Vol.
166,
January
No. 30,
2, 1990
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
STABILIZATION Magdalena
OF MICROTUBULES
R. Mejillano’,
‘Department
November
20,
653-660
BY GTP ANALOGUES
Janice S. Barton2 and Richard H. Himes’
of Biochemistry, Lawrence, Kansas
2Department
Received
AND
1990
University 660452106
of Chemistry, Washburn Topeka, Kansas 66621
of Kansas University
1989
We recently demonstrated that the nonhydrolyzable analogues of GTP (GMPPCP and GMPPNP) and ATP support the elongation phase of tubulin assembly and are incorporated into the E-site of polymerized tubulin. In this report we studied the stability of microtubules containing GTP analogues by examining length redistributions after shearing at polymer steady state. The mean length of a population of microtubules containing GMPPCP increased only by 37% over a 150 min time period after shearing. Microtubules which contained 70% ATP and 30% GDP at the E-site increased in length by 88%. In contrast, the mean length of microtubules assembled in the presence of GTP increased by 410% in the same time period. These results su gest that microtubules containing GMPPCP or ATP at their ends are stabilized f rom depolymerization. 0 1990Academic press, W.
Although in tubulin
it is well known that GTP at the exchangeable
is hydrolyzed
of GTP hydrolysis remain
unclear.
analogues,
to GDP during
in the polymerization
tubulin
reaction and in microtubule
and GMPPNP,
(1). Moreover,
these nucleotides
prevent the disassembly assembly conditions,
which normally
site (E-site) the roles
stability
that the nonhydrolyzable
as well as ATP, support the elongation
the assembly reaction and are incorporated tubulin
assembly to microtubules,
In a recent study, we demonstrated
GMPPCP
nucleotide
in significant
GTP phase of
amounts into polymerized
stabilize microtubules
at steady state and
occurs in the absence of GTP. Under non-
we also showed that these analogues and ATP displace GDP
Abbreviations: GTP, guanosine 5’-tri hosphate; GDP, guanosine 5’diphosphat.e; PCP, guanylylQ3, y-methylene) I ‘phosphonate; GMPPNP, guanylylimidodiphosphonate; ATP, adenosine 5’-tri hosphate; Pipes, piperaxine-N, N’-bis (2ethanesulfonic acid); EGTA, ethylene Pycol bis @uninoethyl ether)-N,N,N’N’tetraacetic acid; HPLC, high pressure 7 iquid chromatography. 0006-291x/90 653
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
166, No. 2, 1990
BIOCHEMICAL
from the E-site of tubulin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and that GMPPCP
binds to tubulin
with an affinity
which
is about lOOO-fold less than that of GTP. The ability dynamic
of these nueleotides
instability
that at polymer
to stabilize
model proposed by Mitch&on
steady state mass, microtubules at the ends. According
microtubules
is consistent
and Kirschner
(2) which postulates
become stabilized
tubulin-GTP
subunits
interconvert
between a growing phase of GTP capped microtubules
agents such as microtubule-associated
(5) apparently microtubules
contain
mainly
examined
core depolymerizes.
Based on this model,
assembled in the presence of nonhydrolyzable dynamic instability
tubulin-analogue
analogues
should be
behavior because these microtubules
along their length (6). To test this hypothesis,
the length dynamics at steady state of microtubules
ATP incorporated
and a shrinking
proteins (MAPS) (3,4) and glycerol
decrease the rate of these interconversions.
stable and not exhibit
by a cap of
to this model, microtubules
phase in which the GTP cap is lost and the labile tubulin-GDP Stabilizing
with the
with GMPPCP
into the polymer and compared them with those assembled
we or
in the
presence of GTP.
