Figure 8.

Long-term down-regulation of actomyosin-II activity disrupts the periodic actin rings and causes the formation of FAS. (a) Knockdown efficiency of predesigned siRNA constructs. PC12 cells transfected with two different siRNAs targeting MHC of NM-IIB (siNMII-1# and siNMII-2#). Cell lysates were used to detect the endogenous MHC level of NM-IIB by Western blot. (b) Representative images of axons in siNMII transfected neurons; arrowheads indicate the FAS. Scale bar = 5 µm. (c and d) Quantification of the NM-II fluorescence intensity (c) and FAS number (d) of siNM-II 1# and 2# transfected axons shown in b. (e) Representative images showing that along the siNM-II transfected axons, periodic actin structures were disrupted. The unevenly distributed F-actin–accumulation and F-actin–absent black patches are marked with arrowheads and brackets, respectively. Scale bar = 1 µm. (f) Quantification of the F-actin accumulation along the siNM-II 1# and 2# axons. (g) Rat hippocampal neurons were cotransfected on DIV12 with the Lifeact-mRFP and either MRLC (WT)-GFP, S19AT18A (MRLC (AA)-GFP), or S19DT18D (MRLC (DD)-GFP) plasmids. (h) SiR-actin labeling shows disrupted periodicity of actin rings in MRLC(AA)-GFP, but not (WT), (DD)-GFP transfected axons, respectively. Scale bars = 1 µm. (i and j) The actin ring diameter fluctuations (i) and the FAS numbers per μm (j) of transfected axons were quantified. Data represent mean ± SEM; n = 12, 15, and 13 for c; n = 11, 13, and 11 for d; n = 10, 11, and 10 for f; n = 28, 45, and 32 for i; and n = 9, 26, and 13 for j (n representing the number of axons analyzed). Data were from two independent preparations (*, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed unpaired t test). MW, molecular weight; n.s., not significant.