To investigate the role of the minus end-directed kinesin-related protein, HSET, in mitotic spindle assembly in mammalian cells, we raised polyclonal antibodies against the COOH-terminal 377 amino acids of the protein. This segment of HSET was expressed as a GST fusion protein, purified by affinity chromatography as described in Materials and Methods, and used to immunize two rabbits. Both rabbits responded similarly to immunization and immunoblot analysis against total HeLa cell protein, showing that these antibodies, αHSET-1 () and αHSET-2 (data not shown), specifically recognized two proteins with equal intensity at 80 and 75 kD. The 80-kD protein identified by our antibodies comigrated with the protein identified by αCTP-2, an antibody raised against the COOH-terminal 11 amino acids (CVIGTARANRK) of XCTK-2, the Xenopus laevis
HSET homologue (Walczak et al. 1997
). Two lines of evidence indicate that the two proteins identified by our antibodies are different isoforms of HSET. First, we have immunoprecipitated sufficient quantities of each protein from a HeLa cell extract to obtain peptide sequence using mass spectrometry. We obtained 12 amino acid peptide sequences from both proteins that were 100% identical to the published HSET sequence. Second, searches of the EST database reveal two classes of HSET cDNAs. The segments of the HSET protein encoded by the two classes of HSET cDNAs are identical, except for the predicted COOH-terminal amino acids. The predicted protein sequence derived from one class of cDNA terminates in the sequence RLPPVSLVRTRGWL, whereas the predicted protein sequence from the other class of cDNA terminates in the sequence NQCVIGTAQANRK. This is consistent with the immunoblot showing that the αCTP-2 antibody is specific for only one of the two isoforms (). The genomic organization of the HSET locus recently reported by Janitz et al. 1999
accounts for only one isoform that terminates in the sequence NQCVIGTAQANRK. Further inspection of the genomic sequence reveals that the COOH terminus of the other isoform is encoded on a distinct exon. We have labeled the 80-kD form HSET-H, and the 75-kD form HSET-L, and with a few specific exceptions as noted, we refer to these proteins collectively as HSET throughout this manuscript. We are currently investigating how the two isoforms are produced and if they differ in any specific functional properties.
HSET Cross-links Microtubules within the Mitotic Spindle
To localize HSET at high resolution within the mitotic spindle, we performed immunogold EM of human CF-PAC1 cells at metaphase. The protocol we have developed involves extraction with a microtubule stabilizing buffer, followed by fixation with glutaraldehyde. This process removes the soluble components of the cells, allowing good penetration of the antibodies, but preserves spindle structure including astral microtubules, centrosomes/centrioles, kinetochore fibers, and chromosomes ( A). Immunofluorescence microscopy showed that HSET localization on the spindle was not detectably different between cells extracted before or after fixation, indicating that its localization was not altered by extraction (data not shown). Using this technique, we obtained good labeling within the metaphase spindle counting 768 gold particles in sections through the long (pole to pole) axis of the half spindle of four different mitotic cells. This labeling was specific because >90% of the gold particles were spindle associated () and no gold labeling was observed when the αHSET-1 antibody was replaced with preimmune antibody (data not shown).
Figure 2 HSET predominately localizes between parallel microtubules in the mitotic spindle of human CF-PAC-1 cells. Cultured CF-PAC-1 cells were fixed and processed for immunogold EM as described in Materials and Methods. A, Low magnification image of a cell processed (more ...)
