(Received for publication, August 14, 1995; and in revised form, November 9, 1995)
From the
Molecular chaperones play an important role in facilitating the proper maturation of many newly synthesized proteins. Here we provide evidence that molecular chaperones also participate in regulating the assembly of the microtubule cytoskeleton. Via indirect immunofluorescence analysis, both hsp 73 and TCP-1 localized within the centrosome in interphase and mitotic cells. These proteins, along with the centrosome-specific protein, pericentrin, were also present within an enriched preparation of centrosomes. Because the centrosome serves as an initiation site for microtubule growth, we examined the ability of cells to regrow their microtubule network in the presence of hsp 73 or TCP-1 specific antibodies. Purified tubulin and GTP were added to cells following the depolymerization and extraction of cellular microtubules. Microtubules were observed to nucleate off the centrosome using this system, even in the presence of anti-hsp 73 antibodies. Incubation with anti-TCP-1 antibodies, however, blocked microtubule regrowth off the centrosome. Similarly, anti-TCP-1 antibodies microinjected into living cells first treated with nocodazole also inhibited the regrowth of the microtubule network following removal of the microtubule poison. Our results complement earlier genetic studies in yeast implicating a role for TCP-1 in microtubule mediated processes, and may help to explain the previously reported mitotic and meiotic abnormalities associated with TCP-1 mutations.
A class of proteins, now being referred to as molecular
chaperones, facilitate various aspects of protein maturation. Perhaps
the best characterized molecular chaperones are members of the
so-called heat shock (hsp) or stress protein family (recently reviewed
in (1) and (2) ). In animal cells, members of the hsp
70 family (DnaK in bacteria) are distributed throughout various
cellular compartments and interact with proteins being synthesized on
polysomes and/or being translocated into organelles. Such an
interaction with the hsp 70 proteins likely serves to stabilize and/or
maintain the nascent polypeptide in a relatively unfolded state until
its synthesis and/or translocation has been completed. Members of the
eukaryotic hsp 60 family (GroEL in bacteria) also bind newly
synthesized and unfolded polypeptides. This class of molecular
chaperones, often called chaperonins, are thought to provide a shielded
environment in which protein folding and/or higher ordered protein
assembly takes place. Although exhibiting only weak homology with other
members of the chaperonin family, a eukaryotic cytosolic chaperonin has
been identified and, like other members of the chaperonin family,
exists as a large (20 S) particle(3, 4) .
Despite structural similarities to other members of the chaperone family, the cytosolic chaperonin exhibits a number of differences as it relates to both its subunit composition, mode of regulation, and apparent substrate specificity. First, at least 8 related subunits, referred to as the TCP-1 family, appear to comprise the cytosolic chaperonin complex in eukaryotic cells. In contrast, chaperonins of both bacteria and plastids contain only 1 or 2 polypeptide subunits. Second, in contrast to the bacterial and plastid chaperones, the family of related TCP-1 proteins do not appear to be be up-regulated after heat shock or other metabolic stresses. Finally, while the bacterial GroEL chaperonin appears to function rather promiscuously, facilitating the folding of most any substrate presented to it, so far the cytosolic TCP-1 containing chaperonin has been observed to interact with only a limited number of substrates. Interestingly such substrates for the TCP-1 containing chaperonins are nucleotide binding proteins and include actin, tubulin, and luciferase(4, 5, 6, 7, 8, 9) .
Previous observations have also provided a connection between the TCP-1 proteins and microtubule related phenomena. In mice, expression of TCP-1 increases markedly during spermatogenesis(10) , a process that requires the assembly of complex macromolecular structures including the microtubule core of the flagellum. In addition, mutations within the TCP-1 locus are associated with both microtubule abnormalities and transmission ratio distortions(11) . Similarly in yeast, temperature-sensitive mutations of the TCP-1 gene result in a number of alterations in microtubule mediated processes. For example, at the non-permissive temperature the cells exhibit abnormal looking microtubules and give rise to the accumulation of multi-nucleated progeny(12) . Taken together, these results suggest that TCP-1 may play an important role in both the folding and the proper assembly of cytoskeleton proteins.
In the course of examining the intracellular locale of hsp 73 and TCP-1, we observed a portion of both proteins to exhibit rather intense staining near the nucleus. Such a locale appeared similar to another protein under study in our laboratory, the so-called pericentrin protein which has recently been shown to be a component of the centrosome(13, 14) . Owing to the fact that the centrosome serves as a primary nucleation site for microtubule growth(15) , along with the aforementioned observations relating TCP-1 mutations in yeast to microtubule abnormalities, we examined the possible relationship of hsp 73 and TCP-1 with the centrosome. We show here by different means that a portion of both hsp 73 and TCP-1 are, in fact, resident proteins of the centrosome. In addition, evidence is presented implicating a role for TCP-1 in mediating the growth of the microtubule network off the centrosome.