MATERIALS
AND METHODS
Preparation of Tubulin. Bovine brain tubulin was isolated by three cycles of assembly-disassembly (7) followed by chromatography on a phosphocellulose (Whatman Pll) - Biogel P-10 pig back column as described by Algaier and Himes (8). The purified tubulin in PEM r uffer (0.1 M Pipes, H 6.9,l mM EGTA and 1 mM MgS04) was drop-frozen in liquid nitrogen and store crat -80°C. Nucleotide Analysis of Tubulin. To determine the nucleotide content of unpolymerized tubulin, the protein was precipitated with perchloric acid and after neutralization, the supernatant was chromatographed on a Partisil SAX-10 anion exchange HPLC column as previously described (1). The peak areas were measured using the Sigma scan software (Jandell Scientific) and compared to nucleotide standards. Depletion of E-Site Nucieotide. For these studies, tubulin was first depleted of GDP and GTP at the E-site to allow for maximum into oration of the analogue during assembly. Tubulin from our pre arations usual ‘p y contains 0.7 to 0.8 mol GDP and 1.2 to 1.3 mol GTP per mol of tfl e dimer. We first exchanged the E-site GDP with GTP because GTP binding is decreased by a factor of 1000 in the absence of Mgz+ (9) making it easier to replace the nucleotide with the analope. Tubulin (30 pM) was incubated with 0.5 mM GTP for 30 min at 4°C. The tubuhn-GTP solution (600 pl) was centrifuged through 5 ml of Sephadex G-50 in 0.1 M Pipes, pH 6.9 to remove free GTP. The tubulin in the eluate contained 0.15 mol GDP and 1.94 mol GTP per mol of protein. The sample was allowed to sit on ice for 20 min and then incubated with 5 n&i GMPPCP or 5 mM ATP for 30 min at 25°C. A second centrifugation through 5 ml of Se hadex G-50 in Pipes resulted in tubulin with 0.10 to 0.15 mol of GDP and 1.0 mol o PGTP per mol of protein. This indicated that all of 654
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the GTP in the E-site had been displaced by GMPPCP or ATP. Binding of the analogues is weak and as a result, they are removed easily by gel filtration. Before assembly the samples were made 1 mM in MgSO4 and 5 mM in GMPPCP or ATP. Microtubule Assembly Reactions. The tubulin-GMPPCP or tubulin-ATP solution was assembled in the presence of microtubule seeds at 37°C in a Peltier temperature-controlled spectrophotometer. Nucleating seeds were prepared from tubulin (25 pM) assembled in the presence of 5 pM tax01 and 0.25 mM GTP and sheared by passing the microtubules several times through a 25 gauge syringe needle. The seeds (wtiwt ratio of 0.1 to unpolymerized tubulin) were added to the prewarmed tubulin solution (25 pM) to initiate the assembly reaction at 37°C. The microtubules were either left unperturbed or sheared just before reaching steady state by passing the solution four times through a 25 gauge needle. Aliquots were removed at different time periods for microtubule length measurements and determination of protein and nucleotide concentrations in the polymer. For a control, assembly was done in a similar manner but in the presence of 1 mM GTP. Determination
of the Amount ofProtein Polymerized and its Nucleotide Content.
To determine the amount of protein in the polymer, an aliquot of the assembled microtubules was centrifuged directly in a Beckman TL-100 ultracentrifuge or through a sucrose cushion for nucleotide analysis as described previously (1). The concentration of the protein was determined by the Bradford assay (10). To determine amount of incorporation of GMPPCP and ATP into polymerized tubulin, the method for nucleotide analysis of unpolymerized tubulin described above was used. Microtubule Length Determinations. An aliquot of the assembly reaction mixture was diluted 25-fold into PEM buffer containing 0.25% glutaraldehyde. The samples (10 pl) were placed on carbon- and Formvar-coated grids (100 mesh), washed three times with water and stained with 2% uranyl acetate. The grids were viewed with a Philips 300 electron microscope and photographed in the scan mode at a magnification of 375x. Microtubule lengths were measured from photographic enlargements using a Houston HiPad digitizing tablet and Sigma Scan software (Jandell Scientific).
RESULTS in the Presence of GMPPCP and ATP. Tubulin-
Assembly of Tub&in
GMPPCPP,
tubulin-ATP
or tubulin-GTP
in the presence of microtubule
assembled to steady state within 20 to 25 min. polymer
The nucleotide
when assembly was done in the presence of GMPPCP
seeds
content of the was 0.21 mol GDP,
0.88 mol GMPPCP
and 0.91 mol GTP, while in the presence of ATP polymerized
tubulin
0.30 mol GDP, 0.68 mol ATP and 0.98 mol GTP per mol of dimer.
contained
Thus, as reported previously the polymer
if tubulin
cold treatment, of non-specific
amount of analogue
had been depleted of E-site nucleotide
the microtubules
depolymerized
completely
was present in
before assembly. Upon suggesting
the absence
aggregates.
