(A, B) Colocalization analysis of mGFP-tagged human-yeast myosin-I chimeras (green) and mCherry-tagged calmodulin-like light chain Cam2 (red) in myo1Δ cells. mGFP alone, mGFP-tagged SpMyo1 and chimeras were expressed from plasmids under control of 3xPnmt1 promoter for 12-18 hours in the absence of thiamine.
(A) Single confocal sections through the middle of the cells. The blue Sp IQ+/− labels indicate the presence or the absence of the SpMyo1 IQ2 motif. Scale bars, 1 μm.
(B) Montages of individual patches at 2-second intervals. Scale bars, 0.5 μm. Numbers representing percent colocalization (mean ± SD) of Cam2 with SpMyo1 or SpM-HsT-1 in dynamic actin patches are shown above montages. N=41-63 patches in 3-6 cells.
Yellow arrowheads indicate colocalization of Cam2 with SpMyo1 and SpM-HsT-1 in actin patches. White arrows indicate colocalization of Cam2 with HsM-SpT-2 and HsM-SpT-3 in cortical aggregates. Red arrowheads indicate Cam2-only puncta that form in the absence of SpMyo1 IQ2 motif.