Figure 4: Arp2/3-mediated nucleation generates the free F-actin network but is not necessary for migration.

(a) Localization of the ARPC4 subunit of the arp2/3 complex in live migrating HL60 cells. Orthogonal views of ARPC4 distribution in cells migrating in confined environments. Scale bar=6 μm. (b) Perspective view of the cell in a. The leading edge, nucleus and uropod are indicated by red, white and blue arrows, respectively. (c) Steady-state ARPC4 distribution in the leading edge. (d) Local application of the arp2/3 inhibitor CK666 to the leading edge of a cell expressing ARP3-GFP. CK666 was applied exclusively to the leading edge at t=0 s. Scale bar=5 μm. (e) Ratio of frontal fluorescence intensity to rear fluorescence intensity for cells expressing ARPC4, ARP3 and ARP3 in the presence of CK666, and ARP3 in the presence of CK869. Data are plotted as box-whisker plots in which the top of the box represents the first quartile of the data, the bottom the third quartile and the line denotes the median. Whiskers indicate the maximum and minimum measurements. The number of cells for each measurement is indicated above each box. Asterisks denote significant differences when comparing populations pairwise with a Student’s t-test (P<0.01). (f) Local application of the arp2/3 complex inhibitor CK666 to the leading edge of a cell expressing actin-GFP. CK666 was applied exclusively to the leading edge at t=0 s. Blebs at the leading edge resulting from CK666 treatment are shown by white arrows. Scale bar=10 μm.