Immunogold Localization Shows that HSET Predominately Associates between Microtubules in the Metaphase Mitotic Spindle of CF-PAC1 Cells
We quantified the localization of the gold particles obtained by staining for HSET in two ways. First, we divided the half spindle into 1-μm sections perpendicular to the long axis of the spindle. We then counted the number of gold particles and microtubules in each of these sections ( E). The average number of microtubules per section is relatively constant, although there are fewer microtubules nearest the centrosome, consistent with previous work (e.g., Brinkley and Cartwright 1971
). The average number of gold particles was also relatively constant, although there are fewer gold particles near the pole. These data indicate that HSET is concentrated within the main body of the half spindle and contrasts sharply with the concentration of NuMA at the spindle pole that we observed previously, using a similar technique (Dionne et al. 1999
Second, we quantified the position of each individual gold particle relative to the microtubules (). More than 82% of the gold particles were localized within the main body of the spindle with <18% being either not spindle-associated or associated with the astral microtubules. Nearly half of all the gold particles were found to be located between adjacent microtubules ( and and ). Individual gold particles can be seen in the high magnification images in B and C, with HSET's association with spindle microtubules most clearly depicted in C. This image shows an uninterrupted length of a pair of microtubules that have several gold particles in the intermicrotubule space. Many of the microtubules labeled for HSET terminate within a mass of chromatin ( and ; black star) or at a kinetochore ( D, arrow). This indicated that the microtubule polymers in those specific images are oriented parallel to one another with respect to their plus and minus ends. Thus, while our data do not address if HSET localized between antiparallel microtubules, they show that a fraction of HSET is localized between parallel microtubules within the spindle during metaphase. These results, in combination with various in vitro data showing that members of this class of kinesin-related protein have two (or more) microtubule binding domains and are capable of bundling microtubules (McDonald et al. 1990
; Meluh and Rose 1990
; Chandra et al. 1993
; Kuriyama et al. 1995
; Sharp et al. 1997
; Walczak et al. 1997
; Karabay and Walker 1999
), suggest that HSET plays a role in cross-linking microtubules within the mammalian metaphase spindle.
Centrosomes Mask HSET Activity during Mitosis in Living Cells
To determine if the HSET-specific antibodies were capable of disrupting mitotic spindle assembly in living cells, we microinjected αHSET-1 into both HeLa and CF-PAC1 cells. Injected cells were then monitored as they progressed through the cell cycle, fixed, and processed for immunofluorescence either in mitosis or after mitosis was completed. The fixed cells were stained for tubulin and for the injected rabbit antibody. Immunofluorescence analysis of 41 mitotic cells showed that 26 had normal bipolar mitotic spindles. The remaining 15 mitotic cells appeared to have abnormal spindles (data not shown), however, the abnormality was subtle in that the spindles were somewhat barrel-shaped with slightly broader poles than usual. This abnormality did not impede normal transit through mitosis, since 100% (32 out of 32) of αHSET-1–injected cells completed mitosis and formed typical pairs of G1 cells within a typical one hour time frame. This efficiency was similar to values obtained with the preimmune control antibody, where ~90% of injected cells completed mitosis normally (n = 18). These results suggest that either our antibody is not effective at perturbing HSET function in vivo or that inhibition of HSET has no severely deleterious effect on spindle morphology or function in vivo.
Previous work on this class of kinesin-related motor had shown it to be involved in meiotic spindle assembly and function (Lewis and Gencarella 1952
; Davis 1969
; Endow and Komma 1996
, Endow and Komma 1997
; Matthies et al. 1996
). To test if perturbation of HSET function blocked meiotic spindle assembly and function in mammalian cells, we injected αHSET-1 antibodies into mouse oocytes (). Immunoblot analysis of total protein from mouse oocytes showed that HSET-H is the predominant isoform in these cells, and that our antibodies were specific for HSET-H in this cell type (data not shown). The oocytes were injected at the germinal vesicle stage and allowed to mature for 16 h, after which metaphase II arrest would normally occur. Injected oocytes were then processed for indirect immunofluorescence where we stained for chromatin, tubulin, and the injected antibody. In mock-injected oocytes, which completed meiosis and arrested at metaphase II, the meiotic spindles were typically barrel-shaped with broad poles and few astral microtubules. Eg5 localized strongly to the spindle poles of these cells ( A) and HSET localized along the length of the body of the spindle (data not shown). There were also numerous cytoplasmic asters (cytasters) scattered throughout the cytoplasm ( A, arrows), but these did not immunostain for HSET or Eg5. Mock injection of oocytes did not