Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
* Author for correspondence (e-mail: fjmcnally{at}ucdavis.edu )
Accepted 25 November 2001
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Summary |
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Key words: Katanin, Microtubule, Mitosis, Centrosome, -Tubulin
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Introduction |
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Microtubules that have been nucleated from -TuRCs in vitro have
minus ends that are physically capped. These
-TuRC caps prevent
minus-end polymerization and depolymerization
(Wiese and Zheng, 2000
;
Keating and Borisy, 2000
).
However, two types of experimental data indicate that microtubule minus ends
do depolymerize in vivo. First, minus end depolymerization has been directly
observed on single microtubules in cytoplasts
(Rodionov and Borisy, 1997
).
Second, the poleward flux of tubulin polymer in mitotic spindles
(Mitchison, 1989
), indicates
that microtubule minus ends are constantly depolymerizing during mitosis. This
minus end depolymerization in the spindle pole may provide one of several
forces to drive anaphase A chromosome segregation.
If the functional minus-end capping by -TuRCs observed in vitro
(Wiese and Zheng, 2000
) is
applicable in vivo, then only three mechanisms can explain microtubule
minus-end depolymerization in vivo. First, some microtubules may nucleate in
the cytoplasm without
-TuRCs. Second,
-TuRCs might detach from
microtubule minus ends either due to a spontaneous exchange rate or due to a
-TuRC uncapping enzyme. Third, a pre-existing microtubule that is
broken or severed should have an exposed minus end that is at least
transiently free to depolymerize. Breakage or severing of microtubules in
interphase cells has been clearly documented
(Waterman-Storer and Salmon,
1997
; Odde et al.,
1999
), whereas microtubule severing in spindles cannot be directly
observed due to the resolution limit of light microscopy.
Katanin has an in vitro microtubule-severing activity
(McNally and Vale, 1993) and
is concentrated at mitotic spindle poles in vertebrates and echinoderms
(McNally et al., 1996
;
McNally and Thomas, 1998
).
Thus katanin might sever microtubules from their
-TuRC caps and allow
minus-end depolymerization during mitosis. Because
-TuRCs cap
pre-existing minus ends in vitro, it is possible that the broad
microtubule-dependent distribution of
-tubulin in vertebrate spindles
(Lajoie-Mazenc et al., 1994
)
is partly due to cytosolic
-TuRCs binding to severed minus ends. Two
lines of evidence are consistent with this model. First, analysis of
vertebrate mitotic spindles by serial EM reconstruction reveals that the minus
ends of many microtubules are found at some distance from the centrosome
(Mastronarde et al., 1993
).
Second, release of microtubules from mitotic centrosomes has been directly
observed in vitro (Belmont et al.,
1990
) and in vivo (Rusan et
al., 2001
).
Testing the hypothesis that katanin severs microtubules from their
centrosomal nucleation sites during mitosis requires appropriate inhibitory
reagents. Katanin is a heterodimer composed of a catalytic 60 kDa AAA subunit
(p60) and an 80 kDa subunit (p80) involved in subcellular targeting
(Hartman et al., 1998). We have
previously demonstrated that transient expression of the C-terminal domain of
the p80 katanin subunit causes dissociation of endogenous p60 subunits from
mitotic spindle poles (McNally et al.,
2000
). We have also previously reported an affinity purified
anti-p60 katanin antibody that is monospecific in cultured vertebrate cell
lines and which inhibits the microtubule-severing activity present in
Xenopus egg extracts (McNally and
Thomas, 1998
). Because katanin is an AAA enzyme and because point
mutants of other AAA enzymes that cannot hydrolyze ATP act as dominant
inhibitors of the wild-type enzyme (Babst
et al., 1998
), we have also developed a point mutant of p60
katanin that inhibits wild-type p60 (see below). These three inhibitors of
katanin activity and localization were used in this study to investigate the
role of microtubule severing at mitotic spindle poles.
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Materials and Methods |
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ATPase assays
200 µl reactions containing 0.075 µM 6-his-wt-p60, varying
concentrations of GFP-P loop K-A p60 and 1.3 mM ATP were incubated at
25°C. At eight 1 minute intervals, 25 µl aliquots were removed and
analyzed for phosphate content by the malachite green method
(Kodama et al., 1986).
