(Received for publication, January 14, 1997, and in revised form, April 28, 1997)
From the Swiss Institute for Experimental Cancer
Research (ISREC), 155, Chemin des Boveresses, CH-1066 Epalinges,
Switzerland and the ¶ Department of Molecular Biology, Sciences
II, University of Geneva, 30, quai Ernest-Ansermet,
CH-1211 Geneva 4, Switzerland
The kinesin-related motor HsEg5 is essential for
centrosome separation, and its association with centrosomes appears to
be regulated by phosphorylation of tail residue threonine 927 by the
p34cdc2 protein kinase. To identify proteins able to
interact with the tail of HsEg5, we performed a yeast two-hybrid screen
with a HsEg5 stalk-tail construct as bait. We isolated a cDNA
coding for the central, -helical region of human
p150Glued, a prominent component of the dynactin complex.
The interaction between HsEg5 and p150Glued was enhanced
upon activation of p34CDC28, the budding yeast homolog of
p34cdc2, provided that HsEg5 had a phosphorylatable residue
at position 927. Phosphorylation also enhanced the specific binding of
p150Glued to the tail domain of HsEg5 in vitro,
indicating that the two proteins are able to interact directly.
Immunofluorescence microscopy revealed co-localization of HsEg5 and
p150Glued during mitosis but not during interphase,
consistent with a cell cycle-dependent association between
the two proteins. Taken together, these results suggest that HsEg5 and
p150Glued may interact in mammalian cells in
vivo and that p34cdc2 may regulate this interaction.
Furthermore, they imply that the dynactin complex may functionally
interact not only with dynein but also with kinesin-related motors.
Microtubule-based motors of the kinesin and dynein families are implicated in many different cellular processes, including organelle transport, mitotic spindle assembly, and chromosome segregation (for recent reviews see Refs. 1-6). Molecular studies have greatly improved our understanding of the structures of these motors, and mechanistic aspects of microtubule-motor interactions are beginning to emerge (for reviews see Refs. 7 and 8). In contrast, it remains largely unknown what mechanisms control the subcellular distribution and activity of individual motors in time and space (9).
Accessory proteins with a potential role in targeting have been identified for both dynein and kinesin family members. Specifically, cytoplasmic dynein has been shown to interact with a multimolecular complex termed dynactin (10-12), and it has been proposed that dynactin might anchor cytoplasmic dynein to specific subcellular structures (9, 13, 14). Dynactin is composed of at least nine polypeptides, including an actin-related protein termed centractin or Arp1 (15-17), a 50-kDa protein termed dynamitin (18), and a 150-kDa protein homologous to the Drosophila gene product Glued (10, 19, 20). Genetic data from Saccharomyces cerevisiae, Neurospora crassa, and Drosophila melanogaster support a functional association between dynein and dynactin complexes (21-28). By immunocytochemistry, both cytoplasmic dynein and dynactin localize to membranous organelles, centrosomes, and kinetochores, indicating that the two complexes display at least partially overlapping subcellular distributions (10, 16, 18, 29-31). Kinesin function also requires accessory proteins, both in vivo and in vitro. One prominent protein interacting with kinesin, termed kinectin (32), may promote the association between kinesin and cargo vesicles (33-35). Many KRPs1 may require accessory proteins for a specific association with appropriate subcellular structures, but the identification of partners for KRPs has proven difficult, most likely because the interactions between these proteins are labile and highly regulated.
Prominent among the KRPs implicated in different aspects of mitotic spindle assembly and function are the BimC family members (reviewed in Refs. 3, 4, and 6). These motors are required for spindle pole separation and hence for bipolar spindle formation in organisms ranging from yeast to human. Named after the BimC gene product of Aspergillus nidulans (36), likely functional homologs of BimC have been described in S. cerevisiae (Cin8p and Kip1p) (37), Schizosaccharomyces pombe (cut7) (38, 39), D. melanogaster (KLP61F) (40), Xenopus laevis (XlEg5) (41, 42), and Homo sapiens (HsEg5) (43). In a previous study, we showed that HsEg5 associates with centrosomes in early prophase (43), and data obtained for both XlEg5 and HsEg5 indicate that this event depends on phosphorylation of tail residue threonine 927 (Thr-927) by p34cdc2/cyclin B (43, 44). Prompted by these findings, we have now used a yeast two-hybrid screen to identify proteins that would interact with the HsEg5 tail domain. We report that the HsEg5 tail interacts specifically with the human homolog of the dynactin component p150Glued. Moreover, we present evidence that this interaction is enhanced by phosphorylation of residue Thr-927 of HsEg5, both in yeast cells in vivo and in vitro. These provocative results have implications not only for the function of HsEg5, but they also provide a new perspective on the role of the dynactin complex.
