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

Abstract

Tropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

Distinct actin–tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes

“…Whereas, in the absence of N-terminal acetylation, the T diss values of the HMW isoforms Tpm1.6 and Tpm2.1 exceeded those measured for the LMW isoforms Tpm3.1 and Tpm4.2 by 1 -6 C, the opposite was true for the isoforms with N-terminal acetylation. Compared with an increase of T diss by 10 -13 C upon acetylation of Tpm3.1 and Tpm4.2, acetylation of Tpm1.6 and Tpm2.1 leads to a reduction of T diss by 1 -2 C. In line with the kinetic binding model of Tpm molecules to actin (Bareja et al, 2020), the greater thermal stability of A-Tpm can be explained by stronger end-to-end contacts of Tpm molecules. Alternative explanations include stronger coordination of Tpm assembly on actin filaments or greater flexibility of Tpm cables.…”

Celebrating 20 years of live single-actin-filament studies with five golden rules

“…In addition, such direct observations further revealed information that seems inaccessible to other methods: that cofilin-induced severing occurred preferentially at the cluster boundary located toward the pointed end of the filament ( 31 , 44 , 45 ), how fast severing per cofilin cluster occurred ( 31 ), and how the action of cofilin is affected by other proteins ( 22 , 44 , 46 – 51 ) or by the oxidation of actin filaments ( 51 , 52 ). The cooperative formation of tropomyosin clusters was also confirmed on single filaments ( 48 , 49 , 53 ), and single-filament studies further revealed that tropomyosins independently decorate the two filament strands ( 48 , 54 ). Another example of key information provided by live single-filament observation is the assessment of formin processivity at the barbed end of a growing filament ( 32 , 55 , 56 ).…”

Rapid exchange of stably bound protein and DNA cargo on a DNA origami receptor

“…Microfluidic devices and coverslips were prepared as described previously 61 . Images were collected on a custom built TIRF microscope described in Bereja et.…”

“…al. 61 with a power density of ∼1-3 W cm -2 (measured at the objective with the laser beam normal to the surface of the coverslip.…”