Velocities were determined for each set of eight data points. Heat inactivated
GFP-P loop K-A p60 was produced by heating for 5 minutes at 75°C.
In vitro microtubule-severing assays
In vitro microtubule severing assays were carried out as previously
described (McNally, 1998;
McNally and Vale, 1993
).
Taxolstabilized, tetramethyl-rhodamine-labeled microtubules were bound to the
surfaces of a flow cell that was coated with a G234A mutant of human kinesin.
Final concentration of polymerized tubulin in the reactions was determined to
be 0.02 µM from the total length of surface-bound microtubules. Reactions
were initiated by perfusing microtubules with a solution of 0.1 µM
6-his-human p60 + GST-con80 and varying concentrations of GFP-P loop K-A p60
in 20 mM K-Hepes, 4 mM MgSO4, 0.2 mM EGTA, 1.8 mM ATP, 20 µM taxol, 100
µg/ml BSA, pH 7.5. Reactions were stopped after 4 minutes by perfusing
microtubules with 0.1% glutaraldehyde in 80 mM K-Pipes, 1 mM MgCl2,
1 mM EGTA, pH 6.8. The ratio of free p60 to tubulin cannot be determined from
these values since analysis of integrated GFP-fluorescence on flow-cell
surfaces indicated that there was 10-times more GFP-p60 bound to the glass
surface than bound to microtubules.
Cell culture and transfections
CV-1 cells were grown in Optimem (Gibco-BRL Life Technologies) medium
supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin.
Cells were plated on coverslips before transfecting plasmid DNAs with
Lipofectamine Plus (Gibco-BRL Life Technologies), always in the presence of
10% FBS. The yellow fluorescent protein (YFP)-tubulin integrated CV-1 cell
line was generated by co-transfecting pEYFP-Tub (Clontech Laboratories) with
pCMV-ouabain (Pharmingen) and selecting for integrated cell lines with 1 µM
ouabain. All plasmids encoding katanin fragments or mutants were constructed
in the vectors pEGFP-C1, pECFP-C1 or pdsRed2-C1 (Clontech Laboratories).
Chariot-based protein transfection
To introduce anti-p60 IgG into CV-1 cells, cells were grown on 25 mm round
coverslips in 30 mm dishes. A mixture of 18 µl Chariot reagent (Active
Motif; Carlsbad, CA) and 36 µg IgG in serum-free Optimem were added to
cells for 2 hours. Media was then replaced with Optimem containing 10% FBS and
cells were fixed 2 hours later. Cells containing cytoplasmic IgG were
identified by staining with anti-rabbit IgG secondary antibody.
Two different regimes were used to ensure that cells were in their first mitosis after transfection. In one regime, CV-1 cells were arrested at G1-S with 2 mM thymidine, transfected, released into thymidine-free medium and fixed 12 hours after release. In the second regime, unsynchronized CV-1 cells were fixed 12-20 hours after transfection.
Determination of -tubulin areas at spindle poles
Fixation
Cells growing on coverslips were fixed by removing culture medium and
adding 3.7% formaldehyde (v/v), 0.25% glutaraldehyde (v/v), 80 mM Pipes, pH
6.8, 100 mM NaCl, 1 mM MgCl2, 5 mM EGTA, 0.2% Triton X-100 (v/v)
for 10-15 minutes at 25°C. Coverslips were post-fixed in 100% methanol at
-20°C. Prior to immunostaining, coverslips were treated with 100 mM
NaBH4 in 50 mM Tris, pH 10.3, 100 mM NaCl, for 5-10 minutes,
25°C, to reduce any remaining aldehydes.
-tubulin immunostaining
Coverslips were immunostained sequentially with a 1:1000 dilution of GTU-88
anti--tubulin monoclonal antibody (Sigma) and a 1:1000 dilution of
Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes) with DAPI and then
mounted in Mowiol mounting medium (Calbiochem) including 2.5% (w/v)
1,4-diazobicyclo[2.2.2]-octane. Cells were never double-labeled with
anti-
-tubulin when
-tubulin areas were determined. Cell cycle
stage was determined from DAPI staining. All cells without a nuclear envelope
and with condensed chromosomes that were not perfectly organized into a
metaphase plate or into two sets of anaphase chromosomes were classified as
`prometaphase'.