A cDNA encoding the stalk and
tail domains of HsEg5 (Eg5ST) was fused to the GAL4 DNA binding domain
(DBD) by inserting a NdeI-SalI fragment of the
original HsEg5 cDNA (43) into plasmid pAS2 digested with
NdeI-SalI (45). Similarly, for fusion of Eg5ST to
the GAL4 activation domain (AD), a (NdeI)-EcoRI
fragment excised from the HsEg5 cDNA was inserted into plasmid
pACT2 digested with SmaI and EcoRI (45). (Some
restriction enzymes are marked in parentheses to indicate that, before
ligation, Klenow polymerase was used to blunt end 5-protruding DNA
fragments). The HsEg5 tail domain (Eg5T) was fused to GAL4 AD by
inserting a HincII-BamHI fragment of the cDNA
into plasmid pACT2 digested with (NcoI) and BamHI. The resulting constructs, Eg5ST and Eg5T, thus
comprise the C-terminal moieties of HsEg5 starting at residues 245 and 764, respectively. For control, the Nek2 protein kinase (46) was fused
to GAL4 DBD by inserting a (NaeI)-(XbaI) fragment
excised from the full-length cDNA into pAS2 digested with
SmaI. Nek2 was also fused to GAL4 AD by inserting a
NcoI-BamHI fragment into pACT2 digested with
NcoI-BamHI. The HsGlu-1 cDNA isolated in the course of the two-hybrid screen was fused to an Myc epitope tag by
inserting an EcoRI-BglII fragment into plasmid
pBSmyc digested with EcoRI and BglII (47). The
whole HsEg5 sequence was fused to the GST protein by inserting the
EcoRI fragment of the cDNA into plasmid pGEX-3X
(Pharmacia Biotech, Inc.).
A yeast two-hybrid screen was
performed essentially as described (45, 48). In brief, yeast strain
Y190 expressing the fusion protein DBD-Eg5ST was transformed with a
human cDNA library constructed in plasmid pACT (48) and plated on
selective medium (lacking tryptophan, leucine, and histidine)
supplemented with 25 mM 3-aminotriazole. From 4 × 106 transformants, 141 His+ colonies were
obtained and assayed for LacZ expression. After being cured from the
bait plasmid, the resulting 50 positive colonies were mated with Y187
yeast strains expressing DBD-Eg5ST, -cdc2, -cdk2 or -lamin fusion
proteins. Seventeen colonies showed a strong specific interaction with
DBD-Eg5ST only. Of 11 colonies without rearrangements in the initial
pACT1 vector, two contained the same insert, termed HsGlu-1. Specific
interactions between HsGlu-1 plasmids and the DBD-Eg5ST bait were also
observed when both plasmids were retransformed into Y190 yeast cells.
For -galactosidase activity assays on filters, yeast colonies were
replica-plated onto nitrocellulose filters, and these were frozen for
20 s in liquid nitrogen. Then they were placed onto a Whatman No.
3MM paper soaked with Z buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM
-mercaptoethanol, pH 7) containing 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (1 mg/ml) and incubated at
30 °C. For
-galactosidase liquid assays, 10 ml of mid-log phase
yeast cultures (A600 ~ 0.5) were centrifuged
and resuspended in 1 ml of Z buffer. Cells were lysed by the addition
of 100 µl of CHCl3 and 100 µl of 0.1% SDS, followed by
a 20-s vortex. After equilibration at 30 °C in a water bath, 0.2 ml
of a 4 mg/ml o-nitrophenyl
-D-galactopyranoside substrate solution was added.
