The Distribution, Abundance ana Subcellular Localization of Kinesin

An antiserum which binds kinesin specifically on Western blots was used to determine the distribution and abundance of chicken kinesin by correlated immunoblotting and immunolocalization. Quantitative immunoblotting showed that the abundance of kinesin varied widely in different cell and tissue types, from 0.039% of total protein in epidermal fibroblasts to 0.309% in sympathetic neurons; of the types examined, only red blood cells lacked detectable kinesin. The molar ratio of tubulin/kinesin varied over a narrower range. To analyze the intracellular distribution of kinesin, cultured fibroblasts were fractionated by sequential extraction with saponin-, Triton X-100-, and SDS-containing buffer. Quantitative blotting of the resulting cell fractions indicated that 68 % of fibroblast kinesin is in soluble form, 32% is membraneor organelle-associated, and none is detectable in cytoskeletal fractions. To visualize this distribution, cells treated by the same extraction protocol were immunofluorescently stained with antikinesin and antitubulin. Without extraction, kinesin staining was located throughout cultured neurons and fibroblasts. However, when fibroblasts were extracted with saponin or Brij 58 before fixation, subsequent staining revealed that the remaining kinesin fraction was colocalized with interphase microtubules, but not with mitotic spindles. Prefixation extraction with Triton abolished antikinesin staining. These data suggest that kinesin may play a role in tubovesicular movement but provide no evidence for a role in mitosis. INESlN is a mechanochemical enzyme which was identified and purified from nervous tissue based upon its microtubule-binding properties (Lasek and Brady, 1985; Vale et al., 1985a; Brady, 1985) and its ability to generate microtubule-based motility in vitro (Vale et al., 1985b). It is a microtubule-activated ATPase (Kuznetsov and Gelfand, 1986; Cohn et al., 1986; Penningroth et al., 1987; Saxton et al., 1988; Bloom et al., 1988) of high molecular mass (Vale et al., 1985b; Bloom et al., 1988), comprising a l10-135-kD polypeptide species that is catalytic (Penningroth et al., 1987; Bloom et al., 1988), along with a 60-65kD species; the native kinesin molecule probably contains two molecules of each species (Bloom et al., 1988). Kinesin has been prepared from nervous tissue of squid (Vale et al., 1985b), chicken (Brady, 1985), cow (Vale et al., 1985; Kuznetsov and Gelfand, 1986), and pig (Amos, 1987), as well as from sea urchin eggs (Scholey et al., 1985) and Drosophila embryos (Saxton et al., 1988), and there is some phylogenetic variation in its reported physical properties (reviewed by Hollenbeck, 1988). Kinesin binds to artificial surfaces such as glass or latex beads in vitro and generates ATP-dependent microtubule (MT) ~ translocation relative to them (Vale et al., 1985b). This in vitro motility has certain kinetic and physiological properties in common with rapid axonal transport; however, kinesin-based movement is unidirectional, such that kinesincoated beads translocate exclusively toward the plus ends of the MTs, and MTs always glide with their minus ends forward (Vale et al., 1985c; Porter et al., 1987; Saxton et al., 1988). Although no direct evidence exists for the function of kinesin in vivo, three roles in cell motility have been suggested. First, the studies summarized above have led to the proposal that kinesin serves as a motor for anterograde organelle movement in axons (Vale et al., 1985c; Vale et al., 1986; Sheetz, 1987), where essentially all MTs are arranged with their plus ends distal to the cell body (Burton and Paige, 1981; Heidemann et al., 1981). Second, based upon the isolation of kinesin from sea urchin eggs and immunostaining of the sea urchin mitotic spindle with a kinesin antibody, a role for kinesin in anaphase chromosome movement has been suggested (Scholey et al., 1985; Vale et al., 1986). However, more recent work indicates that extraction of the MT component of the sea urchin egg spindle does not eliminate anPeter Hollenbeck's present address is Department of Anatomy and Cellular Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. 1. Abbreviations used in this paper: HRP, horseradish peroxidase; MT, microtubule; PBSTx, PBS plus 0.05% Triton X-100; PMED, 0.1 M Pipes, 5 mM MgSO4, 1 mM EGTA, 2 mM DTT, pH 6.9. © The Rockefeller University Press, 0021-9525/89/06/2335/8 $2.00 The Journal of Cell Biology, Volume 108, June 1989 2335-2342 2335 on Jne 1, 2017 D ow nladed fom Published June 1, 1989