Quantitation of -tubulin staining areas
Cells expressing each transfected construct were identified by their GFP
fluorescence and their mitotic stage determined by the organization of their
DAPI-stained chromosomes. Centrosomes and spindle poles were identified by
their intensely fluorescent, spherical areas of -tubulin staining. Each
pole (or centrosome) was brought to sharpest focus, and the image recorded
with a Nikon Microphot SA microscope with a 60x PlanApo 1.4 objective
and a Quantix KAF 1400 CCD camera (Photometrics). Identical exposure times and
gain settings were used to capture the images for all treatments.
To quantitate -tubulin staining areas, regions of cells with
cytoplasmic levels of fluorescence intensity were selected manually. The
average fluorescence intensity of a cytoplasmic region was multiplied by a
factor of 1.11 to yield a threshold fluorescence intensity. IP Lab Spectrum
(Scanalytics) was used to identify all image pixels with fluorescence
intensities greater than the threshold value and then to calculate the total
area of the selected pixels at and around each spindle pole.
Estimation of cytoplasmic concentration of dominant negative katanin
subunits
To establish a relationship between CFP fluorescence intensity and the
molar concentration of CFP-P loop K-A p60, transfected cells were fixed and
processed for immunofluorescence with anti-p60 antibodies. The over-expression
level of p60 was estimated in several dozen transfected cells by determining
the ratio of anti-p60 staining intensity in each transfected cell to that of
an untransfected cell in the same field. The range of CFP fluorescence
intensity in cells analyzed in Figs
5 and
6 correspond to concentrations
10-50-times greater than that of endogenous p60.
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Nocodazole disassembly assay
CV-1 cells with the integrated YFP-tubulin construct were transfected on 25
mm coverslips. 12-20 hours post transfection, coverslips were assembled into
perfusion chambers maintained at 37°C. Cells were imaged with the same
system used for immunofluorescence. Mitotic CFP-expressing cells were
identified quickly using reduced illumination. A single CFP fluorescence image
was captured using fixed neutral density filters, exposure and gain so that
the expression level could be estimated. 50 µl of culture medium containing
80 µM nocodazole was then added gently to the edge of the perfusion chamber
and shuttered, time lapse acquisition of YFP fluorescence images was
initiated. Diffusion of nocodazole to the imaged cell did not appear to be
rate limiting as the fastest changes in YFP fluorescence intensity and spindle
length always occurred in the first 5 seconds of the image sequence.
To analyze the rate of reduction in YFP fluorescence intensity in the spindle, pixels corresponding to each half spindle as well as a cytoplasmic region adjacent to the spindle were highlighted manually. The average pixel values of each of these three regions were determined and the ratio of each half spindle's average pixel value/the average cytoplasmic pixel value was determined for each frame. In control experiments on untreated cells, this ratio remained constant in time lapse sequences, whereas absolute pixel values decreased due to photobleaching. For nocodazole-treated cells, the ratio reached 1.0 when the spindle was no longer visible to the eye (when cytoplasmic and spindle fluorescence were equal). To allow comparison of spindle disassembly rates between cells with different starting ratios, the ratios were converted to a fractional scale where the starting ratio was defined as 100% (of starting ratio) and the ratio at spindle disappearance was defined as 0% (of starting ratio).
Fluorescence recovery after photobleaching
FRAP experiments were carried out on a Zeiss 410 laser scanning confocal
microscope using a krypton/argon laser and the `activate' software for
bleaching of YFP-tubulin. Cells were transiently transfected with dsRed2
fusion proteins.
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Results |
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To determine whether the effect of GFP-P loop K-A p60 on wild-type p60 was
sufficient to prevent microtubule severing in vitro, taxol-stabilized
microtubules were incubated in the presence of purified recombinant wild-type
and P loop K-A human p60 katanin. Purified wild-type human p60 pre-associated
with GST-con80 (a GST fusion to the C-terminal domain of p80 katanin) mediated
complete disassembly of microtubules (Fig.
1; Table 2) as
previously reported (McNally et al.,
2000). Inclusion of a fourfold molar excess of a purified GFP
fusion to P loop K-A human p60 resulted in complete inhibition of microtubule
disassembly by wild-type p60 (Fig.