As an initial step in characterizing the location and
function of members of the hsp 73 and TCP-1 families, antibodies
specific to each protein were prepared. A rabbit polyclonal antibody
was prepared and shown to be specific for hsp 73(18) . For the
preparation of a TCP-1 specific antibody, a peptide corresponding to
amino acids 45-57 of the mouse TCP-1 protein (Fig. 1A) was synthesized and injected into rabbits.
Analysis of the resultant serum was done via immunoprecipitation using
[S]methionine-labeled HeLa cells solubilized in
Laemmli sample buffer containing 1% SDS and heated at 100 °C. Under
such conditions, the rabbit anti-TCP-1 antibody precipitated an
approximately 60-kDa protein (Fig. 1, panel B, lane
2). Preadsorbtion of the serum with an excess of the synthetic
peptide used to elicit the antibody resulted in an inability of the
serum to recognize the 60-kDa protein (Fig. 1B, lane
3). Immunoprecipitation using the rabbit polyclonal antibody to
hsp 73 revealed an approximate 70-kDa band (Fig. 1B, lane
4). Further analysis of this 70-kDa protein by two-dimensional gel
electrophoresis demonstrated the protein to be identical to the
constitutively expressed member of the hsp 70 family, referred to here
as hsp 73 (data not shown, see (18) ).
Figure 1:
Characterization of a rabbit polyclonal
anti-TCP-1 antibody. Panel A, protein sequence alignment of
TCP-1 from mouse, archaebacteria, and yeast (summarized and aligned by
Trent et al., (33) ) reveal relatively high homology
between amino acids 42 and 57. A synthetic peptide identical to those
amino acids shown for the mouse protein was synthesized and injected
into rabbits. Panel B, polyclonal serum was obtained and
characterized by immunoprecipitation using
[S]methionine-labeled HeLa cells solubilized in
Laemmli sample buffer and heated at 100 °C. Lane 1,
molecular weight standards; lane 2, anti-TCP-1
immunoprecipitate; lane 3, immunoprecipitation with anti-TCP-1
serum first preabsorbed with the synthetic peptide used to elicit the
antibody; lane 4, hsp 73 immunoprecipitate; lane 5,
immunoprecipitation using the anti-hsp 73 antibody first preabsorbed
with the TCP-1 synthetic peptide. Panel C, to determine the
native size of the antigen recognized by the TCP-1 synthetic peptide
antibody, [
S]Methionine-labeled HeLa cells were
solubilized by the addition of a buffer containing 0.1% Triton X-100
and applied to a 20-50% glycerol gradient. Following
centrifugation of the gradient (see ``Materials and
Methods''), fractions were collected, subjected to
immunoprecipitation analysis employing the rabbit anti-TCP-1 antibody,
and the immunoprecipitates were then analyzed by SDS-PAGE. Shown is an
autoradiograph of the gel. The majority of the 60-kDa antigen was found
to migrate with an approximate S value of 20 S (as determined in
parallel by analysis of the 20 S bacterial GroEL protein using the same
gradient conditions).
To determine whether
the antigen recognized by the TCP-1 synthetic peptide antibody existed
as an oligomeric species, similar to the situation with other known
members of the chaperonin family,
[S]methionine-labeled HeLa cells were
solubilized by the addition of a buffer containing 0.1% Triton X-100
and applied to a 20-50% glycerol gradient. Following
centrifugation, the gradient was fractionated and aliquots adjusted to
a final 1% SDS and heated at 100 °C for 5 min. Immunoprecipitants
across the gradient revealed the 60-kDa TCP-1 protein to migrate with
an approximate 20 S value (Fig. 1C).
To further
confirm the specificity of our rabbit anti-TCP-1 antibody, we examined
and compared its substrate specificity with that of a previously
described rat monoclonal antibody against TCP-1 (referred to as 23C).
CHO cells were labeled for 16 h with
[S]methionine and cell lysates prepared for
immunoprecipitation analysis. In our first experiment, the cells were
lysed in a buffer containing non-ionic detergents and then used to
examine the substrate specificity of both our rabbit TCP-1 antibody and
23C (Fig. 2). Under these conditions, the rabbit TCP-1 antibody
immunoprecipitated a prominent
60-kDa protein (Fig. 2, lane 2) along with a few other proteins. The 23C antibody
immunoprecipitated a number of polypeptides with molecular masses
between 50 and 70 kDa (Fig. 2, lane 3). Indeed, it is
this kind of analysis utilizing the 23C antibody which has resulted in
the conclusion that the eukaryotic TCP-1 containing cytosolic
chaperonin is comprised of multiple subunits and therefore has been
referred to by some (35) as the TCP-1 ring complex (TRiC).
