Technical Reference #1916

1916.

Nebulin regulates actin filament lengths by a stabilization mechanism Christopher T. Pappas; Paul A. Kreig; and Carol C. Gregorio, University of Arizona, Journal of Cell Biology, 189(1916), (2010)
Link To Paper

Materials & Methods:
FRAP- A Deltavision RT system with a 100× NA 1.3 objective and a CoolSNAP HQ charge-coupled device camera fitted with a 488-nm argon laser and environmental chamber was used for FRAP experiments. Cells were plated on glass-bottom dishes (MatTek) supplemented with 20 mM Hepes and maintained at 37°C for the duration of the experiment. Three prebleach images were recorded followed by photobleaching for 0.1 s at 100% power. To monitor recovery after photobleaching images were captured at 10- or 20-s intervals for 400 s. Analysis of recovery was completed as described previously with minor modifications (Wang et al. 2005b). In brief images were imported into ImageJ 1.37 software (National Institutes of Health) and mean intensities from bleached nonbleached and background regions were recorded. After background subtraction and correction for photobleaching caused by image acquisition intensities were normalized so that prebleach intensity was set to 1 and postbleach intensity was set to 0. Mobile fractions were determined from nonlinear regression curves that were best fit using a two-exponential association equation (R = M(fast) × (1  exp(k1 × t)) + M(slow) × (1  exp(k2 × t))) with Prism 4 software (GraphPad Software Inc.). R is the relative recovery at time t and the total mobile fraction is the combination of M(fast) and M(slow). For the recovery experiments control cells were treated with nebulin-specific siRNA alone or siRNA plus mCherry. 4–11 cells per treatment were analyzed in each experiment and two to three experiments were averaged. All values are means ± SEM and two-tailed unpaired Student’s t tests were used to determine the significance of differences (Excel). After the FRAP experiments the cells were fixed and stained to confirm knockdown of nebulin.

Microscopic Technique
FRAP

Cell Type(s)
Chick skeletal myocytes