1; Table 2).
Heat-denatured GFP-P loop K-A p60 failed to inhibit the microtubule
disassembly activity of wild-type p60. Because the con80 domain of p80 katanin
binds to and activates the p60 subunit
(McNally et al., 2000
), it is
possible that GFP-P loop K-A p60 acts by sequestering GST-con80 away from the
wild-type p60 subunits. In this scenario, addition of excess GST-con80 should
alleviate the inhibition caused by P loop K-A p60. GFP-P loop K-A p60 was
pre-incubated with a slight excess of GST-con80 and tested for inhibition of
wild-type p60. As shown in Table
2, GFP-P loop K-A p60/GST-con80 inhibits wild-type p60 as well as
GFP-P loop K-A p60 alone, indicating that sequestering the con80 domain is not
the mechanism of inhibition. A GFP fusion to a double mutant p60
(GFP-
N-P loop K-A p60), bearing the P loop K-A substitution as well as
a deletion of the N-terminal 29 amino acids previously shown to be required
for association with con80 (McNally et
al., 2000
), did not inhibit wild-type p60 at concentrations
effective for P loop K-A p60 (Table
2). This result demonstrates that GFP-
N-P loop K-A provides
an appropriate negative control for katanin inhibition studies.
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If GFP-P loop K-A p60 is to be useful as a specific in vivo inhibitor of
wild-type katanin, it must inhibit the endogenous wild-type p60 without
binding to microtubules. To test whether GFP-P loop K-A p60 can inhibit
wild-type p60 in vivo and whether microtubule binding occurs in vivo, we
analyzed the activities and localization of katanin subunits expressed in HeLa
cells and CV-1 cells by transient transfection. When HeLa cells were
co-transfected with an epitope-tagged wild-type p60 and GFP, a high percentage
(54±8%, n=552 cells, 4 transfections) of the GFP-positive
cells exhibited extensive microtubule disassembly revealed by a 2-10-fold
reduction in the intensity of anti--tubulin immunofluorescence staining
as previously reported (McNally et al.,
2000
). By contrast, co-transfection of GFP-P loop K-A p60 with
wild-type p60 resulted in more than a 50-fold reduction in the fraction of
co-transfected cells exhibiting microtubule disassembly (0.7±0.55%,
n=1272 cells, 3 transfections). This result indicates that GFP-P loop
K-A p60 is a potent inhibitor of wild-type p60 in vivo.
If in vivo inhibition of wild-type katanin by GFP-P loop K-A p60 is due to
competition for microtubule binding sites, then GFP- P loop K-A p60 should
exhibit some steady state localization to microtubules in vivo. To determine
whether dominant-negative katanin proteins bind to microtubules in vivo, we
examined their subcellular localization in transfected CV-1 cells. In
interphase CV-1 cells, neither CFP-P loop K-A p60 nor CFP-con80 showed any
co-localization with microtubules. This result indicates that in vivo
inhibition of wild-type katanin by GFP-P loop K-A p60 most likely occurs by
direct interaction with wild-type katanin subunits rather than by competing
for microtubule binding sites. Surprisingly, CFP-N-P loop K-A
frequently did show co-localization with interphase microtubules. This result
indicated that any non-specific effects due to microtubule binding in vivo
should be more severe for CFP-
N-P loop K-A than for CFP-P loop K-A p60.
In mitotic CV-1 cells expressing very low levels of CFP fusion protein, CFP-P
loop K-A p60 and CFP-con80 localized in large spindle pole structures, just
like endogenous p60 katanin (McNally and
Thomas, 1998
). By contrast, CFP-
N-P loop K-A p60 never
exhibited localization in large spindle pole structures. The spindle pole
localization of CFP-P loop K-A p60 and CFP-con80 in mitotic CV-1 cells
indicated that these fusion proteins should be able to access the endogenous
katanin in mitotic spindle poles.
