Localization of actin in Chlamydomonas using antiactin and NBD-phallacidin.

P. A. Detmers, J. M. Carboni, John S. Condeelis

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Abstract

We have localized actin in gametes of Chlamydomonas reinhardi by two approaches: (1) indirect immunofluorescence with an affinity-purified antibody and (2) staining with NBD-phallacidin, a fluorescent reagent that binds only to F-actin [Barak et al, 1980, Proc Natl Acad Sci, 77:980-984]. Staining of either mating type "plus" (mt+) or "minus" (mt-) gametes with antiactin antibody resulted in similar fluorescent images: most of the actin was located peripherally along the lateral and posterior aspects of the cells. There was diffuse staining centrally, but the flagella did not stain. No brightly stained spot was observed near the mt+ mating structure, the site where the fertilization tubule elongates with concomitant polymerization of actin [Detmers et al, 1983, J Cell Biol, 97:522-532]. Gametes stained prior to mating with NBD-phallacidin showed no fluorescence above background, indicating that there were no concentrations of F-actin in these cells. This suggested that the cytoplasmic staining observed with antiactin represented primarily a nonfilamentous form of the protein. In mating gametes staining with NBD-phallacidin was detected only in the fertilization tubule, indicating that this was the only dense accumulation of filamentous actin within the cells. Mating gametes stained with antiactin antibody exhibited cytoplasmic fluorescence that was slightly more punctate than prior to mating, and the fertilization tubule was brightly stained. Our observations suggest that the site-specific polymerization of actin within the fertilization tubule occurs in the absence of a concentrated pool of actin subjacent to the mating structure.

Original languageEnglish (US)
Pages (from-to)415-430
Number of pages16
JournalCell Motility
Volume5
Issue number5
StatePublished - 1985

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Chlamydomonas
Actins
Germ Cells
Fertilization
Staining and Labeling
Polymerization
Fluorescence
Antibody Affinity
Flagella
phallacidin
Antibodies
Indirect Fluorescent Antibody Technique
Coloring Agents

ASJC Scopus subject areas

  • Cell Biology

Cite this

Localization of actin in Chlamydomonas using antiactin and NBD-phallacidin. / Detmers, P. A.; Carboni, J. M.; Condeelis, John S.

In: Cell Motility, Vol. 5, No. 5, 1985, p. 415-430.

Research output: Contribution to journalArticle

Detmers, P. A. ; Carboni, J. M. ; Condeelis, John S. / Localization of actin in Chlamydomonas using antiactin and NBD-phallacidin. In: Cell Motility. 1985 ; Vol. 5, No. 5. pp. 415-430.
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