Direct visualisation and kinetic analysis of normal and nemaline myopathy actin polymerisation using total internal reflection microscopy
- 104 Downloads
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 μm/min at 0.1 μM 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 μm min−1 μM−1 (equivalent to 34.5 monomers s−1 μM−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.
KeywordsActin Polymerisation TIRF microscopy Nemaline myopathy Mutation
SBM and J-JF were supported by a Grant from the British Heart Foundation. DU is grateful to the The Bionanotechnology Interdisciplinary Research Collaboration (IRC) for funding.
(MPG 11868 kb)
(MPG 1410 kb)
(MPG 314 kb)
(MPG 716 kb)
- Feng J-J, Marston SB (2006) *Properties of actin mutations D286G, K336E and D292V that cause skeletal muscle myopathy. Biophys J 90:126aGoogle Scholar
- Ilkovski B, Nowak KJ, Domazetovska A, Maxwell AL, Clement S, Davies KE, Laing NG, North KN, Cooper ST (2004) Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms. Hum Mol Genet 13(16):1727–1743. doi: 10.1093/hmg/ddh185 PubMedCrossRefGoogle Scholar
- Ishiwata S, Tadashige J, Masui I, Nishizaka T, Kinosita K Jr (2001) Microscopic analysis of polymerization and fragmentation of individual actin fialments. Results Probl Cell Differen 32:79–94Google Scholar
- Nowak KJ, Wattanasirichaigoon D, Goebel HH, Wilce M, Pelin K, Donner K, Jacob RL, Hubner C, Oexle K, Anderson JR, Verity CM, North KN, Iannaccone ST, Muller CR, Nurnberg P, Muntoni F, Sewry C, Hughes I, Sutphen R, Lacson AG, Swoboda KJ, Vigneron J, Wallgren-Pettersson C, Beggs AH, Laing NG (1999) Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy. Nat Genet 23(2):208–212. doi: 10.1038/13837 PubMedCrossRefGoogle Scholar