However, when the immunoprecipitation analyses were performed using the
labeled cells first solubilized in Laemmli sample buffer containing 1%
SDS and the lysates then heated at 100 °C, slightly different
results were obtained. Now, both our rabbit antibody and the 23C
antibody recognized primarily only a single 60-kDa protein (Fig. 2, lanes 5 and 6, respectively).
Figure 2:
The rabbit polyclonal TCP-1 and a
monoclonal TCP-1 antibody (23C) precipitate similar proteins under
denaturing conditions but not under native conditions. CHO cells were
steady state labeled with [S]methionine for 16 h
and then harvested under denaturing conditions (e.g. Laemmli
sample buffer containing 1% SDS and heated at 100 °C) or native
conditions (cold buffers containing only nonionic detergents).
Immunoprecipitation analyses were performed using either the rabbit
polyclonal anti-TCP-1 or the rat monoclonal 23C TCP-1 specific
antibody. Duplicate immunoprecipitate samples were analyzed by both
one- and two-dimensional gel electrophoresis. Panel A,
analysis of the immunoprecipitates by one-dimensional SDS-PAGE.
Immunoprecipitations from cell lysates solubilized under native
conditions (lanes 1-3) or from cell lysates solubilized
under denaturing conditions (lanes 4-6). Lanes 1 and 4, no added antibody; lanes 2 and 5, the rabbit anti-TCP-1 antibody; lanes 3 and 6, the rat monoclonal 23C anti-TCP-1 antibody. Molecular mass
markers are indicated on the left and the arrowhead indicates the position of the approximate 60-kDa TCP-1 protein. Panels B-E, analysis of the immunoprecipitates by
two-dimensional gel electrophoresis using 70% pH 5-7 and 30% pH
3-10 ampholines (the left side of each panel represents the acid
side of the gel). In panel B is shown the rat monoclonal 23C
immunoprecipitation product obtained from cells lysed under native
conditions; in panel C the rabbit anti-TCP-1
immunoprecipitation product obtained from cells lysed under native
conditions; in panel D the rat monoclonal 23C
immunoprecipitation product obtained from cell lysed under denaturing
conditions; and in panel E the rabbit anti-TCP-1
immunoprecipitation product obtained from cells lysed under denaturing
conditions. The position of the TCP-1 protein(s) is indicated by a bracket while the positions of both actin and tubulin are
indicated by an asterisk (*).
The
above described immunoprecipitation products were analyzed by
two-dimensional gel electrophoresis. The immunoprecipitates obtained
from the nonionic detergent lysed cells using the 23C antibody (Fig. 2, panel B) and the rabbit peptide antibody (Fig. 2, panel C) revealed the presence of numerous
polypeptides. In contrast, the immunoprecipitation products obtained
from the cells lysed in buffers containing SDS, using either the 23C
antibody (Fig. 2, panel D) or the rabbit peptide
antibody (Fig. 2, panel E), revealed only a prominent
60-kDa protein which appeared to be comprised of at least 3 or 4
isoelectric variants (the small amounts of actin and tubulin which also
were present likely represent nonspecific background as evidenced by
their precipitation using preimmune sera, data not shown).
The intracellular locale of hsp 73 and TCP-1 was determined by indirect immunofluorescence methods. While the majority of both proteins appeared to be present throughout the cytosol, and to a lesser extent inside the nucleus, a relatively localized and rather intense staining pattern also was observed within the perinuclear region of the cells (the latter indicated by arrowheads in Fig. 3). This perinuclear staining pattern appeared remarkably similar to that of another protein under study in our laboratory, specifically, the recently described pericentrin protein which has been shown to be a component of the centrosome. To examine whether a portion of both hsp 73 and TCP-1 might be present within the centrosome, simultaneous staining of the cells with antibodies specific for pericentrin and the two molecular chaperones was performed. To facilitate the analysis, HeLa cells growing on glass coverslips were first lightly extracted with buffers containing nonionic detergents to remove the majority of the cytosolic and nuclear forms of hsp 73 and TCP-1. Previous studies have shown that the centrosome, and many of its associated components, are not grossly affected by such extraction with non-ionic detergents(14) . Double label staining using a human autoimmune sera(5051) specific for pericentin, along with either antibodies specific for hsp 73 or TCP-1 revealed that a portion of hsp 73 and TCP-1 were in fact present very near or within the centrosome (Fig. 4). Staining of the centrosome by antibodies to the two molecular chaperones was observed in interphase cells as well as in cells which had entered mitosis. Although not shown, we have observed a similar distribution for hsp 73 and TCP-1 within the centrosome of other animal cells including the two monkey cell lines, CV-1 and COS, rat embryo fibroblasts, baby hamster kidney, mouse 3T3, and Chinese hamster ovary cells.
