Abstract
The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.
Highlights
Data Availability Statement: All relevant data are within the paper
N-terminal (N-Tpm3.1)- and C-terminal (C-Tpm3.1)-tagged Tpm3.1 were transfected into wild type and Tpm3.1/3.2 knockout mouse embryo fibroblasts (MEFs) and the tagged constructs localized predominantly to stress fibers in both cell types (Fig 1A–1D)
The CG3 antibody detects stress fibers in both untransfected and transfected wild type MEFs. These stress fibers co-localize with the tagged proteins, there are regions, at the ends of stress fibers and regions of high tag density that do not co-localize with the antibody staining
Summary
Data Availability Statement: All relevant data are within the paper. We used fluorescent protein-tagged Tpm3.1 and actin and examined Tpm3.1 vs actin recovery in dorsal/ventral stress fibers in mouse embryo fibroblasts (MEFs) and in apical/cortical fibers in rat salivary gland acinar cells. We hypothesised that there are three candidate processes potentially contributing to the recovery: (1) diffusion of unbound tropomyosin molecules in the cytoplasm, (2) exchange of tagged- for untagged-Tpm3.1 on actin filaments which are exposed to the doi:10.1371/journal.pone.0168203.g002
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