The growing microtubules induce transient
(I), a considerable
disassembly.
were sheared shortly before reaching steady state to As shown in Fig. 1, the absorbance decreased by 40% 65.5
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.5 GTP GMPPCP
0.1 , 40
0.0 0
80
I 120
minutes
Figure 1. Effect of shearing microtubules on assembly. Tubulin (25plK) in the presenceof 1 mM GTP, 5 mM GMPPCP or 5 mM ATP and microtubule seedswas polymerized and shearedshortly before steady state (at the arrow).
when GTP was present and significant
repolymerization
shearing as observed previously (2,4). When GMPPCP
occurred with time after or ATP was present, the
absorbance decreased only by 10 to 15% indicating little disassembly of polymer and essentially no reassembly was observed up to 120 min in these two cases. Length Redistribution
Studies. The steady state mean length of representative
populations of microtubules assembled in the presence of GTP, GMPPCP or ATP was measured for b.oth unperturbed or sheared solutions at different time periods. In the unperturbed samples, a small increase in the mean lengths was observed throughout steady
state,
During a 120 min period, the length increase was 8 pm (33%) for
polymerized tubulin-GTP
and 3 urn (16%) for tubulin-GMPPCP
as shown in Figs. 2a
and 2b. In both cases, most of the increase occurred within 20 min after polymer mass steady
state was reached. Similar results have been previously observed
with
unperturbed microtubules assembled in the presence of GTP (31. Upon shearing, however, extensive redistribution
of polymer lengths occurred in the microtubules
assembled with GTP present. The mean microtubule lengths shortened from 24.0 to 7.1 pm as a result of shearing but after 130 min, it had increased to 36.7 pm (410%) with the mean length becoming essentially constant after 100 min (Fig. 3al. In contrast, sheared microtubules assembled from tubulin-GMPPCP such redistribution
did not undergo
of lengths as seen in Fig. 3b. After a decrease in length from
14.3 to 6.2 pm following the shearing process, the mean lengths of the microtubule 656
Vol.
166,
207 10.. 28
No.
a
2, 1990
BIOCHEMICAL
-
AND
BIOPHYSICAL
24 75 * 0.66Ominpm
;;
20 ml" 2921+070pm
3t
RESEARCH
COMMUNICATIONS
20 ml" 19.35 f 0.64 "m
20
l-l 10..
2%
;;I
b f
10-w 2
u
$
.-
O-
20
‘iii x
- 32.69 * 0.64 60minpm
-h-h 10
E
O-
20s
lo--
O20.
1
3"‘
120 m m 32.79 * 0 70 pm
120 ml" 20.44 t 0 53 pm
20..
lo-10.. O+ 0
20
40
4 60
microtubule
04 0
length
10
20
30
1 40
(pm)
Fi
re 2. Length distributions of unsheared microtubules at polymer steady state. of polymerized tubulin in the presence of a) 1 mIvl GTP or b15 mM GMPPCP were taken at the time periods indicated at steady state and prepared for negative stain microscopy. The mean lengths of the microtubules (100-150 for each time point) were measuredas describedunder Materials and Methods. Values are given with standard errors.
--I@=-A iquots
population increased only by 37% during a 150 min period. Similarly,
the mean
lengths of polymerized tubulin containing ATP increased by about 6 pm (88%) during the same time period (Fig. 319,most of this occurring within the first 40 min. In all casesfor both sheared and undisturbed samples, no change in the concentration of tubulin in the polymeric form was detected at different time periods after steady state was reached.
DISCUSSION Previous studies (2-5,11,12) have demonstrated that at steady state, sheared MAP-depleted microtubules assembled with GTP undergo significant
length
Vol.
166, No. 2, 1990
8
BIOCHEMICAL
R
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4 a
658
Vol.