Figure 3: Intracellular locale of hsp 73 and TCP-1 as determined by indirect immunofluorescence. HeLa cells growing at 37 °C on glass coverslips were fixed and permeabilized by immersion in -20 °C absolute methanol for 2 min. The distribution of hsp 73 and TCP-1 were analyzed by indirect immunofluorescence using a rabbit anti-hsp 73 and the rabbit anti-TCP-1 antibody. Primary antibodies were visualized by subsequent incubation with a fluorescein-conjugated goat anti-rabbit antibody. Panels A and B show phase-contrast and fluorescent micrographs of cells incubated with anti-hsp 73. Panels C and D show phase-contrast and fluorescent micrographs of cells incubated with anti-TCP-1. Arrowheads in panels B and D indicate the localized and intense perinuclear staining of either hsp 73 (B) or TCP-1 (D).
Figure 4: A portion of both hsp 73 and TCP-1 co-localize with the centrosome. HeLa cells growing on glass coverslips at 37 °C were lightly extracted with 0.1% Triton X-100 for 2 min and then immediately fixed by immersion in -20 °C absolute methanol. Cells were simultaneously incubated with rabbit anti-hsp 73 plus a human autoimmune anti-centrosome antibody, or with rabbit anti-TCP-1 plus the human autoimmune sera. Primary antibodies were visualized by subsequent incubation with rhodamine-conjugated goat anti-rabbit and fluorescein conjugated-goat anti-human antibodies. Panels A and B show the same cells incubated with anti-hsp 73 (A) and anti-centrosome (B). Panels C and D show the same cells incubated with anti-TCP-1 (C) and anti-centrosome (D). Arrowheads indicate the position of the centrosome. Insets in panels A and C show the staining patterns of hsp 73 and TCP-1 respectively, in mitotic cells.
To further explore the apparent centrosomal
locale of both hsp 73 and TCP-1, an enriched preparation of centrosomes
was prepared and examined for the presence of the two molecular
chaperones(23) . Briefly,
[S]methionine-labeled CHO cells were treated
with the microfilament poison cytochalasin and the microtubule
depolymerizing agent nocodazole. Following lysis of the cells and
subsequent clarification of the lysate, the resultant supernatant was
applied to a discontinuous sucrose gradient, the material centrifuged
at 100,000
g for 1 h and the gradient then
fractionated. As is shown in Fig. 5, panel A, the vast
majority of radioactive material was present within the first half of
the gradient. In addition, a distinct peak of radioactive material was
observed near the bottom of the gradient (fractions 4-7), this
representing material within the 50-70% sucrose interface where
centrosomes have previously been shown to migrate using this procedure (23) . To confirm that the material present within fractions
4-7 contained a relatively dense particle corresponding to the
centrosome, the fractions were pooled and diluted 4-fold with buffer.
The diluted material then was centrifuged at 14,000
g for 20-30 min and the resultant supernatant and pellet
examined by SDS-PAGE. As is shown in Fig. 5B, the vast
majority of the radiolabeled proteins were found within the pellet,
consistent with the proteins being present within a dense particle.
Both hsp 73 and TCP-1, along with a few other proteins including actin
and tubulin (as well as other unidentified proteins) were found to be
present within the enriched preparation of centrosomes (Fig. 5D).
Figure 5:
Isolation and characterization of
centrosomes. CHO cells growing at 37 °C were metabolically labeled
with [S]methionine for 16 h and then used for
the isolation of an enriched fraction of the centrosomes as described
under ``Materials and Methods.'' Panel A, following
the final sucrose gradient centrifugation, 0.5-ml fractions were
collected and a portion of each fraction examined for its radioactive
content via liquid scintillation. Fractions 4-7 (representing
material of the 50-70% sucrose interface) were pooled and used
for all subsequent analysis. Panel B, pooled sucrose fractions (4, 5, 6, 7) were diluted with the
centrosome isolation buffer (1:4) and centrifuged at 14,000
g for 30 min. The material before centrifugation (T),
as well as an equal percentage of the resultant supernatant (S) and pellet (P) then were analyzed by SDS-PAGE.
