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5 ��m/min. We further investigated whether the actin flow in cell protrusion is related to myosin II activity. The myosin II inhibitor blebbistatin was added to MDA-MB-231 cells for 10?min. With blebbistatin treatment, of all measurements within 10 ��m of protrusion tip, 3 out of 12 measurements showed flow faster than 0.5 ��m/min. In contrast, without blebbistatin treatment, we were able to detect actin flow in 25 out of 37 measurements (binomial test P value?[http://www.selleckchem.com/products/CAL-101.html CAL-101 in vitro] was extracted. Interestingly, from Fig.?4B and Fig.?5B, the stable structures appeared to be close to the cell border, whereas the protrusion core has only weak correlation, most likely due to fast diffusing species (G-actin or actin oligomers). In addition, actin flow was also detected near the cell border. Taken together, the most plausible hypothesis is that in cell protrusions, F-actin is distributed close to the cell membrane, whereas G-actin occupies most of the space within the cell protrusion. The actin diffusion coefficient estimated by image correlation of pixels within an orbit is between 0.02 ��m2/s and 0.8 ��m2/s. This wide range may be partially due to colocalization of G-actin and actin polymers in different proportions. As described previously in the experimental design section, we can reconstruct the protrusion cross section based on the intensity profile and the position of F-actin bundle using the pair-correlation map. As [http://en.wikipedia.org/wiki/SKAP1 SKAP1] shown in Fig.?2C, the position of F-actin is close to the [http://www.selleckchem.com/products/AZD0530.html selleck products] protrusion border. Although it is difficult to discern the position of F-actin in 3D by a raster scan of actin-eGFP expressing cells ( Fig.?7A), we imaged MDA-MB-231 cells transfected with Lifeact-eGFP to confirm the position of F-actin in cell protrusions ( Fig.?7B). Lifeact is a short peptide that stains F-actin structures in eukaryotic cells and has been used to visualize F-actin organization in?vivo ( 22). The affinity of Lifeact to F-actin is 8- to 30-fold higher than to G-actin ( 22). In line with the observation from correlation analysis, when cultured in 3D type I collagen matrix, the expression of Lifeact-eGFP in MDA-MB-231 cells shows high intensity along cell periphery, with low expression at the protrusion core. To verify that the long correlation time from two orbits at the same position reflects the F-actin stability, MDA-MB-231 cells were treated with either 1 ��M jasplakinolide, which stabilizes F-actin ( 23), or 1 ��M latrunculin B, which induces actin disassembling ( 24). As shown in Fig.?7C, compare to the untreated cell (control), the long correlation time disappeared after latrunculin B treatment. On the contrary, with jasplakinolide treatment, F-actin stabilization is reflected by long-time stable correlation as indicated by red arrows. The organization of the actin cytoskeleton has a central role in transmitting force and in sensing the mechanical microenvironment of cells.