Reactions were stopped by the addition of 0.5 ml of 1 M
Na2CO3. The
-galactosidase activity was then estimated by measuring the absorbance at 420 nm, and units of activity
were calculated using the expression, 1000 × A420)/(t × v × A600), where t is the reaction time
in minutes and v is the volume of the culture in ml. For nocodazole
arrest, yeasts were grown to mid-log phase. Then cultures were
supplemented with 1% Me2SO and 15 µg/ml nocodazole, and
incubation was extended for another 90 min.
-Galactosidase activity
was then measured as described above. The cell cycle stages of the
cultures were determined by microscopic observation of budding.
Myc-tagged proteins were translated in rabbit reticulocyte lysates according to manufacturer instructions (Promega). For in vitro interaction assays, 100 ng of GST or MalE fusion proteins, purified according to manufacturer instructions (Pharmacia and New England Biolabs, Inc.), were mixed with 10 µl of in vitro translation reactions, and volumes were adjusted to 100 µl with immunoprecipitation buffer (0.25% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8). Samples were incubated for 60 min at 30 °C before 5 µl of monoclonal 9E10 anti-Myc antibody (49) adsorbed to protein G-Sepharose beads were added. After a 60-min incubation at 4 °C on a rotating wheel, beads were rinsed three times in immunoprecipitation buffer, boiled in loading buffer, and run on an SDS-polyacrylamide gel. Immunoblotting was then performed as described, using anti-MalEg5T serum (43). For in vitro phosphorylation of MalEg5T, 10 µg of purified protein (43) was incubated for 1 h at 37 °C with 1 µl of p34cdc2/cyclin B kinase (New England Biolabs) in a total volume of 100 µl according to manufacturer instructions. For coimmunoprecipitation assays, one-hundredth of the sample (i.e. 100 ng of phosphorylated MalEg5T protein) was incubated with in vitro translation products.
Immunofluorescence MicroscopyHeLa cells were seeded onto
glass coverslips and grown for 24 h as described previously (50).
Cells were rinsed with PBS, fixed for 10 min at room temperature with
3.7% paraformaldehyde, 2% sucrose in PBS and permeabilized by a 30-s
treatment with acetone at 20 C°. For co-staining of HsEg5 and
p150Glued, coverslips were first incubated for 1 h
with a mixture of rabbit anti-HsEg5 antibody (Eg5+, 2 µg/ml) (43) and a mouse anti-p150Glued antibody (2 µg/ml; Transduction Laboratories) in PBS, 1% bovine serum albumin.
After several washes in PBS, coverslips were incubated for 45 min with
a biotinylated goat anti-mouse antibody (1:50 in PBS, 1% bovine serum
albumin; Amersham Corp.), washed again, and finally incubated for 45 min with a mixture of Texas red-conjugated streptavidin (1:200,
Amersham Corp.), a 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF)-conjugated goat anti-rabbit antibody (1:500, Pierce), and Hoechst 33258 (0.5 µg/ml, Calbiochem-Novabiochem) in PBS, 1% bovine serum albumin. Coverslips were mounted in 80% glycerol, 3%
n-propyl gallate in PBS, and cells were viewed with a Zeiss
Axioplan 2 microscope using a 63 × oil immersion objective.
To identify
cellular proteins that might interact with HsEg5, a yeast two-hybrid
screen was performed (51, 52). Since KRP function may often require
oligomerization (53-56), we considered it important to use a bait
protein that would be able to oligomerize in yeast. Overexpression of
the full-length HsEg5 protein in S. cerevisiae was toxic;
thus, the motor domain was deleted from HsEg5, and the remaining
stalk-tail moiety (termed Eg5ST) was fused to either the DBD or the AD
of GAL4. Coexpression of DBD-Eg5ST and AD-Eg5ST in yeast activated the
lacZ reporter gene (48), indicating that these two fusion
proteins were able to interact (Fig. 1A).
Deletion of the stalk domain from one of the two proteins (shown for
the pair DBD-Eg5ST and AD-Eg5T) or coexpression of Eg5ST constructs
with control fusion proteins (AD-Nek2 and DBD-Nek2) did not result in
any interaction (Fig. 1A). These data show that HsEg5 fusion
proteins can oligomerize in yeast provided that they contain stalk
domains.