Shown is a fluorograph of the gel. Panel C, two-dimensional
gel analysis of the [
S]methionine-labeled CHO
cells used to isolate the centrosomes. Panel D,
two-dimensional gel analysis of the enriched preparation of
centrosomes. The downward pointing arrow indicates the
position of hsp 73 and the upward pointing arrow the position
of TCP-1. (Note that the pH ampholines used here (100% pH 3-10)
are different than the pH ampholines used in Fig. 2.)
These partially purified centrosomes were centrifuged onto glass coverslips and examined by indirect immunofluorescence, again using antibodies to hsp 73, TCP-1, and the antibody specific for the centrosome protein, pericentrin. As was found with the detergent-extracted cells, coincident staining of pericentrin and hsp 73 (Fig. 6, A and B, respectively), as well as coincident staining of pericentrin and TCP-1 (Fig. 6, C and D, respectively) was observed.
Figure 6: Hsp 73 and TCP-1 co-localize with pericentrin in isolated centrosomes. Centrosome fractions prepared from CHO cells as described in the legend to Fig. 4(fractions 4-7) were pooled, diluted in centrosome isolation buffer, and then centrifuged onto glass coverslips. The coverslips were incubated with antibodies to both hsp 73 (rabbit) and pericentrin (rat), or with antibodies to TCP-1 (rabbit) and pericentrin (rat). Primary antibodies were visualized by subsequent incubation with fluorescein-conjugated goat anti-rabbit and rhodamine-conjugated goat anti-rat antibodies. Panels A and B, centrosomes simultaneously stained for pericentrin (panel A) and hsp 73 (panel B). Panels C and D, centrosomes simultaneously stained for both pericentrin (panel C) and TCP-1 (panel D). Arrowheads indicate representative centrosomes being recognized by both antibodies.
Previous work has established that the centrosome serves both as an anchor for microtubules and as a site for nucleation of microtubule growth(15, 34) . For example, when cells are treated with nocodazole, the microtubules are observed to depolymerize. Upon removal of the drug, the microtubules are observed to regrow with such regrowth emanating from the centrosome(14, 22) . To address the possible role played by either hsp 73 or TCP-1 in facilitating the regrowth of the microtubule network, we initiated ``blocking'' experiments utilizing antibodies to both molecular chaperones. Cells were treated with nocodazole to depolymerize the microtubule network and the cells then injected with either preimmune, anti-hsp 73 or anti-TCP-1 antibodies. Following their injection, the cells were extensively washed to remove the nocodazole, and then further incubated for various times to allow for the regrowth of the microtubule network. In those cells injected with either preimmune or anti-hsp 73 antibodies, regrowth of the microtubules from the centrosome was unaffected (data not shown).
In contrast to the
lack of effects following injection of either the preimmune or hsp 73
antibodies, cells injected with the rabbit anti-TCP-1 antibodies
appeared unable to support new microtubule growth. COS cells growing on
glass coverslips were treated with nocodazole and injected with the
anti-TCP-1 antibody as described above. Ten min after the injections,
the cells were washed with and further incubated in fresh culture
medium in the absence of the microtubule poison. After 30 min, the
cells were immediately placed into fixative and analyzed for the
presence of the injected antibody (via incubation with
fluorescein-conjugated goat anti-rabbit antibody) and the distribution
of the microtubules (via incubation with mouse anti--tubulin
followed by rhodamine conjugated goat anti-mouse antibody). Note that
in the 3-4 cells shown (Fig. 7A), the cell
injected with anti-TCP-1 (Fig. 7B) exhibited some
differences with respect to its pattern of tubulin staining as compared
to the surrounding uninjected cells (Fig. 7C).
Specifically, while all of the cells exhibited intense tubulin
staining, the cell injected with anti-TCP-1 did not exhibit an obvious
filamentous staining of the microtubule network like that observed for
uninjected cells. These apparent differences in the organization of the
microtubules as a function of injected anti-TCP-1 were much more
apparent when the cells were lightly extracted with non-ionic
detergent. For these experiments, nocodazole-treated cells were
injected with anti-TCP-1 as described above, the cells then washed with
and further incubated in fresh culture medium for either 10 or 30 min.
This time, however, the cells were first lightly extracted with
nonionic detergent to facilitate the removal of any nonpolymerized
tubulin. Following their fixation, the identification of the injected
cells, along with the distribution of the microtubules, was determined
by double label staining exactly as described earlier. Note that little
or no microtubule regrowth was observed in those cells injected with
anti-TCP-1 and then allowed either a 10-min (Fig. 7, D-F) or 30 min (Fig. 7, G-I) regrowth period.
