MANS inhibition of chemotaxis analyzed by live-cell TIRF microscopy.

<p>(A) Time-course montage of a GFP-AktPH-expressing NIH 3T3 mouse fibroblast migrating chemotactically in response to a PDGF-BB gradient emanating from an alginate microsphere (open circle). The cell was treated with RNS peptide and monitored by TIRF microscopy, with localization of PI3K signaling displayed using a pseudo-color intensity scale; scale bar = 100 µm. (B) Montage as in part A, except that incubation was in the presence of MANS peptide, which inhibited productive cell movement. Multiple microspheres in this field are indicated by filled circles; scale bar = 100 µm. (C) Chemotactic fidelity was quantified as the fraction of time intervals during which cell movement was aligned with the PDGF gradient (angle within 60°) minus the fraction of time intervals during which movement was misaligned (angle between 120–180°). The histogram compares cells incubated with RNS (<i>n</i> = 32) versus MANS (<i>n</i> = 27) at peptide concentrations of 10 µM (gray, yellow) and 50 µM (black, red). (D&E) For each of the 4 treatment conditions compared in part C, chemotactic fidelity values were sorted into high, medium, and low subpopulations for RNS (D) and MANS (E). Cell centroid translocation paths are plotted with the initial centroid positions located at the origin and the initial PDGF-BB gradient vector aligned along the positive <i>x</i>-axis.