Kinesin is associated with a nonmicrotubule component of sea urchin mitotic spindles.

Abstract

Sea urchin embryos in second division have been lysed into microtubule-stabilizing buffers to yield mitotic cytoskeletons (MCSs) that consist of two mitotic spindles surrounded by a cortical array of filaments. Microtubules have been completely extracted from MCSs by incubation at 0C with Ca2l-containing buffer. An antibody to the microtubule translocator kinesin stains the spindles in MCSs and in MCSs treated with 5 mM ATP and also stains spindle-remnants of the MCSs after the microtubules have been extracted. We conclude that kinesin binds to a nonmicrotubule component in the mitotic spindle. Based on these results, we present several models of kinesin function in the spindle.Kinesin, a mechanochemical protein that mediates ATPdependent motility of microtubules (MTs) in vitro (1, 2) is a good candidate for the motor active in vesicle movement during fast axonal transport (3, 4, 32). Scholey et al. (5) have reported that kinesin localizes in mitotic spindles of sea urchin embryos as determined by staining with antibodies to sea urchin kinesin. This result raises the possibility that kinesin might be involved in some aspect of chromosome movement.Mitosis includes a complex series of movements, any one of which might be mediated by kinesin. For example, the movement of chromosomes away from the spindle poles during prometaphase (6) could result from kinesin bound to the kinetochore working on MT surfaces by a mechanism analogous to that used in bead transport along MTs in vitro (ref.Vale, J.R.M., and J.M.S., unpublished data). Kinesin might act in the overlap between half-spindles to generate shearing forces between adjacent MTs (7,8). Alternatively, kinesin may not act to move chromosomes at all but may move vesicles within the spindle (9-14).To better understand the role of kinesin in mitosis, we need to identify the spindle component(s) with which the protein interacts to generate motile force. For example, kinesin might cross-link MTs to vesicles or kinetochores to generate force for vesicle or chromosome translocation along MTs (6,15). It might cross-link MTs to MTs to generate force for MT-sliding in anaphase B, or it might cross-link MTs to a structural matrix to generate shear forces between MTs and the matrix.We therefore used antibodies to kinesin (5) to probe the location of this protein within spindles of sea urchin embryos. We found that spindles that have been depleted of MTs by extraction with Ca2+-containing buffer at 0°C contain no residual tubulin as determined by immunoblotting or immunocytochemistry. However, when these MT-depleted spindle remnants and the Ca2+ buffer extracts were examined with anti-kinesin antibodies by immunoblotting and immunocytochemistry, we found that kinesin localized to a non-MT component in the spindle and was not coextracted with tubulin. The possible significance of this observation for the role of kinesin in mitosis is discussed. MATERIALS AND METHODS Animals. Sea urchins Strongylocentrotusfranciscanus andLytechinus pictus were collected from the Santa Bar...

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

“…Our aim was to obtain antibodies that might serve as probes for the function of kinesin in blastomeres of early sea urchin embryos, where kinesin may participate in various aspects of cell motility, cell division, and intracellular transport (Scholey et al, 1985Leslie et al, 1987;Porter et al, 1987;Cohn et al, 1987). To this end, the three monoclonal antibodies, SUK 4, 6, and 7 that disrupt kinesin activity in vitro might prove useful for disrupting kinesin function in vivo after their microinjection into living cells (Mabuchi and Okuno, 1977;Kiehart et al, 1982).…”

“…In addition, polyclonal and monoclonal myosin antibodies have been used to inhibit the actin activated ATPase activity of myosin, (Mabuchi and Okuno, 1977;Kiehart and Pollard, 1984b;Peltz et al, 1985) to block myosin-driven motility in vitro (Kiehart and Pollard, 1984b;Flicker et al, 1985) to examine the relationship between myosin polymerization and ATPase activity as well as to probe the conformation and polymerization state of myosin (Citi and KendrickJones, 1987). Inhibitor myosin antibodies have provided evidence for the role of myosin in actin-based organelle transport (Adams and Pollard, 1986) and in cytokinesis (Mabuchi and Okuno, 1977;Kiehart et al, 1982).Kinesin antibodies have been used to establish immunological relatedness between kinesin from various cell types, as well as to identify, purify, and localize their corresponding antigens (Scholey et al, 1985;Vale et al, 1985b;Porter et al, 1987;Leslie et al, 1987;Saxton et al, 1988;Kuznetsov et al, 1988;Neighbors et al, 1988). However, none of these antikinesins was reported to inhibit kinesin activity.…”

“…Kinesin antibodies have been used to establish immunological relatedness between kinesin from various cell types, as well as to identify, purify, and localize their corresponding antigens (Scholey et al, 1985;Vale et al, 1985b;Porter et al, 1987;Leslie et al, 1987;Saxton et al, 1988;Kuznetsov et al, 1988;Neighbors et al, 1988). However, none of these antikinesins was reported to inhibit kinesin activity.…”

“…Bovine brain kinesin is an tt2132 heterotetramer consisting of two 120-kD heavy chains plus two 60-kD light chains, assembled into an elongated molecule (Amos, 1987;Kuznetsov et al, 1988;Bloom et al, 1988). Kinesin is proposed to participate in organelle/vesicle transport, in organizing the endomembrane system, and in mitosis (Vale et al, 1986;Vale, 1987;Leslie et al, 1987;Schroer and Sheetz, 1988), but direct evidence concerning the biological functions of kinesin is currently lacking.Antibodies that inhibit mechanochemical activity have been extremely useful in probing the functions of myosin and 1. Abbreviations used in this paper: MT(s), microtubule(s); PMEG, 0.9 M glycerol, 0.1 M Pipes, pH 6.9, 5 mM EGTA, 2.5 mM MgSO4, 0.5 mM EDTA, 1 mM DTT, 100 Ixg/ml soybean trypsin inhibitor, 1 mg/mlp-tosyl-Larginine methyl ester hydrochloride, 10 Ixg/ml leupeptin, pepstatin, and aprotinin; RT, room temperature.…”

“…Bovine brain kinesin is an tt2132 heterotetramer consisting of two 120-kD heavy chains plus two 60-kD light chains, assembled into an elongated molecule (Amos, 1987;Kuznetsov et al, 1988;Bloom et al, 1988). Kinesin is proposed to participate in organelle/vesicle transport, in organizing the endomembrane system, and in mitosis (Vale et al, 1986;Vale, 1987;Leslie et al, 1987;Schroer and Sheetz, 1988), but direct evidence concerning the biological functions of kinesin is currently lacking.…”