affect the progression of meiosis, as ~70% of cells proceeded through meiosis and arrested at metaphase II within the expected time frame ( E). Oocytes injected with αHSET-1 also progressed through meiosis in the expected time frame, with >70% of αHSET-1–injected oocytes eliciting a first polar body and arresting at metaphase II ( E). Oocytes injected with αHSET-1 and fixed during the first meiotic metaphase showed bipolar spindles in the center of the cell with spindle poles that were broader than in control cells ( B). HSET was observed throughout the length of the spindle, as well as in small aggregates near the microtubule minus ends ( B; arrowheads). The morphology of metaphase II spindles in αHSET-1–injected oocytes, however, was dramatically disrupted compared with either mock-injected cells or metaphase I spindles ( C). The spindle poles were splayed, microtubule minus ends appeared to have lost cohesion, and overall, the spindles had lost bipolarity ( C). In these injected cells, HSET was predominately localized in small aggregates near the microtubule minus ends ( C, arrowheads). To verify that antibody injection was capable of blocking meiosis before metaphase II arrest, we injected antibodies specific to the motor Eg5. Injection of Eg5 antibodies into germinal vesicle stage oocytes blocked the formation of the first bipolar meiotic spindle, with >90% of cells arresting at prometaphase I ( E). In these cells, an astral array of microtubules was assembled around the condensed maternal chromosomes ( D). Therefore, perturbation of Eg5 function blocked the maturation of oocytes at meiosis I. In contrast, perturbation of HSET, while causing obvious defects in the structure of the metaphase I spindle, did not block progression of meiosis until metaphase II. Collectively, these data show that HSET is important for the formation of spindle poles in mammalian oocytes, although the loss of HSET activity was more deleterious to cells in metaphase II than metaphase I of meiosis.
HSET is essential for microtubule organization in metaphase II spindles in mouse oocytes. Mouse oocytes at the germinal vesicle stage were either mock injected (A), injected with antibodies specific for HSET (Β and C), or injected with antibodies specific for Eg5 (D). Injected oocytes were then matured in vitro until metaphase I (7 h; B) or until metaphase II arrest (16 h; A, C, and D). Oocytes were processed for indirect immunofluorescence using antibodies specific for tubulin (red), DNA (blue), and either Eg5 (A) or the injected antibody (B–D; green). Arrows indicate cytoplasmic asters; arrowheads indicate foci of HSET antigen; and the first polar bodies are marked when discernible (1stPB). E, Percentage of microinjected oocytes that mature to metaphase II arrest after 16 h of maturation in vitro postinjection. Oocytes were either mock injected, injected with antibodies specific for Eg5 antibody, or injected with antibodies specific for HSET, as indicated. Bar, 10 μm.
The results of these experiments indicate that our antibodies were capable of perturbing meiotic spindle assembly in mouse oocytes, but that they did not alter the normal assembly and function of the mitotic spindle in cultured cells. Mouse oocytes assemble spindles in the absence of conventional centrosomes (Szollosi et al. 1972
), and we suspected that centrosomes provide additional structural stability to microtubule minus ends at spindle poles that masked any deleterious effect when HSET motor activity was inhibited in cultured cells. To test this hypothesis, we microinjected HSET-specific antibodies into cultured cells and treated those cells with taxol to induce microtubule aster formation. We reasoned that, if centrosomes stabilize the spindle so that HSET function was nonessential, then microtubule asters induced with taxol (many of which lack centrosomes) should be disrupted by our antibodies. For this experiment, we microinjected cells with either the preimmune antibody (control) or αHSET-1, treated the cells with 10 μM taxol, fixed, and processed the cells for immunofluorescence after they entered mitosis (). We stained these cells with antibodies specific for tubulin and NuMA to highlight aster morphology. In cells injected with the preimmune antibody, multiple microtubule asters (13.7 ± 2.6 asters/cell, n
= 10) were observed scattered throughout the cell cytoplasm (, control). Each of these asters had NuMA concentrated at the core, consistent with taxol-induced asters in uninjected cells, indicating that mitotic aster formation was unaffected by microinjection. In contrast, cells injected with αHSET-1 display disorganized microtubule bundles extending throughout the cell cytoplasm, and only a few mitotic asters (1.5 ± 1.3 asters/cell, n
= 10) after taxol treatment (, HSET). NuMA associated with both the asters and the microtubule bundles in these cells, and staining with a human centrosome-specific autoimmune serum (courtesy of J.B. Rattner, University of Calgary, Calgary, Alberta, Canada) verified that each aster observed in these cells contained a centrosome (data not shown). Thus, HSET is essential for meiotic spindle assembly under acentrosomal conditions, but HSET is not essential for spindle assembly in cultured cells because any functional role that it plays is covered by the presence of centrosomes.