166,
No.
increases
2, 1990
BIOCHEMICAL
instability
GTP exposes tubulin-GDP
rapid dissociation. tubulin
findings.
model.
subunits
Regrowth
distribution
towards
more prominent since shearing
leads to enhancement
assembled
ends resulting
in their
occurs as the depolymerized
GTP were sheared.
of shorter
of the depolymerization
eventually
reach a steady state where
these
steady state
Length
changes were
compared to the unperturbed
a larger proportion
cases, microtubules
polymers.
of the length
were observed at polymer
with
unsheared
with
lengths and a spreading
for sheared microtubules generates
the microtubules
studies are in agreement
in microtubule
assembled
were explained
with GTP and add on to the remaining
longer microtubules
mass after microtubules
COMMUNICATIONS
These findings
at the microtubule
of our length redistribution A large increase
RESEARCH
Shearing
of the microtubules
dimers become recharged
The results
BIOPHYSICAL
mass remains constant.
while the polymer
on the basis of the dynamic with
AND
uncapped
samples
microtubules
which
process (3). For both the sheared and
undergo length redistributions
transiently
and
net changes in length redistribution
are no
longer detectable. This dynamic polymerized with
instability
tubulin
a considerable
contained amount
incorporated
at the E-site,
steady state.
The decreased
consistent
into tubulin
previous
GMPPCP,
was not observed
GMPPCP
of GMPPCP
data which
GMPPNP
or ATP (0.7-0.9 mol/mol
did not exhibit
extensive
triphosphate
showed
that microtubules
or ATP, are stabilized
assembled
at polymer
after shearing (GMPPCP
microtubules.
of the
tubulin)
length changes
degree of length redistribution
in the core stabilizes
mass
is
or ATP)
This is also consistent
assembled
in the presence
of
at steady state while in the absence of
added nucleotide,
disassembly
occurs at a slow rate (1,6).
depolymerization
and increase
in length
caused by the small fraction
when the majority
or ATP. Sheared microtubules
with the idea that a nucleoside
incorporated with
behavior
observed
of microtubules
after shearing
containing
from the seeds and from GDP not displaced
The slight were probably
GDP at the E-site
by the analogue.
A larger
that came
increase
in
Figure 3. Length distributions of sheared microtubules at polymer steady state. Microtubules assembled in the presence of a) 1 mM G’I’P, b) 5 mM GMPPCP or c) 5 mM ATP were sheared as indicated in Fig. 1. Length measurements were made at different time points after shearing. 659
Vol.
166, No. 2, 1990
polymer
BIOCHEMICAL
length occurred with microtubules
polymerized
tubulin-GMPPCP
in these microtubules
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
assembled with ATP compared to
since a larger fraction (30%) of polymerized
tubulin
contain GDP.
ACKNOWLEDGMENTS This work was supported by the National M. R. Mejillano
is a Wesley Foundation,
Wichita,
Institutes
of Health
grant GM 36953.
Kansas, Postdoctoral
Scholar.
REFERENCES 1. i: 4. 5. 6. 7. :: 10. 11. 12.
Mejillano, M. R., Barton, J. S., Nath, J. P., and Himes, R. H. (1989) Biochemistry (in press). Mitchison, T., and Kirschner, M. (1984) Nature, Lond. 312.237-242. Farrell, K. W., Jordan, M. A., Miller, H. P., and Wilson, L. (1987) J. Cell. Biol. 104,1035-1046. Keates, R. A. B., and Hallett, F. R. (1988) Science 241,1642-1645. Kristofferson, O., Mitchison, T., and Kirschner, M. (1986) J. Cell Biol. 102, 1007-1019. O’Brien, E. T., and Erickson, H. P. (1989) Biochemistry 28,1413-1422. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U.S.A. 70,765-768. Algaier, J., and Himes, R. H. (1988) Biochim. Biophys. Acta 954,235-243. Correia, J. J., Baty, L. T., and Williams, Jr., R. C. (1987) J. Biol. Chem. 262, 17278-17284. Bradford, M. M. (1970) Anal. Biochem. 72,248-254. Kirschner, T., and Mitchison, M. (1986) Cell 45,329-342. Bayley, P. M., Schilstra, M. J., and Martin, S. R. (1989) J. Cell Science 93,241254.
