; Cohn et al. 1989; Howard et al. 1989).
Kinesin has been isolated from a variety of eukaryotic
organisms (reviewed by McIntosh and Porter, 1989) and
several studies suggest that kinesin may perform a
number of critical functions within the cell (Vale et al.
1986). Possible physiological functions include: antero­
grade transport of vesicles in neurons (Vale et al. 1985a;
Schroer et al. 1988; Brady et al. 1990) and other cell types
(Dabora and Sheetz, 1988a; Pfister et al. 1989), spreading
of tubular membrane structures of lysosomes and the
endoplasmic reticulum along microtubules (Dabora and
Sheetz, 19886; Vale and Hotani, 1988; Hollenbeck, 1989;
Hollenbeck and Swanson, 1990), mitosis (Scholey et al.
1985; Leslie et al. 1987; Endow et al. 1990; Enos and
Morris, 1990; Meluh and Rose, 1990) and using micro­
tubule tracks to move and organize intracellular mem­
branes associated with interphase and mitotic asters of
early sea urchin embryos (Wright et al. 1991).
Sea urchin eggs and early embryos are well suited for
the study of kinesin-driven motile events in vitro and in
vivo. Sufficiently large quantities of eggs and synchronized
dividing embryos can easily be obtained for biochemical
and cytological studies of early embryogenic events such
as chromosome movement, directed transport of vesicles
and organelles and pronuclear migration (see Bloom and
Vallee, 1990 for a review). In addition, microtubules and
motors can be isolated from eggs and embryos and used to
reconstitute microtubule-based motility (e.g. Porter et al.
1987; Cohn et al. 1987,1989), and antibody microinjections
into cells of the early embryo can be used to learn the
function of motor proteins in vivo (Mabuchi and Okuno,
1977; Kiehart et al. 1982).
Journal o f Cell Science, Supplement 14, 109-115 (1991)
Printed in Great Britain © The Company o f Biologists Limited 1991
We have isolated and characterized kinesin from sea
urchin eggs and early embryos (see Table 1 for a summary
of the structural and functional properties of sea urchin
kinesin). The motor binds microtubules in a nucleotidesensitive fashion, and this property has been exploited in
the first two purification procedures described below,
which make use of the fact that kinesin binds to MTs (see
Appendix for abbreviations) in the absence of ATP and
ADP, in the presence of the nucleotide analogue, AMPPNP, or in the presence of the divalent cation chelators,
EDTA and PPPi; subsequently, MT-bound kinesin can be
released from MTs by adding MgATP. An alternative
purification scheme makes use of anti-kinesin monoclonal
antibodies that have been raised against sea urchin
kinesin (Ingold et al. 1988); the latter were used in the last
purification scheme described below which is a rapid
immunoaffinity procedure using one of our anti-kinesin
monoclonal antibodies covalently coupled to a Sepharose
matrix. To assay kinesin mechanochemical activity, our
lab routinely measures two properties of kinesin function:
the MT-activated hydrolysis of ATP and the translocation
of MTs over a surface coated with immobilized kinesin.
The procedures used by our lab to purify and assay sea
urchin kinesin will be described in detail below. Ad­
ditional information can be obtained from articles by
Scholey et al. (1984, 1985,1989); Porter et al. (1987); Cohn
et al. (1987, 1989); Leslie et al. (1987); Ingold et al. (1988);
Johnson et al. (1990).
Collection of sea urchin eggs and early embryos,
and preparation of cytosolic extracts
Most of our studies are performed with sea urchins,
Strongylocentrotus purpuratus, obtained from the Univer­
sity of California Bodega Bay Marine Biology Laboratory
or from Marinus, Inc. (Long Beach, CA). We sometimes
also use S. franciscanus, S. droebrachiensis, Lytechinus
pictus, and L. variegatus. S. purpuratus are transported as
rapidly as possible in styrofoam boxes, packed in layers
with paper well-soaked with sea water and cooled with ice
packs. The urchins can be processed for gamete production
immediately upon their arrival in the lab or they may be
placed in well-aerated tanks and processed at leisure.
Alternatively, the gametes of sea urchins (S. franciscanus
or S. purpuratus) are collected at Bodega Bay and
transported in jars suspended in large containers of
10-15 °C sea water.
1.
Sea urchins are induced to shed their gametes by
injection of approx. 2 ml of 0.56 m KC1 solution into the
coelomic cavity. The yellow eggs are collected by inverting
the shedding females onto beakers filled with sea water for
1-2 h. The white sperm are collected by inverting males
109
onto dry petri dishes. For production of HSS, perform this
and subsequent steps at 0 -4 °C; for developmental studies,
keep the eggs at their physiological temperature of
approx. 15 °C.