Figure 4 HSET is required for the assembly of taxol-induced asters in cultured CF-PAC1 cells. Cultured CF-PAC1 cells were microinjected with either a preimmune antibody (control) or the HSET-specific antibody (HSET) and then treated with 10 μM taxol to (more ...)
HSET and Eg5 Act Antagonistically during the Assembly of Microtubule Asters In Vitro and Mitotic Spindles In Vivo
Previously, we have shown that microtubules induced to polymerize with taxol in extracts prepared from mitotic HeLa cells organize into aster-like arrays (Gaglio et al. 1995
). The organization of microtubule asters in this system requires the motor activity of cytoplasmic dynein and Eg5, and we proposed that a third motor activity was involved, based on the fact that microtubule asters formed in the complete absence of both cytoplasmic dynein and Eg5 (Gaglio et al. 1996
). To determine if HSET has a functional role in organizing microtubule asters in this system, we used our antibodies to specifically perturb HSET activity. Initially, we attempted this by immunodepletion using HSET-specific antibodies. Unfortunately, for reasons that we do not understand, our antibodies maximally depleted only 30% of HSET. This was true regardless of the quantity of antibody used (up to 1 mg). In lieu of immunodepletion, we perturbed the function of HSET by adding our antibodies to the extract. Addition of the preimmune antibody (0.1 mg/ml final concentration) had no effect on the organization of microtubules into asters or on the concentration of NuMA at the aster cores ( and ). In contrast, addition of αHSET-1 (0.1 mg/ml final concentration) to the mitotic extract before or after the induction of microtubule asters blocked the formation of organized aster-like structures. Under these conditions, microtubules were not well-organized and were loosely aggregated in large, disorganized arrays with NuMA diffusely distributed throughout the microtubule aggregates ( and ). In addition to these morphological analyses, we separated the mitotic extract into soluble and insoluble fractions and examined the behavior of known aster components by immunoblot analysis ( E). These blots showed HSET to be a bona fide aster component because a small percentage of HSET-L consistently associated with the insoluble aster-containing fraction. These blots also show that there was no difference in the efficiency with which any of the known aster components associate with the soluble or aster-containing insoluble fractions in the presence of αHSET-1. Thus, consistent with the data of Walczak et al. 1997
showing that XCTK2 is required for spindle assembly in vitro using extracts prepared from frog eggs, HSET is a component of microtubule asters assembled in this cell free system, and is required for both the formation and maintenance of aster-like arrays.
Figure 5 HSET is required for the formation and maintenance of microtubule asters in a cell free mitotic extract. Either the HSET-specific antibody (+αHSET-1; B and D) or a preimmune antibody (+Preimmune; A and C) were added to the mitotic (more ...)