Using the DBD-Eg5ST construct as bait, a human cDNA library was
screened (48), and library clones able to activate both the selection
marker His3 and the lacZ reporter gene specifically in the
presence of DBD-Eg5ST were selected. Among the clones showing a
specific interaction with Eg5ST, two were identical and contained a
1179-base pair cDNA insert. Data base searches revealed that the
corresponding translation product was closely related to a fragment of
the rat p150Glued protein (10, 19); hence, this cDNA
was termed HsGlu-1. As shown in Fig. 1B, AD-Glu-1 activated
the lacZ reporter gene specifically in the presence of
DBD-Eg5ST but not in the presence of unrelated bait constructs
(DBD-cdc2, DBD-cdk2, or DBD-lamin). An alignment between HsGlu-1 and
the central -helical domain (encompassing residues 420-811) of rat
p150Glued is shown in Fig. 1C. Over this region,
the two proteins display 97% identity, indicating that HsGlu-1 almost
certainly represents part of the human homolog of rat
p150Glued. Fig. 1D shows a schematic view of
p150Glued and indicates the regions identified as potential
interaction domains for HsEg5 (this study) and the intermediate chain
of dynein (17, 57). There is potentially a partial overlap between
these two putative binding sites, but it is noteworthy that the HsEg5 interaction site falls outside of the predicted coiled coil domain of
p150Glued.
As shown previously, phosphorylation of tail
residue Thr-927 by the p34cdc2 protein kinase promotes the
association of HsEg5 with early prophase centrosomes (43). To determine
whether the interaction between Eg5ST and HsGlu-1 observed in yeast
might be favored by phosphorylation of Thr-927, DBD-Eg5ST mutants
containing an alanine, serine, or aspartic acid in place of Thr-927
were constructed. These mutants were then coexpressed in yeast together
with AD-Glu-1, and protein extracts were assayed for -galactosidase
activities. As shown in Fig. 2A, the average
-galactosidase activity was higher in yeasts expressing either the
wild-type (T) or the serine mutant (S) of Eg5ST
than in yeasts expressing the corresponding alanine (A) or
aspartic acid (D) mutants. These data indicate that the interaction between Eg5ST and HsGlu-1 is reduced by the mutation of
Thr-927 to a nonphosphorylatable residue (alanine or aspartic acid) but
not by a mutation to another phosphorylatable residue (serine). It is
noteworthy that the replacement of threonine 927 by aspartic acid did
not enhance binding of Eg5ST to HsGlu-1, suggesting that the additional
negative charge introduced by this substitution cannot mimic
phosphorylation of HsEg5. This result is entirely consistent with our
previous finding that a T927D mutation did not confer constitutive
centrosomal association to HsEg5 in vivo (43).
Exponentially growing yeast populations contain only a small proportion
of cells in G2 and M phase (approximately 20%). Therefore, the extent of phosphorylation of the Eg5ST bait proteins by
p34CDC28 protein kinase is expected to be low, and it is
not surprising that analyses of exponentially growing cells revealed
only a modest influence of the identity of residue 927 on the
interaction with HsGlu-1 (Fig. 2A). However, considering
that p34CDC28 protein kinase is maximally active during
mitosis, we predicted that binding of wild-type Eg5ST to HsGlu-1 should
be substantially increased in mitotically arrested cells, whereas
binding of the alanine mutant should not be enhanced. Indeed, compared
with exponentially growing cells (20% in G2 or M),
-galactosidase activity was doubled when yeast cells expressing
wild-type Eg5ST were blocked in mitosis with nocodazole (80% in
G2 or M) but was almost unchanged in the case of the
alanine mutant (Fig. 2B). These results indicate that phosphorylation of Thr-927 by p34CDC28 enhances the
interaction between Eg5ST and HsGlu-1, and this in turn implies that
HsGlu-1 most probably binds to the tail region of the motor.
