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

Abstract

Abstract, We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo ll6-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesininduced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP-sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility.V ARIOUS forms of intracellular transport are thought to depend upon microtubule-associated mechanochemical ATPases (Vale, 1987). One such enzyme, kinesin (Vale et al., 1985a), binds microtubules in a nucleotide sensitive fashion, and displays a microtubule-activated ATPase activity that drives movement of kinesin-coated objects towards the plus end of microtubules (MTs) ~ in vitro (Vale et al., 1985a, b;Scholey et al., 1985;Brady, 1985; Kuznetsoy and Gelfand, 1986;Porter et al., 1987;Cohn et al., 1987;Saxton et al., 1988;Gelles et al., 1988). Kinesin from a variety of animals contains a major polypeptide with an apparent molecular mass of 110-140 kD, encoded by a single gene in Drosophila (Yang et al., 1988), plus copurifying light chains of Mr = 40-80 kD (Vale et al., 1985a,b;Amos, 1987;Kuznetsov and Gelfand, 1986;Saxton et al., 1988). 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. dynein. For example, an antiserum that inhibits the ATPase activity of sea urchin sperm dynein caused a corresponding decrease in the beat frequency of reactivated sea urchin sperm, supporting the hypothesis that the dynein ATPase drives axonemal motility (Ogawa and Mohri, 1975;O...

Two Kinesin Light Chain Genes in Mice

“…8A), suggesting that a large proportion of these proteins are soluble. This pattern corresponded to that seen with the monoclonal antibody, Suk4 (42), that recognizes both uKHC and nKHC. However, when probed with an antibody against synaptophysin (Boehringer Mannheim), the pattern was different.…”

“…Western blotting was done as described above. Anti-synaptophysin (Boehringer Mannheim) and Suk4 (42) antibodies were used at a 1:10 dilution of the manufacturer's stock solution and 1:10 dilution of hybridoma supernatant, respectively.…”

Bidirectional Translocation of Neurofilaments along Microtubules Mediated in Part by Dynein/Dynactin

“…An antibody to the heavy chain of conventional kinesin, SUK4 (Ingold et al, 1988), showed no reactivity in the NF preparation by Western blotting ( Figure 6A) or immuno-EM ( Figure 5G), although reactivity is observed with intermediate fractions resulting from the NF preparation ( Figure 6A). The absence of conventional kinesin, the most prevalent form of kinesin, in the purified NF fraction precludes the possibility of contamination by motor-bound organelles and nonspecific interactions between NFs and soluble kinesins.…”

“…With a broadspecificity kinesin antibody, anti-HIPYR (Sawin et al, 1992), a set of candidate proteins have been identified each of which may or may not contribute to the motility observed. Of the NF-associated kinesins identified by the HIPYR antibody, the 110-kDa polypeptide detected by the anti-HIPYR antibody does not correspond to the conventional kinesin isoform (kinesin-I) recognized by the SUK4 antibody (Ingold et al, 1988) (Figure 5A). The 110-kDa kinesin-like protein may, however, represent one of the other KIF5/kinesin-I isoforms (Nakagawa et al, 1997) of which two, KIF5A and KIF5C, are restricted to neural tissue.…”

Kinesin-1 Regulates Microtubule Dynamics via a c-Jun N-terminal Kinase-dependent Mechanism

“…In the present study, RNAi was used alone or combined with the expression of a dominant-negative construct of the mouse kinesin-1 light chain 2 tetratricopeptide domain (KLC-TPR) to compete out the cargo-binding domain of the endogenous motor (16). Alternatively, microinjection of the Suk-4 antibody was used to block acutely kinesin movement on microtubules (23). To measure polymerization rates, we followed fluorescent EB1 or CLIP-170 in time-lapse experiments.…”