To determine how HSET, cytoplasmic dynein, and Eg5 coordinate microtubule aster formation in this system, we used specific antibodies to perturb the function of each motor individually, as well as to perturb the function of every possible combination of two motors, and all three motors together (). In this experiment, antibodies specific for Eg5 and cytoplasmic dynein were used to deplete those proteins from the mitotic extract, either alone or simultaneously. These depleted extracts, as well as a control extract, were then supplemented with either a preimmune antibody (, control) or αHSET-1 (, +HSET Ab). Microtubule assembly was then induced with taxol, and the resulting structures were fixed and processed for immunofluorescence microscopy using antibodies specific for tubulin and NuMA. The extracts were also separated into soluble, insoluble, and immune pellet fractions, and the behavior of HSET, Eg5, and cytoplasmic dynein within these fractions determined by immunoblot ( I). These immunoblots show that both dynein and Eg5 were depleted to ~100% in each case. These blots also show that none of these motors coimmunoprecipitated with any of the other motors, consistent with our previously published results (Gaglio et al. 1996
). Finally, the immunoblots show that neither the removal of Eg5 and cytoplasmic dynein, nor the addition of αHSET-1, had a detectable effect on the efficiency with which the other motors ( I) or NuMA and dynactin (data not shown) associated with the insoluble microtubule pellet fraction.
Figure 6 HSET, Eg5, and cytoplasmic dynein are required for the formation of microtubule asters in a cell free mitotic extract. Specific antibodies were used to immunodeplete either Eg5 (ΔEg5; C and D), cytoplasmic dynein (ΔDynein; (more ...)
As shown previously, addition of preimmune antibody to the extract had no effect on aster assembly, but addition of αHSET-1 prevented the assembly of mitotic asters ( and and ). Depletion of Eg5 resulted in microtubule asters that were less tightly focused than the controls ( C; Gaglio et al. 1996
). The central core of asters assembled in the Eg5 depleted extract (4.5 ± 0.3 μm, n
= 12) were also expanded, relative to the central core of asters in the control extract (2.3 ± 0.4 μm, n
= 12), as judged by staining for NuMA. Addition of αHSET-1 to an Eg5 depleted extract resulted in microtubule asters that greatly resemble asters formed under control conditions (compare and ). Asters formed in the absence of Eg5 and the presence of αHSET-1 were tightly focused, with NuMA well-concentrated at the central core (2.5 ± 0.4 μm, n
= 12). This result shows that, while addition of αHSET-1 alone prevented microtubule aster formation ( B and 5), the HSET antibody did not block aster formation if Eg5 was absent ( D). This result is consistent with the view that microtubule aster formation in this system requires a balance of forces (Gaglio et al. 1996
). When HSET alone is perturbed, the balance of forces is upset so that asters cannot form. Microtubule aster formation can be restored under conditions where HSET is perturbed if the balance of forces is equilibrated by also removing the motor activity of Eg5. This result indicates that the minus end-directed activity of HSET antagonizes the plus end-directed activity of Eg5 during microtubule aster assembly in this system.
We next tested the effect on microtubule aster assembly if both minus end-directed motors were perturbed. In the absence of cytoplasmic dynein, microtubules fail to organize into aster-like arrays and were randomly dispersed with NuMA distributed along the length of many of the microtubule polymers ( E; Gaglio et al. 1996
). Addition of αHSET-1 to the cytoplasmic dynein depleted extract yielded no microtubule asters and only random microtubule distributions ( F). These results show that microtubule asters fail to form in the absence of HSET alone, cytoplasmic dynein alone, or both HSET and cytoplasmic dynein.
The data presented in and , indicate that the activities of HSET and Eg5 act antagonistically in driving microtubule aster formation in this system. This antagonism is similar to the relationship that we showed previously for cytoplasmic dynein and Eg5 (Gaglio et al. 1996
), which is reproduced here in and . These results show that both of these two minus end-directed motors antagonize Eg5 during microtubule aster formation. However, these results do not discriminate between the possibilities that these two motors act together to antagonize Eg5, or that these two motors act independently, with each antagonizing Eg5. To distinguish between these possibilities, we perturbed the function of all three of these motors, reasoning that if these two minus end-directed motors act together, then the perturbation of both minus end motors in an Eg5 depleted extract should yield results similar to the perturbation of either minus end-directed motor alone in an Eg5 depleted extract (i.e., mitotic asters should form). The results of perturbing HSET in a cytoplasmic dynein and Eg5 depleted extract show that aster-like arrays did not form, and that the microtubules were randomly dispersed ( H). The lack of microtubule aster formation in the absence of all three motors is in stark contrast to the microtubule asters that form in the absence of either Eg5 and HSET ( D) or Eg5 and cytoplasmic dynein ( G). This demonstrates that these two motors act independently of each other in antagonizing Eg5 activity in this system.
