FIG 2.

Gα_i proteins colocalize with F-actin at cell protrusions and at phagocytic cups. (A) Time-lapse confocal microscopy of RAW 264.7 cells expressing YFP-Gα_i2 (green) and RFP-Lifeact (red) and exposed to zymosan focusing on a phagocytic cup. Numbers represent elapsed time (in minutes). For each time point, a bright-field overlay image and two fluorescent images electronically zoomed 2× are shown. Bar, 5 μm. (B) Time-lapse confocal microscopy images of RAW 264.7 cells expressing YFP-Gα_i2 (green) and RFP-Lifeact (red) and exposed to zymosan. Arrowheads indicate Gα_i together with actin condensed at membrane protrusions and ruffles targeting and engulfing zymosan. An overlap and corresponding colocalization map is shown. Imaging began 5 min after zymosan addition. Bar, 10 μm. (C) Confocal microscopy of BMDM exposed to fluorescent zymosan and immunostained for Gα_i2, Gα_i3, and F-actin (phalloidin). Bar, 5 μm. (D) Quantification of YFP-Gα_i2 and RFP-Lifeact colocalization over time. The percent colocalization of RFP-Lifeact and YFP-Gα_i2 above threshold (above) and Pearson's colocalization coefficient (below) are shown. Images acquired at 12 s/frame. (E) Confocal microscopy of RAW 264.7 cells expressing RFP-Lifeact (red) and YFP-Gα_i2 (green) before or after a 15-min exposure to latrunculin B. Individual and overlap images are shown. Bar, 8 μm.