Microinjection of fluorescently labeled alpha-actinin into living fibroblasts

Feramisco, J. R. (1979) Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proceedings of the National Academy of Sciences of the United States of America, 76 (8). pp. 3967-3971. ISSN 0027-8424

URL: http://www.ncbi.nlm.nih.gov/pubmed/291056

Abstract

alpha-Actinin from chicken gizzard labeled with tetramethylrhodamine isothiocyanate has been incorporated into living fibroblast cells by microinjection. Fluorescent labeling of alpha-actinin was carried out such that the conjugated protein was functional in vitro as shown by its ability to bind to F-actin. Within 1-2 hr after injection, diffuse fluorescence was observed throughout the cytoplasm and only faint fluorescence was apparently associated with the stress fibers. During the ensuing 2-15 hr, however, most of the fluorescence was seen as periodicities along the stress fibers and as foci of the microfilament polygonal networks. This distribution of alpha-actinin in the living cells was strikingly similar to that found by indirect immunofluorescence localization of endogenous alpha-actinin in fixed samples of the same cell type. Control studies in which heat-treated (100 degrees C, 2 min) fluorescent alpha-actinin or tetramethylrhodamine isothiocyanate alone was injected into the cells indicated that the stress fiber and polygonal network labeling was specific for "native" fluorescently labeled alpha-actinin. These results suggest that the dynamic properties of proteins and structures in cultured mammalian cells can be studied with the use of microinjection and fluorescence microscopic techniques.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > cytoskeletal proteins
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > fibroblasts
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > fibroblasts
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > fibroblasts
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: 1979
Date Deposited: 02 Jun 2016 18:54
Last Modified: 02 Jun 2016 18:54
PMCID: PMC383957
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32676

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