The impact of tropomyosins on actin filament assembly is isoform specific

Abstract

Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro

“…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. Consistent with earlier work [17], the stress-fiber-associated tropomyosins bound β/γ-actin filaments with high affinity (K d  < 1 μM) and the N-terminal sfGFP fusion did not interfere with F-actin binding (Figure 1C; Figure S1C). This was also supported by in vitro total internal reflection fluorescence (TIRF) microscopy experiments demonstrating that the fluorescent fusions of tropomyosins decorated non-labeled β/γ-actin filaments.…”

Cooperative effects of tropomyosin on the dynamics of the actin filament

“…However, the specific properties of Tpm isoforms essential for the distinct cellular functions are poorly understood. It is only known that co‐sedimentation and pyrene‐actin polymerisation assays reveal a moderate difference in binding of Tpm isoforms to F‐actin and in the effects of Tpm isoforms on actin polymerisation‐depolymerisation . We therefore believe that the enhanced ATPase activity of ECP‐actin may provide a useful tool for comparison of different tropomyosins with regard to their effects on actin properties.…”

Effect of surface chemistry on tropomyosin binding to actin filaments on surfaces

“…The implication of this observation is that building actin-tropomyosin polymers with higher stability may require coordination of the assembly process to avoid breaks in the tropomyosin strand, e.g., by nucleation at defined sites [Hsiao et al, 2015] or catalysis of actin-tropomyosin coassembly by actin nucleators [Johnson et al, 2014]. On the flipside, tropomyosin binding to actin filaments in the cytoskeleton may indeed be more dynamic than previously appreciated as shown by the fast turnover of tropomyosin in stress fibres [Bonello et al, 2016;Tojkander et al, 2011]. Thus, different assembly processes may coexist in the cell for actin-tropomyosin polymer formation.…”

“…Expression, Purification, and Labeling of Tpm1.1 Expression and purification of Tpm1.1 was carried out as described previously [Janco et al, 2016]. The Cys-190 residues of the Tpm1.1 dimer are prone to form a disulfide.…”