Note: Contamination of eggs with even traces of sperm
must be rigorously avoided to prevent unexpected fertiliz­
ation of the eggs. Thorough rinsing of hands, containers,
and tools with tap water will kill unwanted sperm.
2. Decant excess sea water from the egg-containing
breakers, pool the eggs, and pass the eggs 10-15 times
through sea water-moistened Nitex screen (150 um mesh;
Small Parts, Inc.) to remove debris and the eggs’ jelly
coats.
Production o f cytosolic extracts (HSS) (steps 3—5)
3. To produce HSS, transfer the eggs to 50 ml plastic
tubes and gently pellet using a low speed, short duration
(lm in) spin in a clinical centrifuge. Aspirate off the
supernatant and gently resuspend the egg pellets in
approx. 7 vols ‘19:1 buffer’ (see Appendix for buffer
recipes). Repeat the centrifugation and resuspension steps
twice more to remove completely the eggs’ jelly coats and
to minimize the Ca2+ concentration (many sea urchin
proteases are Ca2+-activated and MT polymerization is
Ca2+ sensitive). Then resuspend the eggs in sufficient
PMEG buffer to reduce the NaCl concentration to less
than 0 . 1 m and gently pellet again.
4. Resuspend the eggs in 2 vols PMEG buffer and
homogenize on ice using a chilled Dounce homogenizer
until intact eggs are no longer visible under a stereomicroscope (about 10-20 strokes).
5. Centrifuge the homogenate at 85 000 g for 30min at
4°C. Discard the large pellet and the orange, top lipid
layer; save and centrifuge the clear, intermediate super­
natant at 175 000 g for l h at 4°C. Once again, save the
clear, intermediate, high speed supernatant (HSS). This
material is a cytosolic extract and can be stored for
extended periods if immediately frozen in liquid nitrogen
and then transferred to —80 °C.
Embryo production (steps 3a—6a)
3a. For developmental studies, resuspend the dejellied
eggs in approx. 5 vols sea water (pH 8.1, 15 °C) containing
10 mu p-aminobenzoic acid (PABA) to soften fertilization
membranes. Aspirate off the supernatant when the eggs
have settled. Repeat this step 2 -3 times.
4a. Resuspend the eggs in at least 1 0 vol of previously
well-aerated sea water (containing 1 0 m M PABA, pH 8 . 1 ,
15 °C) and very gently stir with a paddle driven by an
overhead electrical motor. The metabolic rate of eggs
greatly increases upon fertilization. Adequate dilution of
the eggs into well-aerated sea water is necessary to
prevent asphyxiation.
5a. Activate sperm by diluting 2 drops of sperm into
50 ml sea water (use within 15 min). To fertilize eggs, add
approx. 0.0025 vol. of diluted sperm to the diluted eggs.
Caution: different sea urchin species are differentially
prone to polyspermy and the quantity of sperm used must
be varied accordingly (see Leslie and Wilson, 1989).
6a. The extent of successful fertilization can be assessed
within a few minutes by observing an aliquot of eggs
under a stereomicroscope: fertilized eggs will be ringed by
a clearly visible fertilization membrane. Egg development
is improved if the dilute egg suspension is occasionally
gently aerated. Well-fertilized and aerated eggs remain
largely synchronized in their mitotic cycles through two or
three divisions.
110
D. Buster and J. M. Scholey
7a. Aliquots of developing embryos can be taken at
selected points in the mitotic cycle for HSS production or
histological studies. For HSS production, the embryos
must be passed through a sea water-moistened Nitex
screen (~10 times) to remove the fertilization membranes.
The embryos can then be processed into HSS as described
above (steps 3-5).
Purification procedures
Kinesin purification by AMP-PNP-induced MT affinity
binding (Scholey et al. 1985; Cohn et al. 1987; Ingold et
al. 1988)
Depletion ofactomyosin and denatured protein. 1. Thaw
frozen HSS (stored at —80 °C in 50 ml plastic tubes) in a
beaker of tap-fed cool water. Move the thawed HSS to ice
immediately.
2. Incubate HSS with hexokinase (10 units ml-1) and
50 m i glucose (to convert ATP to ADP) at room tempera­
ture for 30 min. Then centrifuge at 100 000 g (30 min, 4°C)
to pellet actomyosin and denatured protein, leaving
kinesin and unpolymerized MT protein in the high speed
supernatant.
Induced MT binding by kinesin. 3. Kinesin can be
removed from the supernatant if kinesin is induced to bind
to MT polymers, and then the kinesin-MT complex is
pelleted. First, add 0.5-1 m M GTP and 20 fiM taxol and
incubate at room temperature (RT) for 15 min to promote
MT assembly in the supernatant. Supplement the solution
with >1 m M AMP-PNP to enhance MT binding by kinesin
and incubate at RT for 20 min. Sometimes we have used
5 units ml-1 apyrase in place of AMP-PNP to induce MTbinding by kinesin.
