Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility

Abstract

Two tropomyosin isoforms, human Tm5 NM1 and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5 NM1 cells, we observed large stress fibres which showed predominant co-localisation of β-actin and low-molecular-weight γ-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the β- and γ-actin isoforms, predominately populated by high-molecular-weight α- and β-tropomyosin isoforms. The stress fibres observed in Tm5 NM1 cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5 NM1 isoform with a human-specific Tm5 NM1 siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.