To determine whether HsEg5 and
p150Glued interact directly, in vitro binding
studies were performed. For this purpose, the entire HsEg5 protein was
fused to GST (GST-Eg5), and the tail domain was fused to the MalE
protein (MalEg5). Both fusion proteins were overexpressed in
Escherichia coli and purified. The HsGlu-1 cDNA was
Myc-tagged (myc-Gl1), and the corresponding RNA was translated in a
rabbit reticulocyte lysate. For control, a second protein isolated
during the two-hybrid screen, and thus potentially interacting with
HsEg5, was also tagged (myc-Q2) and translated. In a first experiment
(Fig. 3A), myc-Gl1 (lane 1) and
myc-Q2 (lane 2) translation products, as well as control
reticulocyte lysate (lane 3), were incubated with GST-Eg5
protein; then immunoprecipitations were performed with anti-Myc
antibodies, and the isolated complexes were probed by immunoblotting
with anti-Eg5 antibodies. Efficient immunoprecipitation of the GST-Eg5
fusion protein by anti-Myc antibodies was observed only in the sample
in which myc-Gl1 protein was present (Fig. 3A, compare
lane 1 with lanes 2 and 3). These results show that an interaction between HsGlu-1 and HsEg5 occurs not
only in yeast cells in vivo, but also in vitro,
implying that HsGlu-1 is able to bind directly to HsEg5. In contrast,
when tested under identical conditions, the protein encoded by
Q2 did not bind to HsEg5 in vitro, suggesting
that its isolation in the yeast two-hybrid screen may have been the
result of an indirect interaction.
To rule out a role of the GST moiety in binding to HsGlu-1 and to determine more precisely which part of HsEg5 interacted with HsGlu-1, a similar type of experiment was performed with MalEg5T using MalE protein for control. Moreover, prompted by the finding that phosphorylation of Thr-927 promotes the interaction of HsEg5 with the spindle apparatus in vivo (43) and the interaction with HsGlu-1 in yeast cells (Fig. 2), we examined the influence of prior phosphorylation of the MalEg5T fusion protein by recombinant p34cdc2/cyclin B on its ability to interact with myc-Gl1 in vitro. In the presence of the myc-Gl1 translation product, anti-Myc antibodies efficiently precipitated the MalEg5T fusion protein but not MalE alone (Fig. 3B, lanes 1 and 2), indicating that p150Glued can interact directly and specifically with the tail domain of HsEg5. Prior phosphorylation by p34cdc2/cyclin B clearly enhanced the association of the MalEg5T fusion protein with myc-Gl1 (Fig. 3B, lane 3). To prove that this enhanced binding involved phosphorylation of residue Thr-927, the same experiment was repeated, this time comparing the effect of phosphorylation on the binding ability of either the wild-type MalEg5T fusion protein or the corresponding T927A mutant construct (Fig. 3C). The wild-type Eg5T protein exposed to p34cdc2/cyclin B displayed strong binding to myc-Gl1 (lane 2), whereas both the mutant construct (lane 3) and the unphosphorylated wild-type tail protein (lane 1) showed markedly lower binding. All our attempts to coimmunoprecipitate HsEg5 and p150Glued directly from mammalian cell extracts or to extract soluble HsEg5·p150Glued complexes from cells have failed (data not shown, see "Discussion"). This is not entirely unexpected, however, considering that similar approaches have also failed to reveal the purported interaction between cytoplasmic dynein and p150Glued (for discussion see Ref. 18). In fact, it may be extremely difficult (and perhaps impossible) to find conditions affording the solubilization of the mitotic spindle apparatus while at the same time preserving particular protein-protein interactions to a sufficient extent for visualization by coimmunoprecipitation. In support of this view, we found that efficient solubilization of p150Glued required treatment of cells with 1% Nonidet P-40, whereas detergent concentrations >0.25% abolished the in vitro interaction between p150Glued and HsEg5 (data not shown).
Co-localization of HsEg5 and p150Glued during MitosisTo further explore the possibility that HsEg5 and
p150Glued might interact in mammalian cells in
vivo, we have performed indirect immunofluorescence microscopy on
HeLa cells using antibodies specific for HsEg5 and
p150Glued. If, as suggested by the above results, HsEg5 and
p150Glued undergo a cell cycle-regulated interaction, one
would in fact predict that the two proteins should co-localize during
mitosis but not during interphase. Immunofluorescent staining of HeLa cells showed that, during interphase of the cell cycle,
p150Glued is associated with centrosomes (Fig.