4. Centrifuge the solution over a 15 % sucrose cushion
(containing I m M GTP, 2.5 ¡u m taxol may be added to
stabilize MTs) in a swinging bucket rotor at 23 000 g,
60 min, 10 °C (or 57 000 g, 20 min, 10 °C, in fixed angle
rotor) to pellet the kinesin—MT complexes.
5. Wash the pellet by homogenizing in 0.1 m M GTP in
PMEG buffer (or in 20 m M EDTA, 2 m M ATP, 0.1 m M GTP,
10 /¿M taxol in PMEG buffer with magnesium sulfate
omitted) and let stand 10 min at 4°C. Centrifuge in a
swinging bucket rotor at 45 000 g, 20 min, 4°C (or at
57 000g, 20 min, 4°C, in a fixed angle rotor).
6. Release kinesin from MTs in the pellet by homogeniz­
ing the pellet in 100 m M KC1,10 m M MgATP (0.1 m M GTP,
5 , u m taxol may be included) and holding overnight at
0 -4 °C. Add a further 5 m M MgATP before centrifuging at
45 000 g, 20 min, 4°C to pellet MTs and leave MAPs in
supernatant.
Biogel A5M chromatography. 7. Fractionate the
extracted MAPs supernatant by gel filtration chroma­
tography (Biogel A5M (Bio-Rad), 1.8cmx 12 cm) in PMEG
buffer containing 1 m M ATP (ATP successfully omitted in
some experiments).
8. Fractions are tested for MT-translocating activity by
video microscopy (see below), and analysed by SDS-PAGE
and Western blotting (using anti-kinesin monoclonal
antibodies). The peak of kinesin activity constitutes those
fractions that will support MT movement at ^0.4/ans_1.
These fractions are pooled for further analysis.
MT binding and release. 9. The pooled kinesin fractions
are further purified by addition of 1.5mgml_1 P ll
phosphocellulose-purified bovine brain MTs (polymerized
by incubation with >10jUM taxol and 0.5 m M GTP for
15 min at RT; tubulin purified according to the method of
Table 1. Biochemical properties of sea urchin kinesin
1. Polypeptide subunits:
►2mol 130x103 Mr heavy chain
►2 mol 78/84 x l 0 3Mr light chain
2. Size/morphology:
►Sedimentation c o e ffic ie n t s .6 S
►2 heads, stalk and tail in EM
►Total length=75nm
►Stalk diameter—3 -5 nm
►Globular head diam eter=9-10nm
►Tails—heterogeneous
3. Globular heads:
►Released by proteolysis as 45x l 0 3Mr fragments
►Formed from N-terminal portion of heavy chain
►Contain ATP and MT binding sites
►Motor domains
4. MT-activated MgATPase:
(nmol min-1 m g-1 )
►Intact kinesin typically—50
►Kinesin proteolysed to 45x l 0 3Mr—520-615
5. Motility:
►Plus-end directed
►Blocked by some monoclonal antibodies that bind heads
►Substrates-, broad specificity for Mg nucleotides
(A TP>GTP>TTP=U TP>CTP>ITP)
MgATP; K m—60 ¿¿m, Vmax—0.6 um s~1
MgGTP; K m—2 m M, Vmax=0.4 um s~1
►Inhibitors; Mg-free ATP and Mg2+ chelators (EDTA, PPPi)
MgATPyS; competitive inhibitor, # ¡= 1 5 //m
MgADP; competitive inhibitor, # ¡= 1 5 0 /<m
AMP-PNP, vanadate; potent/complex inhibitors
AMP-PCP, NEM; weak/complex inhibitors
Williams and Lee, 1982), 20 m M EDTA, and 2 m M ATP, and
incubation for 20 min at RT. Pellet the resulting kinesin-M T complex by centrifugation (45 000^, 20 min,
20 °C).
10. Resuspend the kinesin—MT pellet in PMEG buffer
containing 10 m M ATP, 12.5 m M magnesium sulfate, 30 /im
taxol, and 0.1 m M GTP for 30 min, RT, to release kinesin
from MTs. Centrifuge the solution again at 45 000 g,
20 min, 20 °C to pellet MTs and leave purified kinesin in
the supernatant.
P l l phosphocellulose chromatography. 11. Tubulin and
ATP can be removed from the kinesin-containing super­
natant by P l l phosphocellulose chromatography; kinesin
(but not tubulin or ATP) binds to P l l phosphocellulose
resin (Whatman). Activate P l l phosphocellulose resin
according to manufacturer’s instructions and equilibrate
in PMEG buffer. Mix 1 vol P l l to 10 vols of supernatant
from step 10 (or from step 8 if required) for 30 min, 4°C.
