Monoclonal Antibodies to Kinesin Heavy and Light Chains Stain Vesicle-like Structures, but Not Microtubules, in Cultured Cells

• Published in: The Journal of cell biology Vol. 108; no. 4; pp. 1453 - 1463
• Main Authors: Pfister, K. Kevin, Wagner, Mark C., Stenoien, David L., Brady, Scott T., Bloom, George S.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.04.1989; The Rockefeller University Press
• Subjects: Adenosine Triphosphatases - analysis; Animals; Antibodies; Antibodies, Monoclonal - isolation & purification; Biological and medical sciences; Brain - enzymology; Cattle; Cell Line; Cell structures and functions; Cells; Cells, Cultured; Cellular immunity; Cultured cells; Cytoskeleton, cytoplasm. Intracellular movements; Epithelial cells; Fluorescent Antibody Technique; Fluorescent antibody techniques; Fundamental and applied biological sciences. Psychology; Kinesin; Macromolecular Substances; Microtubule Proteins - analysis; Microtubules; Microtubules - enzymology; Microtubules - ultrastructure; Molecular and cellular biology; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - immunology; Neurons; Organelles; Organelles - enzymology; Organelles - ultrastructure; Ungulates; Brain; Motility; Cell organelle; Purification; Bovine; Rodentia; Tissue culture; Monoclonal antibody; Immunological method; Proteins; Vertebrata; Mammalia; Cell line; Mouse; Immunoblotting assay; Cytoskeleton; Artiodactyla; Microtubule; Immunofluorescence; ATP; Ungulata; Nucleic acid; Fluorescence microscopy
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.108.4.1453

Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.