Localized actomyosin bundles that are generated by MIIA and stabilized by MIIB define the cellular region that forms the rear. (A) CHO.K1 cells were plated on fibronectin and allowed to attach for 10 min. The cells were then fixed and stained for phosphorylated RLC (pRLC), MIIA (using a MHCII-A antibody), MIIB (MHCII-B antibody), or F-actin (rhodamine-phalloidin). Images were captured using a confocal microscope (FV300; Olympus). Z projections are shown. Representative examples are shown of cells exhibiting an accumulation of these proteins (“proto-bundles,” marked with arrows). The asterisks mark the leading edge in the Z projection and corresponding 3D reconstruction. (B) CHO.K1 cells were plated on PLL and allowed to attach for 45 min. The cells were stained as in A, and they display similar proto-bundles (marked with arrows). Representative examples are shown. (C and D) Cells were transfected with RLC-D,D–GFP and either control (C) or MHCIIB-shRNA (D) to inhibit MIIB expression. Representative time points of their spreading on fibronectin are shown. In C, arrows point to the region of the cell initially primed by RLC-D,D to form the rear, where protrusion does not occur. The complete movie is shown in Video 1. In D, arrows point to clusters, or proto-bundles, denoted by the accumulation of MIIA (the only isoform present in the knockdowns, labeled with the RLC-D,D–GFP) that form and disassemble rapidly. Images were captured in TIRF mode using an inverted microscope (IX70; Olympus) coupled to a CCD camera (Retiga Exi; Qimaging). The complete movie is shown in Video 2. (E) CHO.K1 cells knocked down for MIIA or MIIB were transfected with wild-type RLC (control, black bars) or RLC-D,D (gray bars) coupled to GFP, adhered for 10 min to fibronectin, fixed, and quantified for presence of proto-bundles as for those shown in A. Data are the mean ± SD of three experiments (error bars) with >200 cells scored in each experiment. Bars, 10 µm.