Kinesin is a mechanochemical ATPase that induces translocation of latex beads along microtubules and microtubule gliding on a glass surface. This protein is thought to be a motor for the movement of membranous organelles in cells. Recently Hollenbeck and Swanson [Hollenbeck, P. J. & Swanson, J. A. (1990) Nature (London) 346, [864][865][866] showed that kinesin is involved in the positioning of tubular Iysosomes in macrophages. However, the role of this protein in the movement of organelles was not yet clear. We used a polyclonal antibody against the kinesin heavy chain that inhibited kinesindependent microtubule gliding in vitro to study the role of kinesin in the movement of pigment granules in melanophores of the teleost black tetra (Gymnocorymbus ternetzi). Microinjection of the antibody into cultured melanophores did not produce any specific effect on the aggregation o pigment granules in melanophores, but it did result in a strong dosedependent inhibition of the dispersion. Immunoblotting of melanophore extracts showed that the kinesin antibody reacted in these cells with a single protein component with a molecular mass of 135 kDa. Thus, kinesin is responsible for the movement of pigment granules from the center to the periphery of the melanophore.

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1-3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

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Abstract. One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before, this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.