4b), whereas HsEg5 is distributed throughout
the cytoplasm (Fig. 4a), consistent with previous data (10,
43, 58). Beginning in early prophase, however, HsEg5 also associates
with the centrosomal region, presumably as a consequence of
phosphorylation of Thr-927 by p34cdc2/cyclin B (43, 44). As
a result, the distributions of HsEg5 and p150Glued overlap
at least partially as soon as mitosis begins (Fig. 4, c-e).
Both proteins then remain associated with the mitotic spindle throughout mitosis (Fig. 4, f-k). Although of limited
resolution, these immunocytochemical data support the idea that HsEg5
and p150Glued may interact not only in yeast cells and
in vitro but also in mammalian cells in vivo;
furthermore, they are consistent with the hypothesis that the
interaction between the two proteins is controlled by cell
cycle-dependent phosphorylation of Thr-927 in the tail
domain of HsEg5.
In human cells, the KRP HsEg5 is required for centrosome separation and formation of a bipolar spindle (43). At the onset of mitosis, the association of this mitotic motor with the spindle apparatus is promoted by phosphorylation of a critical residue (Thr-927) in the tail domain of HsEg5, and this phosphorylation is most likely brought about by p34cdc2 (43, 44). Thus, HsEg5 provides an interesting example for studying the regulated targeting of a microtubule-dependent motor to a particular subcellular structure. Here, we have used a yeast two-hybrid screen to search for proteins interacting with HsEg5 and have isolated a cDNA encoding the central part of a human homolog of p150Glued, a subunit of the dynactin complex. Moreover, a recombinant HsEg5 tail protein could also bind to p150Glued in vitro, indicating that the two proteins are able to interact directly. We may conclude, therefore, that the central domain of p150Glued (corresponding to residues 420-811 in the rat sequence) contains a binding site for the tail domain of HsEg5 (residues 764-1057). Most remarkably, the association between the two proteins was enhanced by phosphorylation of Thr-927, both in vitro and in yeast cells in vivo. Finally, immunofluorescence microscopy showed that HsEg5 and p150Glued display a striking co-distribution during mitosis, whereas, as expected, they do not co-localize during interphase of the cell cycle.
Taken together, these observations raise the intriguing possibility that HsEg5 may undergo a cell cycle-regulated interaction with the dynactin complex in mammalian cells. However, we caution that the available data do not constitute definitive proof for a direct in vivo interaction between HsEg5 and p150Glued. All our attempts at coimmunoprecipitating the two proteins directly from mammalian cell extracts have been unsuccessful. This negative result may be explained by our observation that the in vitro association between HsEg5 and p150Glued is readily broken under the conditions that are required to efficiently extract these proteins from cells. Also, we note that similar difficulties have been encountered in the case of the purported association between dynein and dynactin (58). Support for a physiologically significant interaction between dynein and dynactin rests not only on biochemical data (12, 57) but also on genetic studies showing that mutations in the two complexes produce similar phenotypes (21-28). Both HsEg5 and cytoplasmic dynein are clearly required for bipolar spindle formation (43, 59), but we are not aware of genetic evidence that suggests a direct interaction between BimC family members and dynactin. One possible explanation for the lack of such genetic data is that mitotic KRPs display a significant degree of functional redundancy (60). Thus, genetic interactions between KRPs and dynactin may be difficult to reveal without mutational inactivation of multiple genes. Our present findings suggest that a deliberate search for genetic interactions between BimC homologs, other KRPs, and dynactin components might be rewarding.
