Analysis of biophysical and functional consequences of tropomyosin–fluorescent protein fusions

Abstract

The dynamic nature of actin polymers is modulated to facilitate a diverse range of cellular processes. These dynamic properties are determined by different isoforms of tropomyosin which are recruited to distinct subpopulations of actin polymers to differentially regulate their functional properties. This makes tropomyosin an attractive target for labelling discrete actin populations. We have assessed the effect of different fluorescent labelling strategies for this protein. Although tropomyosin–fluorescent fusions decorate actin in vivo, they are either nonfunctional or perturb regulation of actin nucleation and cell cycle timings. Thus, conclusions and physiological relevance should be carefully evaluated when using tropomyosin fusions.

Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro

“…In the fluorescent fusion proteins, sfGFP/mCherry was linked to the N termini of the tropomyosins because such fusion proteins localize properly to the stress fiber network in cells and can rescue the knockout phenotype ( Figure S1 B) [ 7 ]. Moreover, a recent biochemical study demonstrated that an amino-terminal S. pombe tropomyosin fluorescent fusion binds actin filaments and forms similar end-to-end interactions compared to endogenous acetylated tropomyosin [ 16 ]. The actin filament co-sedimentation assay was first applied to examine the binding of non-tagged and sfGFP-tagged tropomyosins to non-muscle β/γ-actin filaments.…”

Dynamics of Tpm1.8 domains on actin filaments with single-molecule resolution

“…Fluorescence labeling of tropomyosins can impair their function, so that the effects of different labeling strategies depend on the isoform and experimental system. Fusion of a fluorescent protein tag to the N-terminus of Cdc8, the sole tropomyosin in fission yeast, does not perturb its assembly on actin filaments in vitro or localization in the cell ( Brooker et al , 2016 ) but leads to severe functional defects, in particular misregulation of actin nucleation and cell division ( Wu et al, 2003 ; Brooker et al , 2016 ). These defects can be alleviated by using certain cysteine mutants of Cdc8, and Cdc8 labeled at the engineered cysteine can assemble on actin ( Christensen et al, 2017 ).…”

Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks

“…However, fluorescently labeling tropomyosins is generally problematic as mutations or insertions within the protein can potentially disrupt its coiled-coil structure ( Greenfield and Hitchcock-DeGregori, 1995 ). Additionally, tropomyosins labeled on the N- or C-terminus are not fully functional, as the presence of a label blocks end-to-end associations between tropomyosin molecules ( Brooker et al, 2016 ). Therefore, we created three distinct Cdc8 mutants, each containing an engineered cysteine mutation that could be labeled for visualization by TIRFM ( Figure 1—figure supplement 1 , Methods).…”