660
166,
January
No. 30,
2, 1990
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
STABILIZATION Magdalena
OF MICROTUBULES
R. Mejillano’,
‘Department
November
20,
653-660
BY GTP ANALOGUES
Janice S. Barton2 and Richard H. Himes’
of Biochemistry, Lawrence, Kansas
2Department
Received
AND
1990
University 660452106
of Chemistry, Washburn Topeka, Kansas 66621
of Kansas University
1989
We recently demonstrated that the nonhydrolyzable analogues of GTP (GMPPCP and GMPPNP) and ATP support the elongation phase of tubulin assembly and are incorporated into the E-site of polymerized tubulin. In this report we studied the stability of microtubules containing GTP analogues by examining length redistributions after shearing at polymer steady state. The mean length of a population of microtubules containing GMPPCP increased only by 37% over a 150 min time period after shearing. Microtubules which contained 70% ATP and 30% GDP at the E-site increased in length by 88%. In contrast, the mean length of microtubules assembled in the presence of GTP increased by 410% in the same time period. These results su gest that microtubules containing GMPPCP or ATP at their ends are stabilized f rom depolymerization. 0 1990Academic press, W.
Although in tubulin
it is well known that GTP at the exchangeable
is hydrolyzed
of GTP hydrolysis remain
unclear.
analogues,
to GDP during
in the polymerization
tubulin
reaction and in microtubule
and GMPPNP,
(1). Moreover,
these nucleotides
prevent the disassembly assembly conditions,
which normally
site (E-site) the roles
stability
that the nonhydrolyzable
as well as ATP, support the elongation
the assembly reaction and are incorporated tubulin
assembly to microtubules,
In a recent study, we demonstrated
GMPPCP
nucleotide
in significant
GTP phase of
amounts into polymerized
stabilize microtubules
at steady state and
occurs in the absence of GTP. Under non-
we also showed that these analogues and ATP displace GDP
Abbreviations: GTP, guanosine 5’-tri hosphate; GDP, guanosine 5’diphosphat.e; PCP, guanylylQ3, y-methylene) I ‘phosphonate; GMPPNP, guanylylimidodiphosphonate; ATP, adenosine 5’-tri hosphate; Pipes, piperaxine-N, N’-bis (2ethanesulfonic acid); EGTA, ethylene Pycol bis @uninoethyl ether)-N,N,N’N’tetraacetic acid; HPLC, high pressure 7 iquid chromatography. 0006-291x/90 653
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
166, No. 2, 1990
BIOCHEMICAL
from the E-site of tubulin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and that GMPPCP
binds to tubulin
with an affinity
which
is about lOOO-fold less than that of GTP. The ability dynamic
of these nueleotides
instability
that at polymer
to stabilize
model proposed by Mitch&on
steady state mass, microtubules at the ends. According
microtubules
is consistent
and Kirschner
(2) which postulates
become stabilized
tubulin-GTP
subunits
interconvert
between a growing phase of GTP capped microtubules
agents such as microtubule-associated
(5) apparently microtubules
contain
mainly
examined
core depolymerizes.
Based on this model,
assembled in the presence of nonhydrolyzable dynamic instability
tubulin-analogue
analogues
should be
behavior because these microtubules
along their length (6). To test this hypothesis,
the length dynamics at steady state of microtubules
ATP incorporated
and a shrinking
proteins (MAPS) (3,4) and glycerol
decrease the rate of these interconversions.
stable and not exhibit
by a cap of
to this model, microtubules
phase in which the GTP cap is lost and the labile tubulin-GDP Stabilizing
with the
with GMPPCP
into the polymer and compared them with those assembled
we or
in the
presence of GTP.