Katanin's activity is not required for spindle assembly, anaphase or
cytokinesis
To test whether inhibition of katanin with CFP-P loop K-A p60 would prevent
spindle assembly or function, CV-1 cells with an integrated
YFP--tubulin construct were transiently transfected with CFP or CFP-P
loop K-A p60. Mitotic spindles were monitored by time lapse imaging of
YFP-tubulin fluorescence. Six out of six CFP-transfected cells that were
initially observed with a mono-astral array of microtubules assembled a
bipolar spindle of metaphase length (9-12 µm) in 9-24 minutes. Likewise,
four of five CFP-P loop K-A-transfected cells that were initially observed
with a mono-astral array of microtubules assembled a bipolar spindle of
metaphase length in 10-21 minutes. Only one out of five CFP-P loop
K-A-transfected cells failed to assemble a bipolar spindle of metaphase length
before filming was stopped at 244 minutes. Thus, the majority of CFP-P loop
K-A-transfected cells are able to assemble a bipolar spindle with normal
kinetics.
If katanin inhibition causes a change in the occupancy or tension of
microtubules at kinetochores, a delay in the metaphase-anaphase transition
would be expected (Rudner and Murray,
1996). Thirteen out of fourteen CFP-transfected cells initially
observed with metaphase length bipolar spindles completed anaphase and
cytokinesis in 17-22 minutes. These cells spent 2-58 minutes with a metaphase
length spindle before initiating anaphase. One CFP-transfected cell completed
anaphase after spending 199 minutes with a metaphase length spindle and one
failed to enter anaphase after 189 minutes of filming. Nine out of thirteen
CFP-P loop K-A transfected cells initially observed with metaphase length
spindles completed anaphase and cytokinesis in 10-30 minutes. The majority of
these cells (8/9) spent 22-105 minutes with a metaphase length spindle. One
cell spent 244 minutes with a metaphase length spindle before completing
normal anaphase and cytokinesis. A minority (3/13) of CFP-P loop
K-A-transfected cells spent 287-317 minutes with a metaphase length spindle
before filming was stopped. These results indicate that the majority of CFP-P
loop K-A-transfected cells did not exhibit a delay in the metaphase-anaphase
transition and exhibited normal anaphase and cytokinesis.
The area occupied by -tubulin at prometaphase spindle poles is
dependent on microtubules
To test the hypothesis that recapping of microtubule ends generated by
katanin-mediated severing is partly responsible for the broad distribution of
-tubulin during mitosis
(Lajoie-Mazenc et al., 1994
),
we first devised an objective assay for the area occupied by
-tubulin
in the spindle. CV-1 cells were fixed and stained with anti-
-tubulin
monoclonal antibody and fluorescent secondary antibody. For each cell, the
average pixel value of anti-
-tubulin staining in the cytoplasm was
determined. The area occupied by pixels with a value greater than 1.11 times
the cytoplasmic value was determined as described in Materials and Methods. As
shown in Table 3, the area
occupied by
-tubulin increases dramatically at the interphase-prophase
transition and again at the prophase-prometaphase transition. If this increase
in
-tubulin area during M phase is really an indirect consequence of
the generation of microtubule minus ends, then this increase in area should be
sensitive to microtubule depolymerization with nocodazole. CV-1 cells were
treated with 20 µM nocodazole for either 30 minutes or 12 hours. Most
mitotic cells in the 30 minute treatment are likely to have entered M phase
before microtubule depolymerization whereas most mitotic cells in the 12 hour
treatment are likely to have entered M phase without microtubules. (Both
treatments allow complete microtubule disassembly in mitotic CV-1 cells as
assayed by anti-
-tubulin immunofluorescence.) As shown in
Table 4, nocodazole treatment
had no effect on
-tubulin areas at interphase centrosomes but reduced
-tubulin areas in prophase and prometaphase cells close to those values
observed in interphase. The histogram analysis in
Fig. 2 shows the sensitivity of
prometaphase
-tubulin area to a 30 minute nocodazole treatment and
illustrates the variability of this area in control cells. These results are
consistent with two models explaining the increased
-tubulin area
observed in mitotic CV-1 cells, association of
-TuRCs with dynein
complexes on the sides of microtubules
(Young et al., 2000
) or the
presence of microtubule minus ends distributed throughout the spindle.