In contrast, by 10 min, the uninjected cells had initiated microtubule
aster formation (Fig. 7F) and by 30 min had regrown
their complete microtubule network (Fig. 7I).
Figure 7: Microinjection of anti-TCP-1 antibodies into living cells first treated with nocodazole prevents the regrowth of the microtubule network. COS cells, first treated with nocodazole for 90 min, were injected with anti-TCP-1 antibodies. The medium containing nocodazole was removed and the cells extensively washed with and further incubated in fresh medium for either 10 or 30 min. In one case the cells were immediately placed into fixative, while in the other cases the cells were first lightly extracted with microtubule stabilization buffer supplemented with nonionic detergent prior to their fixation by methanol. Identification of the injected cells along with the distribution of the microtubules was determined by double label indirect immunofluorescence as described in the methods. Panels A-C, cells 30 min after the removal of nocodazole and then immediately placed into fixative. Panels D-F, cells 10 min after removal of the nocodazole, detergent extracted and then placed into fixative. Panels G-I, cells 30 min after removal of the nocodazole, detergent extracted, and then placed into fixative. Panels A, D, and G (left-hand column) show the phase-contrast micrographs; panels B, E, and H (middle column) show the identity of the injected cells; panels C, F, and I (right-hand column) show the distribution of the microtubules. The arrowheads in panels C, F, and I indicate the antibody injected cells.
We next performed similar in vitro blocking experiments using permeabilized cells. In our first experiment, COS cells were treated with nocodazole to depolymerize the microtubule network and the cells then detergent extracted to remove the free 6 S tubulin. Antibodies (preimmune as a control, anti-hsp 73 or anti-TCP-1) were added to the extracted cells, and the ability of the cells to support microtubule regrowth following addition of purified tubulin was examined. In those cells incubated with preimmune antibodies, new microtubule growth (as assayed by staining with the anti-tubulin antibody) was seen emanating from a single site, likely the centrosome (Fig. 8A). Incubation of the cells with the anti-hsp 73 antibody again had no obvious effect on microtubule regrowth (Fig. 8B). In contrast, in two separate experiments, incubation of the extracted cells with the anti-TCP-1 antibody blocked the regrowth of the microtubules (Fig. 8, C1 and C2). Note, however, that prior incubation of the extracted cells with the anti-TCP-1 antibody did not prevent subsequent staining of the centrosome with the anti-tubulin antibody (note the localized perinuclear staining in Fig. 8C2). Such staining likely represents that portion of tubulin present within the centriolar region of the centrosome.
Figure 8:
Incubation of detergent permeabilized and
nocodazole-treated cells with anti-TCP-1 antibody blocks the re-growth
of the microtubules. HeLa cells growing on glass coverslips were
treated with nocodazole (20 µg/ml) for 90 min at 37 °C. The
medium was removed and the cells extracted with microtubule
stabilization buffer supplemented with 0.1% Triton X-100 for 2 min at
room temperature. Following their extraction and subsequent washing
with MSB, the cells were incubated with rabbit preimmune sera, rabbit
anti-hsp 73, or the rabbit anti-TCP-1 antibody. Following a 10-min
incubation at room temperature, the antibody solution was removed, the
cells washed with MSB buffer and then incubated with MSB containing 1
mM GTP, 2 mg/ml tubulin, and 20% glycerol. After 12 min at 37
°C the cells were washed with MSB, fixed with 2% glutaraldehyde in
MSB, and processed for immunofluorescence using the anti--tubulin
antibody followed by rhodamine conjugated goat anti-mouse antibody.
Shown are the immunofluorescent photographs of the tubulin staining. Panel A, cells incubated with rabbit pre-immune serum; panel B, cells incubated with rabbit anti-hsp 73; panel
C, cells incubated with rabbit anti-TCP-1 (C1 and C2 represent two
different experiments using the anti-TCP-1 antibody). Note the
formation of microtubule asters only in those cells incubated with
either the preimmune or hsp 73 antibodies.
To further confirm these results we examined the effects of incubating the cells with the rat monoclonal antibody (23C) specific for TCP-1 (21) . In vitro regrowth experiments, identical to those shown in Fig. 8, were performed using Chinese hamster ovary cells (CHO). Similar to our results using the rabbit polyclonal anti-TCP-1 sera, addition of the 23C antibody prior to the addition of purified tubulin resulted in an inhibition of microtubule growth (Fig. 9B).