12. Wash the resin batch-wise (by centrifuging in a
clinical centrifuge) 2 times with 5 vols PMEG buffer, then
2 times with 5 vols PMEG containing 0.1 M KC1 to remove
non-binding material. Pack the resin into a column and
then elute kinesin using PMEG buffer containing 3=0.5 m
NaCl or KC1. Collect 25 drop fractions and use SDS-PAGE
to locate kinesin-containing fractions. Dialyze the pooled
fractions against PMEG buffer (0-4°C ) to remove salt.
The purified kinesin has both ATPase and MT translo­
cating activities and can be stored for extended periods in
liquid nitrogen. Using this method, we obtain 50-500 ,ug
kinesin from 100 ml HSS (see Fig. 1A).
Kinesin purification by PPPi-induced M T affinity
binding (Johnson et al. 1990)
P ll phosphocellulose chromatography o f HSS. 1. Thaw
150 ml of HSS (stored at -8 0 °C ) and supplement with
fresh protease inhibitors (see Appendix). Add HSS to
approx. 20 ml of activated P l l phosphocellulose resin and
gently mix the slurry for l h (all steps at 0 - 4 °C). Remove
non-binding proteins by washing the P l l resin 3 times
with about 4 vols of PMEG buffer, then once with 4 vols of
A
B
C
Fig. 1. SDS polyacrylamide gel electrophoresis of purified sea
urchin kinesin. The three lanes are o f kinesin samples
analyzed on different, Coomassie-stained gels. Kinesin was
purified by (A) AMP-PNP-induced MT binding (procedure I),
(B) PPPi-induced MT binding (procedure II), and (C)
immunoaffinity chromatography (procedure III). Arrows
indicate the 130 x 10s Mr kinesin heavy chain polypeptides.
Brackets mark the 84 and 78 X 103Mr kinesin light chain
polypeptides. Procedure I yields active kinesin with variable
light chain content, whereas procedure II yields kinesin with
similar MT-translocating properties and a consistent light
chain content (1 mole heavy chain: 1 mole light chain doublet).
Procedure III yields kinesin that is usually inactive (because of
contaminating SUK4) but has consistent light chain content (1
mole heavy chain: 1 mole light chain doublet). Contaminating
polypeptides can be recognised because they also cosediment
with control IgG resins.
PMEG buffer containing 0.1 m KC1. A clinical centrifuge is
used to pellet the resin after each wash.
2. Bound proteins (including kinesin) are eluted with 2
vols of PMEG buffer containing 0.75 m KC1. The eluate is
passed through glass wool to remove any residual P l l
resin - the presence of P l l resin in subsequent steps
Purification and assay of sea urchin kinesin
111
greatly decreases kinesin yield. Dilute eluate with 1 vol of
PMEG buffer.
Ammonium sulfate precipitation. 3. Precipitate kinesin
from the eluate by adding 0.39 g ammonium sulfate per ml
eluate (60 % final concentration) and stirring overnight at
4°C.
4.
Precipitated material is pelleted by centrifugation at
17 250# for 10min, 4°C. Resuspend the pellet in 3 ml
PMEG buffer and clarify by centrifugation at 17 250 g,
10 min, 4°C.
Biogel A1.5M chromatography. 5. The clarified super­
natant is fractionated and desalted by gel filtration
chromatography on a Biogel A1.5M (Bio-Rad) column
(2.5 cmx 15 cm) equilibrated with PMEG buffer containing
2mM ATP and 100 u m (instead of 2.5 h i m ) magnesium
sulfate. Kinesin-containing fractions are identified by
SDS-PAGE and immunoblotting, pooled, and then sup­
plemented with 2 0 / i m (final concentration) taxol.
PPPi-induced, MT binding: 6. Polymerize MTs by
mixing a previously prepared, concentrated solution of
P ll phosphocellulose-purified bovine brain tubulin (suf­
ficient tubulin is added to make the tubulin final
concentration 1-2 mg ml-1 after the pooled Biogel frac­
tions are added at step 7), with roughly equimolar taxol
(10 jUM per lm g m l-1 tubulin), and 0.5 m M GTP. Incubate
at 37 °C for 30 min.
7. Chill and then mix the MT solution with the pooled,
taxol- and kinesin-containing Biogel fractions and incu­
bate for 10 min, 4°C. Add 20-30 m M (final concentration)
PPPi (or EDTA) to promote kinesin binding to MTs and
incubate for a further 10 min, 4°C. The kinesin-MT
complexes are pelleted by centrifugation at 85 000 g,
20 min, 4°C.
8. Kinesin is released from the MTs by resuspending
the pellet in 2 ml of cold resuspension buffer using a
chilled Dounce homogenizer and incubating for 1 h at 4°C.
