We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at ϳ30 m/min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at ϳ75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of ϳ5 m/min, and microtubules glide for an average of 3 m before ceasing movement. Finally, we purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.Conventional kinesin and kinesin-related proteins (KRPs) 1 constitute a rapidly expanding superfamily of microtubuleassociated motor proteins that perform a variety of cellular functions (1, 2). Conventional kinesin was originally identified as a fast moving (ϳ30 m/min) microtubule-based motor in squid giant axons (3), and it is believed to function as a heterotetramer consisting of two heavy chains and two light chains (reviewed in Ref. 4). It has since been identified in virtually all cell types and has multiple roles in vesicle trafficking (5-9); however, it is not yet clear if conventional kinesin has a role in animal cell mitosis. Microinjection of antibodies specific to kinesin heavy chain had no effect on mitotic progression of early sea urchin embryos (10), and no mitotic defects in kinesin heavy chain mutants have been detected in Drosophila melanogaster (11). However, severe defects in early mitoses were found in C. elegans kinesin heavy chain mutants (12). In contrast to conventional kinesin, many KRPs are known to have mitotic functions, including roles in spindle formation, spindle maintenance, and chromosome movement. These mitotic KRPs include members of the BimC subfamily, the Cterminal subfamily, the MKLP1 subfamily, chromokinesins, and others (reviewed in Refs. 13 and 14).More than 200 kinesins and KRPs have been identified and catalogued.2 Whereas most of the biochemical characterization performed on these motors has been on recombinantly expressed protein or protein fragments, the characterizations of the biochemical properties of kinesins and KRPs in their native forms from natural host cells have been limited. In the case of conventional kinesin, which has been the most extensively studied kinesin in native form, the cell types from which it has been isolated and thoroughly studied are restricted for the most part to brain tissue and early embryonic cells (reviewed in Ref. 4). Furthermore, only two KRPs have been isolated from n...
The dynamic behavior of mammalian microtubules has been extensively studied, both in living cells and with microtubules assembled from purified brain tubulin. To understand the intrinsic dynamic behavior of mammalian nonneural microtubules, we purified tubulin from cultured HeLa cells. We find that HeLa cell microtubules exhibit remarkably slow dynamic instability, spending most of their time in an attenuated state. The tempered dynamics contrast sharply with the dynamics of microtubules prepared from purified bovine brain tubulin under similar conditions. In accord with their minimal dynamic instability, assembled HeLa cell microtubules displayed a slow treadmilling rate and a low guanosine-5-triphosphate hydrolysis rate at steady state. We find that unlike brain tubulin, which consists of a heterogeneous mixture of -tubulin isotypes ( II ,  III , and  IV and a low level of  I ), HeLa cell tubulin consists of  I tubulin (ϳ80%) and a minor amount of  IV tubulin (ϳ20%). The slow dynamic behavior of HeLa cell microtubules in vitro differs strikingly from the dynamic behavior of microtubules in living cultured mammalian cells, supporting the idea that accessory factors create the robust dynamics that occur in cells.
Mitotic-centromere-associated kinesin (MCAK) is a member of the KIN I (internal motor domain) subfamily of kinesin related proteins. MCAK and its homologues destabilize microtubules both in cells and in vitro. Here, we analyzed the effects of MCAK in the presence and absence of ATP on the dynamic instability behavior of steady state microtubules assembled from purified HeLa cell tubulin. In the presence of ATP, substoichiometric levels of full length MCAK and a segment (A182) consisting of the motor and neck domains strongly increased the catastrophe frequency of the microtubules. These data demonstrate that MCAK is a microtubule-catastrophe promoting factor in vitro, and support the hypothesis that MCAK may serve as a catastrophe-promoting factor in cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.