Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells

K. K. Pfister, Mark Wagner, D. L. Stenoien, S. T. Brady, G. S. Bloom

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)1453-1463
Number of pages11
JournalJournal of Cell Biology
Volume108
Issue number4
StatePublished - 1989
Externally publishedYes

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Kinesin
Microtubules
Cultured Cells
Coloring Agents
Organelles
Monoclonal Antibodies
Light
Antibodies
Cytoplasmic Structures
Centrosome
Spindle Apparatus
Membranes
Membrane Transport Proteins
Cilia
Interphase
Octoxynol
Hybridomas
Tubulin
Fluorescence Microscopy
Adenosine Triphosphatases

ASJC Scopus subject areas

  • Cell Biology

Cite this

Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. / Pfister, K. K.; Wagner, Mark; Stenoien, D. L.; Brady, S. T.; Bloom, G. S.

In: Journal of Cell Biology, Vol. 108, No. 4, 1989, p. 1453-1463.

Research output: Contribution to journalArticle

Pfister, K. K. ; Wagner, Mark ; Stenoien, D. L. ; Brady, S. T. ; Bloom, G. S. / Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. In: Journal of Cell Biology. 1989 ; Vol. 108, No. 4. pp. 1453-1463.
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AU - Brady, S. T.

AU - Bloom, G. S.

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