MATERIALS
AND METHODS
Preparation of Tubulin. Bovine brain tubulin was isolated by three cycles of assembly-disassembly (7) followed by chromatography on a phosphocellulose (Whatman Pll) - Biogel P-10 pig back column as described by Algaier and Himes (8). The purified tubulin in PEM r uffer (0.1 M Pipes, H 6.9,l mM EGTA and 1 mM MgS04) was drop-frozen in liquid nitrogen and store crat -80°C. Nucleotide Analysis of Tubulin. To determine the nucleotide content of unpolymerized tubulin, the protein was precipitated with perchloric acid and after neutralization, the supernatant was chromatographed on a Partisil SAX-10 anion exchange HPLC column as previously described (1). The peak areas were measured using the Sigma scan software (Jandell Scientific) and compared to nucleotide standards. Depletion of E-Site Nucieotide. For these studies, tubulin was first depleted of GDP and GTP at the E-site to allow for maximum into oration of the analogue during assembly. Tubulin from our pre arations usual ‘p y contains 0.7 to 0.8 mol GDP and 1.2 to 1.3 mol GTP per mol of tfl e dimer. We first exchanged the E-site GDP with GTP because GTP binding is decreased by a factor of 1000 in the absence of Mgz+ (9) making it easier to replace the nucleotide with the analope. Tubulin (30 pM) was incubated with 0.5 mM GTP for 30 min at 4°C. The tubuhn-GTP solution (600 pl) was centrifuged through 5 ml of Sephadex G-50 in 0.1 M Pipes, pH 6.9 to remove free GTP. The tubulin in the eluate contained 0.15 mol GDP and 1.94 mol GTP per mol of protein. The sample was allowed to sit on ice for 20 min and then incubated with 5 n&i GMPPCP or 5 mM ATP for 30 min at 25°C. A second centrifugation through 5 ml of Se hadex G-50 in Pipes resulted in tubulin with 0.10 to 0.15 mol of GDP and 1.0 mol o PGTP per mol of protein. This indicated that all of 654
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the GTP in the E-site had been displaced by GMPPCP or ATP. Binding of the analogues is weak and as a result, they are removed easily by gel filtration. Before assembly the samples were made 1 mM in MgSO4 and 5 mM in GMPPCP or ATP. Microtubule Assembly Reactions. The tubulin-GMPPCP or tubulin-ATP solution was assembled in the presence of microtubule seeds at 37°C in a Peltier temperature-controlled spectrophotometer. Nucleating seeds were prepared from tubulin (25 pM) assembled in the presence of 5 pM tax01 and 0.25 mM GTP and sheared by passing the microtubules several times through a 25 gauge syringe needle. The seeds (wtiwt ratio of 0.1 to unpolymerized tubulin) were added to the prewarmed tubulin solution (25 pM) to initiate the assembly reaction at 37°C. The microtubules were either left unperturbed or sheared just before reaching steady state by passing the solution four times through a 25 gauge needle. Aliquots were removed at different time periods for microtubule length measurements and determination of protein and nucleotide concentrations in the polymer. For a control, assembly was done in a similar manner but in the presence of 1 mM GTP. Determination
of the Amount ofProtein Polymerized and its Nucleotide Content.
To determine the amount of protein in the polymer, an aliquot of the assembled microtubules was centrifuged directly in a Beckman TL-100 ultracentrifuge or through a sucrose cushion for nucleotide analysis as described previously (1). The concentration of the protein was determined by the Bradford assay (10). To determine amount of incorporation of GMPPCP and ATP into polymerized tubulin, the method for nucleotide analysis of unpolymerized tubulin described above was used. Microtubule Length Determinations. An aliquot of the assembly reaction mixture was diluted 25-fold into PEM buffer containing 0.25% glutaraldehyde. The samples (10 pl) were placed on carbon- and Formvar-coated grids (100 mesh), washed three times with water and stained with 2% uranyl acetate. The grids were viewed with a Philips 300 electron microscope and photographed in the scan mode at a magnification of 375x. Microtubule lengths were measured from photographic enlargements using a Houston HiPad digitizing tablet and Sigma Scan software (Jandell Scientific).
RESULTS in the Presence of GMPPCP and ATP. Tubulin-
Assembly of Tub&in
GMPPCPP,
tubulin-ATP
or tubulin-GTP
in the presence of microtubule
assembled to steady state within 20 to 25 min. polymer
The nucleotide
when assembly was done in the presence of GMPPCP
seeds
content of the was 0.21 mol GDP,
0.88 mol GMPPCP
and 0.91 mol GTP, while in the presence of ATP polymerized
tubulin
0.30 mol GDP, 0.68 mol ATP and 0.98 mol GTP per mol of dimer.
contained
Thus, as reported previously the polymer
if tubulin
cold treatment, of non-specific
amount of analogue
had been depleted of E-site nucleotide
the microtubules
depolymerized
completely
was present in
before assembly. Upon suggesting
the absence
aggregates.