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The area occupied by -tubulin at prometaphase spindle poles is
dependent on katanin's activity and spindle pole localization
To test whether the increase in area occupied by -tubulin in mitosis
is dependent on katanin-mediated microtubule severing,
-tubulin areas
were determined in CV-1 cells expressing GFP-P loop K-A p60 due to transient
transfection. As shown in the histogram in
Fig. 3C, nearly 40% of cells in
their first prometaphase after expression of the dominant katanin inhibitor
exhibited interphase-like
-tubulin areas (<1.6 µm2)
compared with only 5% of control GFP-expressing cells. Because 60% of
prometaphase GFP-P loop K-A-expressing cells had normal
-tubulin areas,
simple comparison of average values was not informative. However, analysis of
these data sets using the Mann-Whitney nonparametric test revealed that the
difference between GFP-expressing and GFP-P loop K-A p60-expressing cells was
highly significant (P=0.0001). Prolonged (48 hour) expression of
GFP-P loop K-A p60 resulted in a more severe reduction in
-tubulin
areas with many prometaphase cells exhibiting no detectable foci of
-tubulin staining (D.B. and F.J.M., unpublished). The apparent loss of
-tubulin foci in some cells may indicate a physiological response to
long term expression of this particular fusion protein or that our imaging
system is not adequate to distinguish very small, dim foci from bright
cytoplasmic staining. Expression of GFP-
N-P loop K-A p60, which was a
poor inhibitor of katanin in vitro, did not cause a significant reduction in
prometaphase
-tubulin areas (Fig.
3D). To further verify that the reduced
-tubulin areas in
prometaphase cells were due to inhibition of katanin, a previously described
anti-human p60 katanin antibody that inhibits microtubule severing in
Xenopus extracts (McNally and
Thomas, 1998
) was introduced into CV-1 cells using a viral fusion
peptide (see Materials and Methods). Cells were treated with either anti-p60
antibody or a control IgG isolated from the same serum but affinity depleted
of anti-p60 antibodies; cells were fixed 4 hours after initially adding
antibodies. Anti-rabbit IgG immunofluorescence confirmed that the anti-p60
antibody but not the control antibody localized to mitotic spindle poles (D.B.
and F.J.M., unpublished). Analysis of prometaphase
-tubulin areas in
anti-p60 containing cells compared with control IgG-containing cells revealed
a significant (P=0.02) reduction due to katanin inhibition
(Fig. 3E). Inhibition of
katanin did not have a statistically significant effect on
-tubulin
areas in interphase, metaphase or anaphase cells relative to cells expressing
GFP alone (D.B. and F.J.M., unpublished; see Discussion). The observed
reduction of
-tubulin areas by inhibition of a microtubule-severing
protein is consistent with the model in which the broad distribution of
-tubulin in spindles is at least partly due to the presence of
microtubule minus ends.
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To test the importance of katanin's localization at spindle poles, we
analyzed the effects of a GFP fusion to the C-terminal con80 domain of human
p80 katanin. Over-expression of this domain of p80 causes the mislocalization
of endogenous p60 from mitotic spindle poles but does not inhibit microtubule
severing activity of wild-type p60
(McNally et al., 2000).
Analysis of prometaphase
-tubulin areas in GFP-con80-expressing CV-1
cells 12-20 hours after transfection revealed no significant decrease relative
to GFP expressing cells (P=0.18) as indicated in
Fig. 4A. However, analysis of
-tubulin areas 48 hours post-transfection
(Fig. 4B) revealed that
GFP-con80 did cause a significant reduction in prometaphase
-tubulin
areas relative to GFP expressing cells (P=0.0067) after prolonged
expression. Thus, eliminating the local concentration of katanin at spindle
poles has the same effect as inhibiting katanin's severing activity, although
with slower kinetics.
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The reduction in -tubulin areas by katanin inhibition was not
accompanied by gross abnormalities in spindle structure. Analysis of GFP-P
loop K-A p60-expressing cells by anti-
-tubulin staining and anti-NuMA
staining revealed that these spindles exhibited a normal morphology (D.B. and
F.J.M., unpublished). One trivial explanation for the extreme reduction in
-tubulin area might be that targeting of GFP-P loop p60 to centrosomes
causes the degeneration of centrioles as has been observed in cells
microinjected with anti-glutamylated tubulin antibodies
(Bobinnec et al., 1998
). To
test this possibility, GFP-P loop K-A p60 expressing cells were fixed 48 hours
post-transfection and stained with anti-centrin antibody. Normal centriole
staining was observed in prometaphase cells (D.B. and F.J.M., unpublished),
indicating that the reduction in
-tubulin area is not due to centriole
degeneration. Thus katanin inhibition appears to have a specific effect on
-tubulin distribution in prometaphase spindles.
