In addition, Y-27632 restored lamellipodial ruffling at the leading edge (Figure 6D, indicated with white arrowhead, and Movie S9).
Supporting our previous results demonstrating SLK-tubulin co-precipitation [13], SLK co-localized with the microtubule network at the leading edge (figure 1G I).
In MOSE-E cells, PKCβII (Figure 5B, red) appeared as distinct punctae throughout the cytoplasm, co-localizing with actin stress fibers and actin at the leading edge (Figure 5B, merge).
Interestingly, line scan analysis of the distribution of CXCR2 and endogenous LASP-1 mirror each other in the area marked by F-actin at the leading edge (Figure S3, B).
The sudden increase of the mean filament length in the frontal zone can be understood by analyzing the filament length histograms at various distances from the leading edge (Figure 4C).
After the induction of cell polarization with 1 µM fMLP on immobilized fibrinogen, the majority of the control cells translocated PI3K p110δ (86.3%) and PI3K p110γ (84.6%) to the leading edge (Figure 5A, B and C).
Active Rac localized to the membrane at the leading edge (Figure 3C), as well as in the uropod, where it was predominantly in vesicular structures around the MTOC and not on the plasma membrane.
The spatio-temporal control of PI3K p110δ activity by Syk may be involved in the amplification of migratory signals and may be critical for the localization of this process at the leading edge (Figure 6).
The experiments with rapamycin-inducible linkers revealed an increased density of ER PM junctions near the leading edge (n=27, Figure 2B and Supplementary Figure S5 at http://www.biochemj.org/bj/451/bj4510025add.htm) confirming the conclusion from the experiments with YFP STIM1 regarding the preferential clustering of ER PM junctions in this region.
Thus, the mean reflection point calculated with the leading edge filter (Figure 5A: meanLE level) is located on the leading slope of the data distribution.
We obtained an estimate of D from the error surfaces associated with the leading edge data Figure 4G.