Pellet MTs by centrifugation at 85 000#, 20 min, 4°C.
P ll phosphocellulose chromatography. 9. Mix the kin­
esin-containing supernatant with 1—2 ml of activated P ll
phosphocellulose resin and gently rock the slurry at 4°C
for 1 h. Wash the P ll resin 2 times with about 8 vols of
PMEG buffer, and wash twice more with PMEG buffer
containing 0.1m KC1 to remove non- and weakly-binding
proteins. After each wash, gently pellet the resin in a
clinical centrifuge before resuspending in the next wash
buffer.
10. Pack the washed P ll resin into a disposable 11ml
polystyrene column (Bio-Rad), and elute kinesin by
developing the column with PMEG buffer containing 0.6 m
KC1 and collecting 25 drop fractions.
Kinesin-containing fractions can be identified by
SDS-PAGE and dialyzed against PMEG buffer to remove
KC1. This procedure yields —0.1 mg active kinesin from
100 ml HSS (see Fig. IB).
Kinesin purification by monoclonal antibody
immunoaffinity chromatography (Johnson et al. 1990)
For immunoaffinity chromatography, we routinely use the
monoclonal anti-kinesin, SUK4, prepared from mouse
ascitic fluid using Protein A Affigel and the MAPs II kit
(Bio-Rad) as described previously (Ingold et al. 1988;
Johnson et al. 1990). Nonspecific mouse IgG and rat IgG
purchased from Sigma are routinely used as controls for
non-specifically sedimenting polypeptides. We usually
immobilize the SUK4 to cyanogen bromide-activated
Sepharose 4B (Pharmacia) to produce an effective immunoadsorbent. We have used SUK4 Sepharose immuno112
D. Buster and J. M. Scholey
adsorbents on fresh and frozen HSS from eggs and
embryos, and we have observed no significant differences
in the kinesin preparations obtained. Our current
immunoaffinity protocol is described below.
Preparation o f HSS. 1. Thaw frozen HSS and sup­
plement with fresh protease inhibitors (see Appendix).
Clarify by centrifugation at 150000 g", lh , 4°C, to remove
any denatured protein.
Elimination o f non-specific IgG-binding proteins.
2. Gently mix the HSS with 1/20 vol rat IgG-Sepharose
for 1—2h to remove nonspecific IgG-binding proteins (all
steps at 4°C). The resin is pelleted by brief centrifugation
in a clinical centrifuge and the HSS saved. The resin is
regenerated with successive washes of 1 m NaCl in 10 m M
phosphate buffer, pH 8.5, then I m NaCl in 0.1 m
diethylamine buffer pH 11.5, and then PMEG buffer.
Immunoprécipitation. 3. The preadsorbed HSS is div­
ided and mixed with either control (non-specific mouse
IgG) or SUK4 resin that has been incubated with an equal
volume of 10 mg ml-1 SBTI for 1-2 h at 4°C to saturate
nonspecific protein binding sites. Approximately 1 vol of
resin is incubated with 50 vols HSS for 1-2 h (4°C) with
gentle mixing.
4. Wash the resins 3 times with 30 vol PMEG buffer.
Then wash with 30 vol of 1 m NaCl, 10 m M ATP (pH 7) to
remove kinesin-associated proteins (see Schroer et al.
1988), and then with 30 vol I m NaCl in 10 m M phosphate
buffer pH 8.5 (to get the resin into a higher pH solution in
preparation for elution).
5. A 50 fil sample of a resin may be boiled in SDS sample
buffer (Laemmli, 1970) for PAGE analysis. The remainder
of the resin (approx. 400-450 ¡A) is treated with at least 2
vols of 1 m NaCl, 0.1 m diethylamine pH 11.5 to elute the
bound kinesin. The eluate can be neutralized by immedi­
ate addition of 0 . 1 m sodium acetate (pH4) or I m dibasic
phosphate. Alternatively, kinesin can be precipitated from
the eluate using 20 % trichloroacetic acid and washed with
cold acetone.
Kinesin purified by this procedure lacks MT translocat­
ing activity, possibly as a result of a small amount of the
function-blocking monoclonal, SUK4, leaching from the
column into the eluate or because the elution conditions
denature the kinesin. In some experiments using the same
elution conditions, we were able to recover active kinesin
using immunoadsorption with SUK2-Sepharose, although
the efficiency of adsorption was lower than with SUK4Sepharose. Approximately 0.5-1 mg kinesin can be immu­
noaffinity purified from 100 ml HSS (Fig. 1C). The resins
can be re-used if they are immediately neutralized with
PMEG buffer and regenerated with the same washing
protocol as is used with the rat IgG-Sepharose (above). We
have found that some kinesin (presumably denatured)
becomes irreversibly bound to re-used SUK4-Sepharose
resin. This residual kinesin does not elute from the resin
and, therefore, presumably does not contaminate sub­
sequent immunoaffinity purification experiments.
