Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: Evidence for distinct microfilament populations

Justin M. Percival, Gethin Thomas, Terri Anne Cock, Edith M. Gardiner, Peter L. Jeffrey, Jim J.C. Lin, Ron P. Weinberger, Peter Gunning

Research output: Contribution to journalArticle

58 Scopus citations

Abstract

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the α Tm(fast) and β-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and α(f)9d (detects specific Tms from the α Tm(fast) and β-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by α(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and α(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the α Tm(fast) and β-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to α Tm(fast) and β-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo. (C) 2000 Wiley-Liss, Inc.

Original languageEnglish (US)
Pages (from-to)189-208
Number of pages20
JournalCell Motility and the Cytoskeleton
Volume47
Issue number3
DOIs
StatePublished - Nov 15 2000

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Keywords

  • Actin compartments
  • Cell cycle
  • Isoform sorting
  • Microfilaments
  • Tropomyosin

ASJC Scopus subject areas

  • Cell Biology

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