The rate of spindle disassembly in nocodazole is dependent on
katanin's activity and katanin's localization at spindle poles
Because microtubules polymerize and depolymerize from their ends, a spindle
with an increased number of plus and minus ends due to katanin-mediated
severing should disassemble faster when polymerization is blocked with
nocodazole. To test this hypothesis, we monitored the microtubule disassembly
rate in mitotic CV-1 cells expressing an integrated YFP-tubulin construct and
transiently transfected CFP fusion proteins. The rate of microtubule
disassembly was determined by monitoring the rate of decrease in YFP-tubulin
fluorescence intensity by time-lapse imaging of living cells perfused with
high concentrations of nocodazole (estimated 20 µM final concentration; see
Materials and Methods). As shown in Fig.
5, control spindles perfused with nocodazole shortened to about
half their starting length before the fluorescence intensity of YFP-tubulin in
the spindle decreased to background levels. A plot of YFP-tubulin fluorescence
intensity decrease over time reveals a rapid rate of spindle disassembly in
control cells (Fig. 6A). Cells
in which katanin was inhibited by CFP-P loop K-A p60 exhibited reduced rates
of spindle disassembly, but spindles eventually disassembled completely. A
large difference between control and CFP-P loop p60-expressing cells was
observed in the time required to achieve 80% loss in YFP-tubulin fluorescence
intensity (20% of initial intensity in Fig.
6A). Times required to achieve 80% loss of YFP-tubulin spindle
intensity are displayed as histograms in
Fig. 6B-F. In roughly 60% of
cells in which katanin was inhibited (CFP-P loop K-A p60) or mislocalized from
spindle poles (CFP-con80), the time to 80% loss of spindle intensity was much
greater than is ever observed in control spindles. CFP-N-P loop K-A
p60, which was a poor inhibitor of severing and had no effect on
-tubulin areas, had very little effect on the rate of spindle
disassembly in nocodazole (Fig.
6F).
Tubulin turnover in the spindle is not grossly affected by katanin
mislocalization
It is possible that increasing the number of microtubule plus and minus
ends in prometaphase by microtubule-severing might contribute to the increased
rate of turnover between polymerized and unpolymerized tubulin pools that is
observed in mitotic cells (Saxton et al.,
1984; Zhai et al.,
1996
). To test this hypothesis, we monitored the rate of
YFP-tubulin fluorescence recovery after photobleaching (FRAP) in spindles of
CV-1 cells transiently expressing either dsRed2 (Discosoma red
fluorescent protein) or dsRed2-con80 (to displace katanin from spindle poles).
One half spindle was completely photobleached and recovery of the bleached
half spindle was monitored by time-lapse confocal microscopy. To compensate
for photobleaching during the recovery period, recovery was expressed as a
ratio of the intensity of the bleached half spindle to that of the unbleached
half spindle. This ratio should reach a value of 1.0 when the bleached tubulin
is completely replaced by unbleached YFP-tubulin. As shown in the examples in
Fig. 7, fluorescence of the
bleached half spindle typically plateaued at a ratio lower than 1. This could
be due to the slow turnover reported for kinetochore microtubules
(Zhai et al., 1995
) or it
could be because a fraction of spindle microtubules was destroyed during
photobleaching of the YFP. In these experiments, recovery could not be
monitored for longer periods of time to distinguish between these
possibilities. Cells expressing dsRed2-con80 or dsRed2 alone showed no
differences in the half time to the plateau ratio, t
-p, or
in the predicted half time to a ratio of 1.0, t
(Fig. 7). There was also no
difference in the degree of recovery at the observed plateau (dsRed2:
0.84±0.06 (n=10), dsRed2-con80: 0.87±0.09
(n=13)). Thus mislocalization of katanin from spindle poles did not
have a discernable effect on tubulin turnover even though it slowed the rate
of nocodazole-mediated spindle disassembly
(Fig. 6E).
|
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Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The increase in -tubulin concentration and area of occupation during
mitosis is probably due to multiple mechanisms. Khodjakov and Rieder concluded
that the increase in
-tubulin concentration at the centrosome did not
require microtubules suggesting that an increased number of cytosolic
-TuRCs can bind directly to the pericentriolar material during mitosis
(Khodjakov and Rieder, 1999
).