The growing microtubules induce transient
(I), a considerable
disassembly.
were sheared shortly before reaching steady state to As shown in Fig. 1, the absorbance decreased by 40% 65.5
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.5 GTP GMPPCP
0.1 , 40
0.0 0
80
I 120
minutes
Figure 1. Effect of shearing microtubules on assembly. Tubulin (25plK) in the presenceof 1 mM GTP, 5 mM GMPPCP or 5 mM ATP and microtubule seedswas polymerized and shearedshortly before steady state (at the arrow).
when GTP was present and significant
repolymerization
shearing as observed previously (2,4). When GMPPCP
occurred with time after or ATP was present, the
absorbance decreased only by 10 to 15% indicating little disassembly of polymer and essentially no reassembly was observed up to 120 min in these two cases. Length Redistribution
Studies. The steady state mean length of representative
populations of microtubules assembled in the presence of GTP, GMPPCP or ATP was measured for b.oth unperturbed or sheared solutions at different time periods. In the unperturbed samples, a small increase in the mean lengths was observed throughout steady
state,
During a 120 min period, the length increase was 8 pm (33%) for
polymerized tubulin-GTP
and 3 urn (16%) for tubulin-GMPPCP
as shown in Figs. 2a
and 2b. In both cases, most of the increase occurred within 20 min after polymer mass steady
state was reached. Similar results have been previously observed
with
unperturbed microtubules assembled in the presence of GTP (31. Upon shearing, however, extensive redistribution
of polymer lengths occurred in the microtubules
assembled with GTP present. The mean microtubule lengths shortened from 24.0 to 7.1 pm as a result of shearing but after 130 min, it had increased to 36.7 pm (410%) with the mean length becoming essentially constant after 100 min (Fig. 3al. In contrast, sheared microtubules assembled from tubulin-GMPPCP such redistribution
did not undergo
of lengths as seen in Fig. 3b. After a decrease in length from
14.3 to 6.2 pm following the shearing process, the mean lengths of the microtubule 656
Vol.
166,
207 10.. 28
No.
a
2, 1990
BIOCHEMICAL
-
AND
BIOPHYSICAL
24 75 * 0.66Ominpm
;;
20 ml" 2921+070pm
3t
RESEARCH
COMMUNICATIONS
20 ml" 19.35 f 0.64 "m
20
l-l 10..
2%
;;I
b f
10-w 2
u
$
.-
O-
20
‘iii x
- 32.69 * 0.64 60minpm
-h-h 10
E
O-
20s
lo--
O20.
1
3"‘
120 m m 32.79 * 0 70 pm
120 ml" 20.44 t 0 53 pm
20..
lo-10.. O+ 0
20
40
4 60
microtubule
04 0
length
10
20
30
1 40
(pm)
Fi
re 2. Length distributions of unsheared microtubules at polymer steady state. of polymerized tubulin in the presence of a) 1 mIvl GTP or b15 mM GMPPCP were taken at the time periods indicated at steady state and prepared for negative stain microscopy. The mean lengths of the microtubules (100-150 for each time point) were measuredas describedunder Materials and Methods. Values are given with standard errors.
--I@=-A iquots
population increased only by 37% during a 150 min period. Similarly,
the mean
lengths of polymerized tubulin containing ATP increased by about 6 pm (88%) during the same time period (Fig. 319,most of this occurring within the first 40 min. In all casesfor both sheared and undisturbed samples, no change in the concentration of tubulin in the polymeric form was detected at different time periods after steady state was reached.
DISCUSSION Previous studies (2-5,11,12) have demonstrated that at steady state, sheared MAP-depleted microtubules assembled with GTP undergo significant
length
Vol.
166, No. 2, 1990
8
BIOCHEMICAL
R
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4 a
658
Vol.