Assay procedures
Video microscopic assay o f sea urchin kinesin-driven
microtubule motility
We have adapted the original MT gliding assay described
by Vale et al. (1985a) to allow us to measure conveniently
the speed of kinesin-driven MT movement over glass
surfaces in real time. This is a highly reproducible,
quantitative assay that allows us to characterize motor
enzymology and screen for probes that inhibit kinesindriven transport using very small quantities of the motor
(on the order of 1 fig per assay).
The motility assay (Porter et al. 1987; Cohn et al. 1987,
1989)
1. Spread a solution of purified kinesin (15 fA) onto a No.
1 coverslip and allow it to adsorb for 20 min in a
humidified chamber.
2. After adsorption, 2 ul of approx. 0.2 mg ml-1 MTs
(bovine brain tubulin purified by P ll phosphocellulose
chromatography and polymerized in 1 mM GTP and 20 [im
taxol) are added, along with 0.5-2.0 /¿I each of nucleotide
and/or inhibitor solution (made up as stock solutions of
1-100 mM) to obtain a final volume of approx. 20/A.
3. Invert the coverslip and seal the edges to a
microscope slide with molten VALAP (vaseline/lanolin/
paraffin, 1:1:1 (w/w/w)).
4. The sealed slide is mounted on the microscope,
taking care to avoid bubbles or dust in the immersion oil.
Focus onto the coverslip surface looking through the
ocular, adjust field iris and condenser with lamp set for
critical illumination and differential interference contrast
(DIC) optics set at extinction. Redirect image to monitor,
then turn slider away from extinction until MT-covered
coverslip surface is visible. ‘Fine tune’ contrast on monitor
using optics and camera control box by trial and error. Use
the computer mouse to track and measure velocities of
10-30 MTs, and finally command the computer to print
out the velocity histogram, with the mean MT velocity and
the standard deviation.
In our lab, kinesin-driven MT translocation is visualized
by use of a video-enhanced microscope system consisting of
a Zeiss standard research microscope with a X100 (NA
1.25) objective, x2 optavar, a wide-band-pass green
interference filter (wavelength 546 nm), and standard DIC
optics. The microscope is equipped with a 100 W mercury
arc lamp set at a critical illumination and is connected to a
MTI Series 68 Newvicon video camera (equipped with
remote gain and black offset level) whose output is
directed to a RCA monochrome monitor. Using this set up,
we find that no computer background subtraction is
needed to visualize the MTs (Porter et al. 1987; Cohn et al.
1987, 1989). The velocities of MTs ‘gliding’ across the
coverslip are measured in real time using computerassisted analysis (Cohn et al. 1987,1989). The video output
of an Amiga 1000 computer is mixed with the output of the
MTI video camera using a video mixer installed by DAGE
Inc. - allowing the image of the computer’s mousecontrolled cursor (an arrow) to be superimposed on the
image of the MTs. Alternatively, a Genlock-type video
mixer can be connected to the Amiga computer directly,
for better resolution and greater versatility. A computer
program (Appendix) monitors the x and y coordinates of
the cursor, as well as the time measured by an internal
computer clock. In practice, the cursor is moved to the site
of a MT end and the mouse input button triggered to mark
its position and the time of the observation. After an
arbitrary period (usually 10-30 s), the cursor is moved to
the new site of the same MT end and again the mouse
button triggered. The program calculates the distance
between the two x,y coordinates (using scalar factors
between x,y coordinates and actual distances measured
previously with a stage micrometer) and divides this value
by the time difference to obtain the velocity of the
translocating MT. After the measurement of an arbitrary
number of MT velocities (usually 10-30), the program will
calculate the mean velocity and standard deviation and
also print out a histogram of MT velocities.
We have also used the assay to monitor the velocity of
MT-gliding induced by 21S dynein prepared by sucrose
density gradient centrifugation from sea urchin sperm
flagellar axonemes. We find, however, that the dynein
often moves MTs too fast for us to accurately find MT ends,
causing greater errors in the measured velocity. There­
fore, we would recommend the restricted use of this assay
for accurately measuring velocities ^2/im s“ 1. Other
advantages and limitations of this assay are discussed by
Cohn et al. (1989).
Use of MT motility assays for analysis o f steady state
kinetic parameters of kinesin (Cohn et al. 1989)
Using the procedure described above, MT motility assays
and their analyses can be performed to determine the
kinetic parameters of kinesin-driven MT translocation.
