In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts

Pittenger, M. F., Helfman, D. M. (1992) In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts. J Cell Biol, 118 (4). pp. 841-58. ISSN 0021-9525 (Print)0021-9525 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1500427
DOI: 10.1083/jcb.118.4.841

Abstract

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.

Item Type: Paper
Uncontrolled Keywords: Actin Cytoskeleton/*metabolism Actins/*metabolism Amino Acid Sequence Animals Cell Line Cloning, Molecular Escherichia coli/genetics *Interphase Microscopy, Fluorescence Molecular Sequence Data Tropomyosin/chemistry/genetics/*metabolism
Subjects: Investigative techniques and equipment > cloning
Investigative techniques and equipment > assays > cloning

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

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > myosin
CSHL Authors:
Communities: CSHL labs > Helfman lab
Depositing User: Matt Covey
Date Deposited: 30 Sep 2015 18:59
Last Modified: 30 Sep 2015 18:59
PMCID: PMC2289570
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31804

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