Figure 9: The previously characterized rat monoclonal antibody specific for TCP-1 also inhibits in vitro microtubule regrowth in CHO cells. In vitro microtubule regrowth experiments, identical to those described in the legend to Fig. 8, were performed using either a control antibody (preimmune) or a purified preparation of the rat monoclonal antibody (23C) specific for TCP-1. Shown is the pattern of tubulin staining. Panel A, cells incubated with the control antibody; panel B, cells incubated with the rat monoclonal 23C anti-TCP-1 antibody.
Our studies, in summary, demonstrate that a subpopulation of two molecular chaperones, hsp 73 and TCP-1, co-localize with and likely are integral components of the centrosome in eukaryotic cells. This conclusion was reached, in part, on the basis of double label immunofluorescence experiments showing that antibodies to these two molecular chaperones stained perinuclear structures in a fashion identical to that using antibodies which recognize a defined centrosomal antigen, pericentrin. Using this approach, we previously observed the presence of hsp 73 in centrosomes of both mitotic and interphase cells(24) . Likewise, using an antibody that recognized both the constitutive (hsp 73) and stress inducible (hsp 72) forms of hsp 70, Rattner (25) showed what appeared to be centrosomal staining in mitotic cells. Here, we have performed additional experiments to verify that hsp 73 and TCP-1 are indeed centrosomal components. For example, both hsp 73 and TCP-1 were found to be present within an enriched preparation of centrosomes isolated by conventional means. Moreover, we have found that treatment of the isolated centrosomes with high concentrations of salt did not result in the release of the centosomal antigen pericentrin, hsp 73, or TCP-1, results indicative that all three of these proteins likely are integral components of the organelle (data not shown). Finally, for at least TCP-1 we have shown that both our own antibody, as well as a previously described TCP-1 antibody (23C) appeared effective in inhibiting the growth of microtubules off the centrosome, be it in detergent permeabilized cells or in living cells microinjected with the anti-TCP-1 antibody.
Some discussion regarding the properties of our polyclonal TCP-1 antibody merits inclusion here. As was shown in Fig. 1and Fig. 2, our antibody, when used in immunoprecipitation studies of cell lysates first subjected to SDS and heating, precipitated the same TCP-1 antigen as that recognized by the previously described 23C anti-TCP-1 antibody. However, when the analysis was performed using cells lysed under ``nondenaturing'' conditions (e.g. nonionic detergents and no heating) the 23C antibody precipitated the TCP-1 antigen along with a number of other proteins. This collection of co-precipitating proteins has been suggested to represent a heteromeric TCP-1 containing chaperonin complex (35) and as such has been given the name TRiC. In contrast, our rabbit antibody did not precipitate a TRiC-like complex from the cells lysed under nondenaturing conditions. One possible explanation for this difference is that the binding of our antibody to the TRiC somehow results in the dissociation of the heteromeric TCP-1 containing particle. Alternatively, there may exist two forms of the TCP-1 protein, one present within TRiC, and the other perhaps present in monomeric form. As was shown in Fig. 1, we do routinely observe what appears to be two forms of the TCP-1 protein, one which migrates in the velocity sedimentation gradients with an apparent mass of 20 S, and the other which remains at the top of the gradient and therefore which might represent the ``free'' or monomeric form of the protein. If so, one wonders what form of TCP-1 is present within the centrosome. For example, does the centrosome contain the entire TCP-1 containing chaperonin complex TRiC, or simply the TCP-1 protein itself?
Could our results showing compromises in the ability of the cells to initiate growth of the microtubules off the centrosome following addition/injection of anti-TCP-1 antibodies be explained by simple steric hindrance? For example, upon binding of the TCP-1 antibody to the centrosome might the subsequent recruitment of 6 S tubulin (or other essential components such as microtubule-associated proteins) be hindered from reaching the centrosome? Although difficult to rule out, we did show that in cells first incubated with either anti-hsp 73 or anti-TCP-1 (either of which bound to the centrosome) we could subsequently stain the centrosome with the antibody to tubulin (Fig. 8). Hence, the tubulin antibody presumably is still able to bind to that portion of tubulin within the centriolar component of the centrosome, even when the organelle had first been engaged with the antibodies to the two different molecular chaperones. Similarly, we have performed all of the other possible permutations involving sequential addition of the three different antibodies that were shown here to stain components present within the centrosome. For example, incubation first with antibodies to pericentrin did not prevent subsequent staining of the same centrosome with either hsp 73 or TCP-1 antibodies, and vice versa. Thus we think it unlikely that the TCP-1 antibody, when engaged with the centrosome, is simply preventing the access of other components, like tubulin, to the centrosome.
