Direct visualisation and kinetic analysis of normal and nemaline myopathy actin polymerisation using total internal...

Actin filaments were formed by elongation of pre-formed nuclei (short crosslinked actin-HMM complexes) that were attached to a microscope cover glass. By using TIRF illumination we could see actin filaments at high contrast despite the presence of 150 nM TRITC-phalloidin in the solution. Actin filaments showed rapid bending and translational movements due to Brownian motion but the presence of the methylcellulose polymer network constrained lateral movement away from the surface. Both the length and the number of filaments increased with time. Some filaments did not change length at all and some filaments joined up end-to-end (annealing). We did not see any decrease in filament length or filament breakage. For quantitative analysis of polymerisation time course we measured the contour length of all the filaments in a frame at a series of time points and also tracked the length of individual filaments over time. Elongation rate was the same measured by both methods (0.23 microm/min at 0.1 microM actin) and was up to 10 times faster than previously published measurements. The annealed filament population reached 30% of the total after 40 min. Polymerisation rate increased linearly with actin concentration. K(on) was 2.07 microm min(-1) microM(-1) (equivalent to 34.5 monomers s(-1) microM(-1)) and critical concentration was less than 20 nM. This technique was used to study polymerisation of a mutant actin (D286G) from a transgenic mouse model. D286G actin elongated at a 40% lower rate than non-transgenic actin.