1. Adsorb 14-15 fA kinesin to a glass coverslip for
20 min, then add the desired concentrations of substrates,
inhibitors and phosphocellulose-purified bovine brain MTs
(to 20 /ugml-1) to a final volume of 20 fA. Nucleotide stock
solutions are made up as 100 mM solutions of 0.1 m Tris,
pH 7. Inhibitor solutions are made up as 100mM stocks in
either 0.1 m Tris pH 7 or PMEG buffer. Mg-nucleotides (or
analogs) are prepared by adding 100 mM MgS04 to the
stock solutions. Dilutions of working solutions are made in
PMEG buffer.
2. Measure the velocities of at least 10 MTs for each
data point and each experiment should be performed on at
least two different preparations of kinesin. For determi­
nations of Km and K[, kinesin is used at a concentration of
250-300 fig ml-1, which is at least 4-5 times the critical
concentration necessary for MT motility (Cohn et al. 1987,
1989).
Note: In order to eliminate any decrease in ATP
concentrations during the motility assays due to hydroly­
sis, perform all experiments using ATP at concentrations
= S 1 0 0 um (except those involving measurements of ADP) in
the presence of an ATP regenerating system of
10 units ml-1 creatine-phosphokinase and 50 mM phosphocreatine. Samples containing creatine-phosphokinase and
phosphocreatine retain MT motility for several hours,
even when using ATP concentrations as low as 10-25 fiM.
3. The Vmax and Km of substrates that support kinesindriven MT-motion can be obtained from Lineweaver-Burk
plots (Fig. 2). To study compounds interfering with MT
translocation, the type of inhibition is determined by
plotting the reciprocal of MT velocity versus the reciprocal
of substrate concentration at a constant inhibitor concen­
tration (Lineweaver and Burk, 1934) or the reciprocal of
MT velocity versus inhibitor concentration at constant
substrate concentration (Dixon, 1953).
Use of motility assays for identifying function-blocking
kinesin antibodies (Ingold et al. 1988)
The ability of antibodies to inhibit the MT-translocating
activity of kinesin can be assayed using video-enhanced
microscopy and computer-assisted velocity analysis as
described above.
1.
Adsorb 14/d of 100-300 /igml“ 1 kinesin solutions
onto coverslips for 10-20 min at RT in a humidified
chamber before addition of the test antibodies. Then add
antibody solutions to the coverslips for an additional
10-20 min. Routinely we use solutions of monoclonal
antibodies purified from ascitic fluid. These can be
Purification and assay of sea urchin kinesin
113
l/[M g ATP] (fiM -')
Fig. 2. A Lineweaver-Burk plot of kinesin-driven microtubule
gliding rate as a function of MgATP concentration. Values for
the maximal velocity (Vmax) and the Michaelis constant (K m)
can be obtained from this plot (Table 1; see Cohn et al. 1989 for
details).
2
[IgG] (Mgml ')
Fig. 3. Effects of two monoclonal antibodies on kinesin-driven
microtubule gliding. SUK2 is an IgG that binds the stalk of
kinesin and does not significantly inhibit microtubule
movement, whereas SUK4 is an IgG that binds to the kinesin
head and blocks kinesin-driven motility. (See Ingold et al. 1988
for details).
obtained at sufficiently high concentrations that only
small (1 -2 /il) volumes are needed.
2. Add polymerized, P l l phosphocellulose-purified, bov­
ine brain MTs (20 fig m l-1) and MgATP (10 m M ) to the
coverslip, and seal the coverslip to a slide using VALAP.
3. Determine the velocity of a gliding MT over an
interval of at least 10 s and randomly select 10-20 MTs
from several microscopic fields to be used for each data
point. Each set of data points and standard deviations in
Fig. 3 is from single typical experiments that have been
reproduced many times.
The ATPase assay (Cohn et al. 1987)
ATPase assays are performed at RT by modification of the
method of Seals et al. (1978). The assays themselves are
quantitative and reproducible (even though we have
observed variability in the specific ATPase activity of our
kinesin preparations; see Cohn et al. 1987), but require
larger quantities (—100 ¡.ig per assay) of kinesin. The
specific ATPase activities of our tubulo-kinesin prep­
arations (<100 nmol min-1 mg-1 at 25°C) are usually
114
D. Buster and J. M. Scholey
much lower than those found by other workers, but the
activity can be stimulated markedly by addition of
function blocking antibodies (Ingold et al. 1988) or
proteolysis (Table 1).
1. Kinesin (prepared by purification procedure 1 or 2,
above) in 200-300 «1 PMEG buffer is mixed with 40 «1
phosphocellulose-purified bovine brain MTs (lO m gm l-1)
or with 40 /A buffer plus 1 ¡A of 10 mM taxol (as a control).
2. Add sufficient PMEG buffer to each assay tube to
give a final volume of 380 /A, and incubate the mixture for
approx. 10 min at RT.