We estimated the microtubule aster forming capacity of the mitotic extracts during these various depletion experiments by counting the total number of microtubule asters in 20 randomly selected microscope fields (). These counts demonstrate that the microtubule aster forming capacity of the extracts depleted for Eg5, Eg5 and cytoplasmic dynein, or Eg5 with the addition of the HSET antibody were comparable to that of a control extract. On the other hand, if the extract was depleted of cytoplasmic dynein, or if the HSET antibody was added to the extract alone, extract depleted of cytoplasmic dynein, or extract depleted of both cytoplasmic dynein and Eg5, then virtually no microtubule asters were observed. Thus, the images shown in are representative of the populations of microtubule structures observed under each condition tested.
Figure 7 Microtubule aster forming capacity of mitotic extracts depleted of various components. The average number of microtubule asters in 20 randomly selected microscope fields (400×) from three separate experiments is shown and each is normalized to (more ...)
Collectively, the results from the experiments presented in and lead to three conclusions. First, the minus end-directed activity of HSET opposes the plus end-directed activity of Eg5 in a way that is similar to the opposition between cytoplasmic dynein and Eg5. Second, the minus end-directed activities of HSET and cytoplasmic dynein oppose the plus end-directed activity of Eg5 independently of each other. Third, although we cannot rule out a minor role played by other motors, the lack of microtubule organization in the absence of all three of these motors indicates that HSET, Eg5, and cytoplasmic dynein are most likely the primary motors responsible for building microtubule asters in this system.
Finally, we tested if HSET functionally opposes Eg5 activity in vivo. For this experiment, we microinjected human CF-PAC1 cells with either antibodies specific for Eg5 or a combination of HSET antibodies and Eg5 antibodies. We monitored the injected cells and fixed and processed them for indirect immunofluorescence using antibodies specific for γ-tubulin to detect centrosomes and for the injected rabbit antibody. Cells injected with the Eg5 antibody alone formed monopolar spindles and arrested in mitosis (, αEg5; Blangy et al. 1995
; Gaglio et al. 1996
). More than 75% of Eg5-injected cells had centrosomes that had not separated to any measurable degree (). In contrast, >68% of cells injected with both HSET and Eg5 antibodies displayed separated centrosomes (). Many of the double injected cells did not have a symmetric spindle at the time of fixation, as judged by the lack of a well-organized metaphase plate (, middle) or the location of both centrosomes on the same side of the chromosomes. The centrosomes in these cells were clearly separated, but were frequently in different focal planes within the cell, which accounts for the variable intensity of each centrosome shown in . In some instances, symmetric, bipolar spindles formed under these conditions (, αEg5/αHSET, and ), and we observed a small fraction of cells (4%) complete mitosis normally, forming pairs of G1 cells with recognizable midbodies (data not shown). These results show a statistically significant (x2
= 50.19, P
≤ 0.0001) increase in centrosome separation in cells injected with antibodies to both HSET and Eg5, compared with cells injected with Eg5 antibodies only. Thus, with respect to centrosome separation, these data indicate that HSET and Eg5 oppose each other in vivo. Furthermore, these results indicate that centrosome separation can proceed under conditions where Eg5 function is blocked.
Figure 8 HSET antagonizes the plus end-directed activity of Eg5 during centrosome separation in vivo. Cultured CF-PAC1 cells were injected with antibodies specific for Eg5 (αEg5) or simultaneously with two antibodies, one specific for HSET and the other (more ...)
Centrosome Separation Can Occur When the Activities of HSET and Eg5 Are Inhibited Simultaneously In Vivo by Antibody Microinjection