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

The nonmuscle actin cytoskeleton consists of multiple networks of actin mic rofilaments. 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 GO and G1 phases and whether c o-expressed isoforms could be sorted into different compartments. Protein l evels of Tms 1, 2, 5a, 6, from the alpha Tm-fast and beta -Tm genes increas ed approximately 2-fold during mid-late G1. Tm 3 levels did not change appr eciably 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 facto r of three during this time. Comparison of the staining of the antibodies C G3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from th e Tm 5NM gene, Tm 5NM-1 and -2) and alpha (f)9d (detects specific Tms from the alpha Tm-fast and beta -Tm genes) antibodies revealed 3 spatially disti nct microfilament systems. Tm isoforms detected by alpha (f)9d were dramati cally sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 a nd Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM i soforms, and marked a discrete, punctate, and highly polarised compartment in NM: 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibre s at 8 h into GI. However, Tms detected by the CG3 and alpha (f)9d antibodi es 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 depolymer isation than filaments containing isoforms from the alpha Tm-fast and beta -Tm genes. This suggests that Tm 5NM isoforms may be in different microfila ments to alpha Tm-fast and beta -Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sor ting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demo nstrate 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 e xpression and localisation of Tm isoforms are differentially regulated in G O and G1 phase of the cell cycle. Tms mark multiple microfilament compartme nts with restricted tropomyosin composition. The creation of distinct micro filament 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.