What could be the physiological significance of a cell cycle-regulated interaction between HsEg5 and p150Glued? Previously, the function of p150Glued has been considered primarily in the context of the purported interaction between dynactin and cytoplasmic dynein, a minus end-directed motor (13). Our identification of p150Glued as a potential partner of a plus end-directed KRP therefore constitutes a surprise. Potentially, our findings have important implications not only for the functions of HsEg5 but also for those of the dynactin complex. The exact function of dynactin in relation to cytoplasmic dynein is not definitively established, and several possibilities have been considered (reviewed in Refs. 9, 13, and 14). One intriguing, albeit poorly explained observation is that both cytoplasmic dynein and the p150Glued component of dynactin are able to bind to microtubules. Specifically, the N terminus of p150Glued harbors a putative microtubule binding domain that is clearly able to mediate binding to microtubules in vitro (17). Interestingly, this domain resembles a motif identified in CLIP-170, a protein implicated in linking endosomes to microtubules (61). Thus, it has been proposed that dynactin might act as a tether among cytoplasmic dynein, microtubules, and cargoes (e.g. vesicles or kinetochores) during those stages of the dynein ATPase cycle when the motor domain is not in contact with microtubules (for discussion see Refs. 14, 17, and 18). Conceptually, it is not difficult to extend this model to mechanochemical motors other than cytoplasmic dynein (see below).
An essential role for HsEg5 and other BimC family members in centrosome and spindle pole body separation is well established, but the precise function of these motors remains unknown, and different scenarios have been considered (for discussion see Refs. 4 and 42). One specific model proposes that plus end-directed motors, while attached to some structure in the vicinity of one centrosome, might move toward the plus end of a microtubule emanating from the other centrosome, thereby promoting spindle pole separation (42). Thus, p150Glued might serve as an anchor for HsEg5, mediating its association with near-centrosomal structures during prophase and perhaps with additional elements of the spindle during later stages of mitosis. Potentially relevant to this model, the dynein-dynactin complex has recently been reported to functionally interact with the protein NuMA, and this protein may also contribute to tether microtubules to the poles (62). An alternative model proposes that BimC family motors could act as tetramers and promote centrosome separation by moving toward the plus ends of microtubules while being bound to two microtubules emanating from distinct centrosomes (4). In this scenario, p150Glued might mediate the association of HsEg5 oligomers with microtubules throughout their ATPase cycles, as proposed previously for the dynein-dynactin complex (see above).
The idea that accessory proteins might function in relation to both dynein- and kinesin-related motors is not without precedent. Specifically, we note that kinectin, an accessory protein implicated in targeting of KRPs to endomembranous vesicles, has been postulated to interact also with cytoplasmic dynein (32, 33). If, as proposed here, dynactin complexes are able to interact with both minus and plus end-directed motors, this would imply that the role of dynactin is more general than hitherto surmised. One obvious possibility is that dynactin complexes might tether multiple classes of microtubule-dependent motors to microtubules, as discussed above. Another possibility would be that dynactin-motor interactions might be important for the recycling of motor complexes. In the context of spindle assembly, for example, the available evidence is consistent with the idea that dynein-dynactin complexes may function at kinetochores (reviewed in Refs. 6, 9, 13, and 14). However, migration toward the minus ends of microtubules will in time be expected to cause these complexes to accumulate at centrosomes, a scenario supported by immunocytochemical data (10, 18, 29, 30, 58). It is attractive to speculate, therefore, that one role of HsEg5 (and perhaps of other centrosome-associated plus end-directed motors) could be to prevent the excessive accumulation of dynein-dynactin complexes at centrosomes by moving them back toward microtubule plus ends. Consistent with such a model, recent studies suggest that mitotic aster formation in vitro requires precisely balanced plus and minus end-directed motor activities (63).
In conclusion, the results described here raise the intriguing possibility that the multiprotein complex dynactin may interact not only with cytoplasmic dynein but also with at least one member of the KRP family, HsEg5. Thus, much like the integral membrane protein kinectin may play a dual role in mediating the association of both dynein and kinesin-related motors with membrane vesicles (33), we propose that the dynactin complex may also play a dual role in mediating the association of cytoplasmic dynein and HsEg5 (and perhaps other KRPs) with the mitotic spindle apparatus.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U90445.
We thank Drs. Stephen Elledge (Baylor College, Houston, TX) and Richard Vallee (Worcester Foundation, Shrewsbury, MA) for generous gifts of yeast two-hybrid reagents and anti-p150Glued antibodies, respectively. We also thank members of the laboratory for helpful discussions and M. Allegrini and N. Roggli for help with the artwork.