Feng Juan-Juan, Ushakov Dmitry S, Ferenczi Michael A, Laing Nigel G, Nowak Kristen J, Marston Steven B
NHLI, Cardiovascular Science and Molecular Medicine, Imperial College London, London, UK.
J Muscle Res Cell Motil. 2009;30(1-2):85-92. doi: 10.1007/s10974-009-9178-9. Epub 2009 May 6.
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.
肌动蛋白丝由附着在显微镜盖玻片上的预先形成的核（短的交联肌动蛋白 - 重酶解肌球蛋白复合物）伸长形成。通过使用全内反射荧光（TIRF）照明，尽管溶液中存在150 nM的TRITC - 鬼笔环肽，我们仍能以高对比度看到肌动蛋白丝。由于布朗运动，肌动蛋白丝表现出快速弯曲和平移运动，但甲基纤维素聚合物网络的存在限制了其远离表面的横向运动。丝的长度和数量都随时间增加。一些丝的长度根本没有变化，一些丝端对端连接在一起（退火）。我们没有看到丝长度的任何减少或丝的断裂。为了对聚合时间进程进行定量分析，我们在一系列时间点测量了一帧中所有丝的轮廓长度，并随着时间跟踪单个丝的长度。两种方法测得的伸长率相同（在0.1 microM肌动蛋白时为0.23微米/分钟），比之前发表的测量结果快达10倍。40分钟后，退火丝群体达到总数的30%。聚合速率随肌动蛋白浓度线性增加。K（on）为2.07微米·分钟⁻¹· microM⁻¹（相当于34.5个单体·秒⁻¹· microM⁻¹），临界浓度小于20 nM。该技术用于研究来自转基因小鼠模型的突变肌动蛋白（D286G）的聚合。D286G肌动蛋白的伸长速率比非转基因肌动蛋白低40%。