Both our experimental approach and some of our results examining the
locale and function of TCP-1 within the centrosome appear similar to
previous studies examining a newly identified member of the tubulin
family, -tubulin(26) . Via indirect immunofluorescence
methods
-tubulin was shown to be present within the centrosome of
both interphase and mitotic cells. In addition, injection of
-tubulin antibodies into living cells was shown to block the
re-growth of the microtubules off the centrosome. Interestingly, like
TCP-1,
-tubulin exists in vivo as a large complex, on the
order of 25 S (27) . Moreover, both
-tubulin as well as
another centrosomal component, the vertebrate actin-related protein are
dependent on the TCP-1 chaperonin for their folding and/or higher
ordered assembly(8) . While we do not yet know how all of these
observations fit together, we suspect that they are more than
coincidental.
Molecular chaperones are important components in the pathways by which newly synthesized proteins acquire their properly folded state. Therefore, we did consider the possibility that microtubule regrowth might require new protein synthesis and that we were simply inhibiting the proper maturation of such proteins as a result of the injected TCP-1 antibodies. However, a number of observations are inconsistent with this notion. First, we found that prior addition of the protein synthesis inhibitor cycloheximide had no effect on the ability of nocodazole-treated cells to regrow their microtubule network following subsequent removal of the microtubule poison (data not shown). Second, cells permeabilized via the addition of nonionic detergents were still competent to support microtubule growth off the centrosome upon addition of purified tubulin and GTP. Finally, at least in vitro, an enriched preparation of 6 S tubulin is competent to assemble into microtubules provided that an energy source is supplied. Indeed, it is this latter result which might lead one to question why molecular chaperones would be needed at all to facilitate the growth of microtubulues in vivo. While we still do not have a satisfactory answer to this fundamental question, it is important to point out that a similar question was raised when molecular chaperones were first suggested to be important components in the pathways of protein folding inside the cell. Here a significant amount of work had established that a purified protein, when first rendered unfolded via treatment with protein chaotropes, could spontaneously refold upon removal of the chaotrope(36) . In vivo, however, the possibility that an unfolded protein (e.g. newly synthesized protein) will interact with other macromolecules during its folding process, thereby leading to nonproductive folding or aggregation, seems quite high. Consequently, molecular chaperones have been suggested to participate in the pathway of protein folding in vivo, not by providing any direct information for the folding process, but rather by reducing the probability of inappropriate intra- or intermolecular interactions which might lead to nonproductive folding and/or aggregation. Exactly how this scenario might apply to the apparent role of TCP-1 in mediating the growth of the microtubules off the centrosome is still unclear to us at this time. Using various in vitro approaches, we hope to begin addressing these types of questions in the near future.
We should mention that there are a number of other observations which link members of the molecular chaperone family with the cytoskeleton, and in particular the microtubules. For example, we and others have shown previously that hsp 73 co-purifies with microtubules during repeated rounds of tubulin assembly/disassembly in vitro(29, 30, 31) . Whether hsp 73 is in fact a true microtubule-associated protein (i.e. a MAP), required for microtubule growth, is not known, but is now being addressed in our laboratory. We now have preliminary evidence that TCP-1 is present at modest amounts in three-time cycled preparations of microtubules. Even more intriguing are recent studies implicating an involvement of the TCP-1 containing chaperonin in facilitating the folding of both newly synthesized actin and tubulin(6, 7, 8) . Finally and most germane to the results presented here are previous genetic studies showing a connection between TCP-1 and microtubule-related phenomenon. In the mouse, where the TCP-1 locus was first identified, mutations in the locus were always associated with transmission ratio distortions(11) , perhaps indicative of microtubule abnormalities. In addition, the TCP-1 protein was shown to be expressed at high levels during spermatogenesis, a process that requires the assembly of complex macromolecular structures, including the microtubule core of the flagellum. Perhaps more in line with the observations presented here are those studies in yeast showing that mutants harboring a cold-sensitive mutation in TCP-1 exhibited a number of microtubule-related abnormalities(12, 32) . Such cells, at their restrictive temperature, failed to grow exponentially, had an impaired generation time, and exhibited rather striking abnormalities in normal cell division events. Moreover, these TCP-1 mutants exhibited a large number of abnormal microtubule staining patterns, and gave rise to both anucleate and multinucleate progeny. Thus, these earlier genetic observations, coupled with the biochemical studies shown here, are indicative that a functional TCP-1 protein may be important for the normal function of the centrosome, especially as it relates to the establishment of a proper mitotic/meiotic spindle. Perhaps one or more molecular chaperones, like TCP-1 and hsp 73, participate directly in facilitating various macromolecular rearrangements which accompany the establishment of mitotic and meiotic spindles and the organization of cytoplasmic microtubule arrays in general (see the following paper (28) for further discussion).