Tropomyosin Isoforms Segregate into Distinct Clusters on Single Actin Filaments

Area of Science:

• Cell Biology
• Biochemistry
• Biophysics

Background:

• Tropomyosins (Tpms) polymerize on actin filaments, regulating muscle and non-muscle cellular functions.
• The spatio-temporal sorting mechanisms of different Tpm isoforms on actin remain poorly understood.
• Tpm isoforms differ in their C-terminal 27 amino acids, potentially influencing their interactions.

Purpose of the Study:

• To investigate the early assembly intermediates of actin-tropomyosin filaments.
• To determine if Tpm isoform recruitment to actin filaments is influenced by actin isoform (muscle vs. cytoskeletal).
• To test for isoform specificity in Tpm incorporation during early nucleation stages.

Main Methods:

• Utilized fluorescently labeled Tpm1.1 (skeletal/cardiac) and Tpm1.6 (cytoskeletal) isoforms.
• Exposed actin filaments to low Tpm concentrations in solution.
• Visualized Tpm-actin assembly patterns using Total Internal Reflection Fluorescence (TIRF) microscopy and developed automated image analysis.

Main Results:

• Tropomyosin isoforms formed distinct "clusters" on actin filaments during early assembly.
• Tpm isoform sorting was not driven by preference for specific actin isoforms.
• A higher probability of incorporating the same Tpm isoform into nascent clusters was observed, indicating self-recognition.
• The terminal 27 amino acids of Tpm isoforms facilitate homotypic interactions, promoting homogeneous cluster formation.

Conclusions:

• Actin-Tpm filament assembly involves spatio-temporal sorting of Tpm isoforms.
• Homogeneous Tpm isoform clusters form due to self-recognition mediated by terminal amino acid sequences.
• This mechanism ensures Tpm isoform specificity in regulating actin filament function.