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.

Actin filament oxidation by MICAL1 suppresses protections from cofilin‐induced disassembly

“…We thus measured the disassembly rates of neonGreen‐Tpm1.8 domains in the presence of G‐actin only (to prevent filament depolymerization). As was reported previously, Tpm1.8 disassembly is asymmetric, faster at the domain boundary located toward the filament barbed end (Bareja et al , 2020). We found that Tpm domains disassembled equally fast from non‐oxidized and oxidized filaments (Fig 3C and D).…”

“…We recently characterized the dynamics of Tpm1.8 domains. We found that Tpm1.8 saturates the two strands of the actin filaments independently and that the domain assembly and disassembly is asymmetric: faster at the domain border closer to the filament barbed end (Bareja et al , 2020).…”

“…neonGreen‐Tpm1.8 was constructed as a protein with N‐terminal His6‐tag followed by mNeonGreen and a peptide linker (GGGSGGGSGTAS) fused to the N‐terminus of Tpm1.8, as described in Bareja et al (2020). The alanine–serine fused directly at the N‐terminus of Tpm1.8 mimics its acetylation.…”

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. Single particle photobleaching was first performed using purified, pre-assembled DNA origami template and DNA cargo in DNA imaging buffer immobilized on a coverslip to a density of ∼1000 particles per field of view (FOV).…”