The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle.
We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.
PubMed ID: 10525540
PMC ID: PMC2174226
Article link: J Cell Biol.
Grant support: GM51542 NIGMS NIH HHS , R37WD12913 PHS HHS
Genes referenced: adcyap1r1 dync1li1 kif11 kifc1 numa1
Antibodies referenced: Kifc1 Ab1
Article Images: [+] show captions
|Figure 1. HSET is expressed as two isoforms in HeLa cells. Total HeLa cell protein was separated by SDS-PAGE and then was blotted with the HSET-specific antibody (αHSET-1) or a COOH-terminal peptide antibody (αCTP-2; Walczak et al. 1997). The two isoforms of HSET detected by αHSET-1 are designated HSET-H and HSET-L. Migration positions of myosin (200), β-galactosidase (116), phosphorylase B (97), and albumin (66) are shown in kD.|
|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 for EM under these conditions. Bar, 1 μm. B–D, High magnification images showing typical HSET localization between microtubules within the spindle. Frequently, HSET localized to microtubules that terminated within a mass of chromatin (B and C; black star) or within a kinetochore (D; arrow). Arrowhead in D indicates a gold particle. Bars, 0.1 μm. E, Sections through the half spindle of four independent cells were divided into 1-μm regions perpendicular to the long axis of the spindle, with the first region (1 μm) spanning the centrosome and the last region (6 μm) close to the chromosomes. The total number of microtubules and gold particles (generated by immunolabeling for HSET) were counted in each section. The total values were averaged over the number of sections, and the average number of gold particles and microtubules plotted as a function of the region of the spindle.|
|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 induce the assembly of cytoplasmic microtubule asters. The injected cells were monitored by phase-contrast microscopy until they entered mitosis and then analyzed by indirect immunofluorescence microscopy using antibodies specific for tubulin (αTubulin), NuMA (αNuMA), and the DNA-specific dye DAPI, as indicated. Bar, 10 μm.|
|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 extract either before (PRE) or after (POST) the formation of microtubule asters. The resulting structures were analyzed by indirect immunofluorescence microscopy using antibodies specific for tubulin and NuMA as indicated. E, These extracts were also separated into 10,000 g soluble (S) and insoluble (P) fractions and subjected to immunoblot analysis using antibodies specific for NuMA, dynactin, Eg5, cytoplasmic dynein, HSET, and tubulin as indicated. Bar, 10 μm.|
|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; E and F), or both Eg5 and cytoplasmic dynein (ΔEg5/ΔDynein; G and H) from a HeLa cell mitotic extract. Untreated extracts (A and B) or the depleted extracts were then supplemented with preimmune antibodies (control) or HSET-specific antibodies (+HSET Ab). The formation of microtubule asters was stimulated by the addition of taxol, ATP, and by incubation at 31°C. The resulting structures were analyzed by indirect immunofluorescence using antibodies specific for tubulin and NuMA, as indicated. I, Eg5-, Dynein-, and Eg5/Dynein-depleted mitotic extracts supplemented with preimmune or HSET-specific antibodies were separated into 10,000 g soluble (S) and insoluble (P) components and the immune pellet fraction (PAb), and were subjected to immunoblot analyses using antibodies specific for Eg5, cytoplasmic dynein, and HSET, as indicated. Bar, 10 μm.|
|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 100% using the control extract.|
|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 specific for Eg5 (αEg5/αHSET). Cells were monitored by phase-contrast microscopy until they entered mitosis, after which they were fixed and processed for indirect immunofluorescence using antibodies specific for γ-tubulin, the injected antibody/antibodies, and with the DNA-specific dye DAPI, as indicated. Arrows indicate separated centrosomes observed in double injected cells. Bar, 10 μm.|