Integrin-Ligand Interactions Control Migration Figure 4. STICS measurements of the retrograde fluxing of paxillin and integrin in CHO.B2 cells plated on FN, LN and ICAM-1 with or without Mn2+. Cells were doubly transfected with the appropriate integrin (left) and paxillin-mCherry (right). (A) a5- expressing cells seldom show slow retrograde flux of paxillin or integrin in adhesions in protruding regions. (B) a6b1 fluxes retrograde in protrusions and is inhibited from 1.6860.09 to 1.060.1 mm/min (P = 0.0051) by addition of Mn2+. Paxillin fluxes are inhibited from 1.6460.09 to 1.060.2 mm/min (P = 0.0051) (C) aLb2 fluxes retrograde fluxing in protrusions is inhibited by almost a factor of 3 (from 2.860.3 to 1.060.1 mm/min, P = 0.0016) and that for paxillin are inhibited from 4.160.4 to 1.760.1 mm/min (P = 0.0016). Scale Bar = 5 mm. Protein velocity is represented using the rainbow color scale bar. (D). Average fluxing velocity for each condition is presented as the mean 6 SEM. aL fluxes faster than a6, along with paxillin (P = 0.0025). The results are from analyses of 29 cells. doi:10.1371/journal.pone.0040202.g004 Figure 5. The efficiency of the ECM-actin linkage differs between a5b1 and a6b1 or aLb2. CHO.B2 cells were doubly transfected with paxillin-Cherry and the indicated integrin (with GFP), then either plated on different concentration of substrates or cross-linked or poly-lysine treated substrates or treated with Mn2+. (A) Fluxing is not affected by covalent cross- linking (x-link) or poly-L-lysine attachment (pLys). CHO-B2 cells doubly transfected with paxillin-Cherry and the indicated integrin (with GFP), then either plated on control, cross-linked or poly-L-lysine attached PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40202