Surface Immobilization of Cardiac Thin Filaments

Abstract

Our objective is to directly observe a single troponin (Tn) complex exchange among its conformational substates. Here, we report experimental conditions for immobilizing thin filaments (TF) to glass coverslips. TF were reconstituted from F-actin, tropomyosin (cTm), and cTn, and were sonicated to produce defined lengths. PEG-functionalized, neutravidin-coated coverslips were prepared, and biotinylated gelsolin was sparsely immobilized to neutravidin. TF were captured from solution in a flow chamber, and imaged under oxygen scavenger solution. Labeled TF cTn(TnC-C89∗AF488) (blue), cTm-C190∗AF546 (green), phalloidin∗AF647 (red) were assessed by three-color widefield fluorescence. Colocalization of blue, green, and red confirmed that thin filaments contained Tn, Tm and actin. The number of dye-labeled cTn per TF was quantified by the number of photo bleaching events per thin filament. We used photobleaching to confirm our ability to prepare TF with one or fewer dye-labeled cTn per filament. These results demonstrate our ability to reconstitute fluorescent-labeled thin filaments immobilized in PEG-coated flow chambers.