Because their analysis was restricted to a region of interest (ROI) of fixed
area, their conclusion does not bear directly on our analysis of
-tubulin areas. In agreement with our results, Young et al. found that
microtubules were involved in recruitment of
-tubulin to mitotic
centrosomes (Young et al.,
2000
). They also found (in agreement with our unpublished results)
that dynein/dynactin is required for recruitment of
-tubulin to the
spindle. They concluded that
-TuRC/pericentrin complexes were
transported down the sides of microtubules by dynein/dynactin before docking
in the pericentriolar material. Our finding that inhibition of microtubule
severing causes a reduction in
-tubulin areas at spindle poles strongly
supports a model in which cytosolic
-TuRCs cap new microtubule minus
ends generated by severing. These new microtubules might be held in place in
the spindle by the combined action of dynein/dynactin and NuMA
(Merdes et al., 1996
;
Gaglio et al., 1997
). Thus the
effect of dynein inhibition is consistent with both models.
Multiple mechanisms behind the microtubule-dependent distribution of
-tubulin may explain why katanin inhibition had a dramatic effect on
the area occupied by
-tubulin during prometaphase but had very little
effect on metaphase or anaphase spindles. Kinetically slower mechanisms may be
able to substitute for katanin by metaphase. Slower mechanisms for generating
free microtubule minus ends around the centrosome might include other
microtubule-severing proteins (Shiina et
al., 1994
) and transport of chromatin/GTP-Ran-induced microtubules
to the spindle pole (Heald et al.,
1997
). Transport of
-TuRC/pericentrin complexes down the
sides of microtubules during metaphase
(Young et al., 2000
), even in
the absence of uncapped microtubule minus ends, might also obscure the effects
of katanin inhibition. Redundant mechanisms are also suggested by our finding
that katanin inhibition only affects
-tubulin areas in about half of
prometaphase spindles, whereas nocodazole reduces
-tubulin areas in
nearly all spindles.
The finding that spindle microtubules disassemble more slowly in nocodazole
when katanin is inhibited or mislocalized provides further support for a model
in which katanin normally generates an increased number of microtubule ends in
the spindle. Two 5 µm microtubules would be expected to depolymerize in
half the time required for one 10 µm microtubule. However, the consequences
of microtubule severing on steady state microtubule dynamics (without
nocodazole) are less clear. Analysis of the exchange rate between polymerized
and unpolymerized tubulin by FRAP revealed no gross differences between
control cells and those in which katanin was mislocalized. Microtubule
severing may have effects on tubulin turnover that are too subtle to be
detected by FRAP; microtubule severing may not contribute to the rapid
turnover of tubulin in mitotic cells or cells may compensate for a reduced
number of microtubule ends by upregulating other proteins such as Kin I
kinesins (Desai et al.,
1999).
Severing of microtubules in the mitotic spindle could result in an
increased number of non-centrosomal microtubules, uncapping of microtubule
minus ends, an increase in microtubule number and a decrease in microtubule
length. Because katanin is essential for C. elegans meiotic spindles,
which are composed entirely of non-centrosomal microtubules
(Srayko et al., 2000), it is
unlikely that detachment of microtubules from the centrosome is the sole
purpose of severing. If uncapping minus ends from
-TuRCs is an
important function of severing, katanin inhibition may result in a reduction
in the rate of poleward tubulin flux. Conversely,
-tubulin inhibition
might lead to an increase in the rate of flux. Poleward tubulin flux may be a
minor component of anaphase A in PtK cells
(Mitchison and Salmon, 1992
)
and a major component in Xenopus extract spindles
(Desai et al., 1998
). Our data
are consistent with an increase in microtubule number due to severing in the
mitotic spindle. Future work will focus on determining the functional
importance of regulating microtubule number and length in mitotic and meiotic
spindles.
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Acknowledgments |
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