3. To start the reaction, add 20 iA of 50 mM [y32P]MgATP (specific activity 10 000-40 000 cpm nmol-1
ATP). Remove aliquots (100 ,ul) at a minimum of three time
points (0, 5, 10, 15, 20, 30 or 60 min), and quench the
reaction in the sample by addition of 30 iA 10 % SDS.
4. Add 75,id phosphate reagent (4 vols 5 n sulfuric
acid/5% ammonium molybdate: 1 vol 0.1m silicotungstic
acid) to react with liberated phosphate; extract the
resulting 32P-labelled phosphomolybdate complex with
lm l of a 65%: 35% (v/v) xylene: isobutanol mixture.
5. After centrifugation in a microfuge to separate
organic and aqueous phases, analyze a 0.7 ml aliquot of the
organic phase (containing the P-phosphomolybdate) of
each sample by scintillation counting.
6. To calculate ATPase specific activity, aliquots of the
[y-32P]MgATP stock solution are scintillation counted to
determine cpm nmol-1 ATP for the stock solution. The
concentration of liberated phosphate for each sample from
step 5 can be calculated by dividing the sample’s cpm by
the value for cpm nmol-1 ATP. The ATPase specific
activity (nmol phosphate min-1 m g-1) is calculated by
dividing the slope of plots of free phosphate concentration
as a function of time by the protein concentration of the
kinesin sample.
Conclusion
It is clear that kinesin performs a number of vital
functions within the cell, all of which are a consequence of
kinesin’s ability to translocate cellular structures along
MT tracks. Sea urchin eggs and early embryos have
proved to be a rich source of kinesin and also serve as
amenable experimental subjects for cytological investi­
gations of kinesin function in fixed cells and in vivo. Our
lab has concentrated on isolating and characterizing sea
urchin kinesin, and has developed the purification and
assay procedures described here. This work has provided
us with basic information on the structure and in vitro
functions of the sea urchin kinesin motor molecule;
Table 1 summarizes our current understanding of these
properties of sea urchin kinesin. The isolation and assay
protocols described in this paper may assist other workers
who wish to study kinesin-like proteins isolated directly
from their natural host cells, or expressed in vitro or in
transformed cells from cloned and manipulated genes.
Appendix
Abbreviations
AMP-PNP: 5'-adenylyl imidodiphosphate.
HSS: high speed supernatant o f cytosolic extract.
MAP: microtubule associated protein.
MT: microtubule.
PAGE: polyacrylamide gel electrophoresis.
PPP;: tripolyphosphate.
SBTI: soybean trypsin inhibitor.
SUK: sea urchin kinesin (notation used for monoclonal
antibodies).
Buffer solutions (all concentrations are final)
1. Sea water: 760 g Instant Ocean sea salts brought to
20 liters with tap water (check specific activity=
1.025-1.026, 4°C).
2. 19:1 buffer: 530 m M NaCl, 28 m M KC1, I m M EDTA,
5 m M Tris HC1 in distilled water, pH 7.
3. PMEG buffer: 0.1m potassium Pipes, 2.5 m M mag­
nesium sulfate (promotes MT assembly), O .lm M EDTA
(chelates divalent metal ions which catalyze sulfhydryl
oxidation), 5 m M EGTA (chelates Ca2+ which inhibits MT
assembly and activates some proteases), 0.9 m glycerol
(establishes correct osmolarity for eggs) in distilled water,
pH 6.9. Stored at 4°C.
Protease inhibitors: l/zg rn l'1 pepstatin and leupeptin,
2 ,u g m l- 1 aprotinin, 100,ugml
SBTI, O .lm M phenyl
methyl sulfonyl fluoride, lm g m l-1 tosyl arginine methyl
ester, I m M dithiothreitol, 20/igml-1 benzamidine, I m M
sodium azide. Protease inhibitors are added to PMEG
buffer immediately before use.
4. Resuspension buffer: 0.5 m M GTP, 30 /j m taxol, 10 m M
ATP, 15 m M magnesium sulfate, 0.33 mg ml-1 bovine
serum albumin, 0.1m KC1 in PMEG buffer. Made
immediately before use.
The computer programs used to analyze MT translocation were
developed by Dr S. A. Cohn while working in the lab of J.M.S.
Current updates of the programs can be obtained from SAC
(current address: De Paul Univ., Dept of Biological Sciences, 1036
W. Belden Ave, Chicago, IL 60614, USA). SAC was recipient of an
American Cancer Society Postdoctoral Fellowship no. PF-2925.
This work was supported by American Cancer Society Grant no.
BE-46C and March of Dimes Birth Defects Foundation Grant no.
1-1188 to J.M.S. Thanks to Dr S. A. Cohn for reviewing an early
version of this paper.
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Purification and assay of sea urchin kinesin
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