166,
No.
increases
2, 1990
BIOCHEMICAL
instability
GTP exposes tubulin-GDP
rapid dissociation. tubulin
findings.
model.
subunits
Regrowth
distribution
towards
more prominent since shearing
leads to enhancement
assembled
ends resulting
in their
occurs as the depolymerized
GTP were sheared.
of shorter
of the depolymerization
eventually
reach a steady state where
these
steady state
Length
changes were
compared to the unperturbed
a larger proportion
cases, microtubules
polymers.
of the length
were observed at polymer
with
unsheared
with
lengths and a spreading
for sheared microtubules generates
the microtubules
studies are in agreement
in microtubule
assembled
were explained
with GTP and add on to the remaining
longer microtubules
mass after microtubules
COMMUNICATIONS
These findings
at the microtubule
of our length redistribution A large increase
RESEARCH
Shearing
of the microtubules
dimers become recharged
The results
BIOPHYSICAL
mass remains constant.
while the polymer
on the basis of the dynamic with
AND
uncapped
samples
microtubules
which
process (3). For both the sheared and
undergo length redistributions
transiently
and
net changes in length redistribution
are no
longer detectable. This dynamic polymerized with
instability
tubulin
a considerable
contained amount
incorporated
at the E-site,
steady state.
The decreased
consistent
into tubulin
previous
GMPPCP,
was not observed
GMPPCP
of GMPPCP
data which
GMPPNP
or ATP (0.7-0.9 mol/mol
did not exhibit
extensive
triphosphate
showed
that microtubules
or ATP, are stabilized
assembled
at polymer
after shearing (GMPPCP
microtubules.
of the
tubulin)
length changes
degree of length redistribution
in the core stabilizes
mass
is
or ATP)
This is also consistent
assembled
in the presence
of
at steady state while in the absence of
added nucleotide,
disassembly
occurs at a slow rate (1,6).
depolymerization
and increase
in length
caused by the small fraction
when the majority
or ATP. Sheared microtubules
with the idea that a nucleoside
incorporated with
behavior
observed
of microtubules
after shearing
containing
from the seeds and from GDP not displaced
The slight were probably
GDP at the E-site
by the analogue.
A larger
that came
increase
in
Figure 3. Length distributions of sheared microtubules at polymer steady state. Microtubules assembled in the presence of a) 1 mM G’I’P, b) 5 mM GMPPCP or c) 5 mM ATP were sheared as indicated in Fig. 1. Length measurements were made at different time points after shearing. 659
Vol.
166, No. 2, 1990
polymer
BIOCHEMICAL
length occurred with microtubules
polymerized
tubulin-GMPPCP
in these microtubules
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
assembled with ATP compared to
since a larger fraction (30%) of polymerized
tubulin
contain GDP.
ACKNOWLEDGMENTS This work was supported by the National M. R. Mejillano
is a Wesley Foundation,
Wichita,
Institutes
of Health
grant GM 36953.
Kansas, Postdoctoral
Scholar.
REFERENCES 1. i: 4. 5. 6. 7. :: 10. 11. 12.
Mejillano, M. R., Barton, J. S., Nath, J. P., and Himes, R. H. (1989) Biochemistry (in press). Mitchison, T., and Kirschner, M. (1984) Nature, Lond. 312.237-242. Farrell, K. W., Jordan, M. A., Miller, H. P., and Wilson, L. (1987) J. Cell. Biol. 104,1035-1046. Keates, R. A. B., and Hallett, F. R. (1988) Science 241,1642-1645. Kristofferson, O., Mitchison, T., and Kirschner, M. (1986) J. Cell Biol. 102, 1007-1019. O’Brien, E. T., and Erickson, H. P. (1989) Biochemistry 28,1413-1422. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U.S.A. 70,765-768. Algaier, J., and Himes, R. H. (1988) Biochim. Biophys. Acta 954,235-243. Correia, J. J., Baty, L. T., and Williams, Jr., R. C. (1987) J. Biol. Chem. 262, 17278-17284. Bradford, M. M. (1970) Anal. Biochem. 72,248-254. Kirschner, T., and Mitchison, M. (1986) Cell 45,329-342. Bayley, P. M., Schilstra, M. J., and Martin, S. R. (1989) J. Cell Science 93,241254.
660
