Figure 6.
Figure 6. RLC-D,D restricts Rac activation and GEF localization to the front of polarized cells. (A) CHO.K1 cells were transfected with constitutively active Rac1 (myc-V12Rac1, top) or Tiam1 (HA-Tiam1 C1199, bottom) and RLC-D,D–GFP, plated onto fibronectin-coated coverslips (2 µg/ml, 60 min), stained for myc or HA, and imaged using a confocal microscope (FV300; Olympus). Representative morphologies are shown. (A, bottom) Axis ratio as defined in Fig. 2. Data are the mean ± SD of three independent experiments (error bars; n > 200). (B) CHO.K1 cells were transfected with RLC-D,D–mCherry (magenta) and mVenus-tagged PA-Rac (green), plated onto 5 µg/ml fibronectin for 15–30 min (initial phases of spreading and polarization), and photoactivated in the rear; e.g., region of RLC bundles (marked with a box). Photoactivation and imaging were done with a confocal microscope (FV1000; Olympus). Protrusion was observed by differential interference contrast to define the edge of the cell (bottom, white line). Image on the right depicts a color overlay before (green) and after (magenta) photoactivation (5 min) using mVenus fluorescence. Inset shows the detail of a fragment of the photoactivated area that displays the most robust protrusion. A representative experiment is shown. (C) CHO.K1 cells were transfected with RLC-D,D–mCherry (green) and PA-Rac (magenta), plated onto fibronectin as in Fig. 6 B, and photoactivated globally. Representative examples are on the left. (C, right) Data are the mean ± SEM (error bars) of the polarity axis scored in three independent experiments. P represents significance using a Student’s t test. (D) CHO.K1 cells were cotransfected with RLC wild type (top left) or RLC-D,D (bottom left) coupled to mCherry and the Raichu-Rac FRET sensor (right). Localized activation of Rac was visualized in a confocal microscope (FV1000) by imaging the intensity ratio image of YFP and CFP, which represents FRET efficiency. Note the lower FRET index at the rear as defined by the RLC-D,D bundles (arrows). FRET values outside of the cell contour were set to zero for representation. Note that the scales are different because of differences in expression levels and imaging parameters, resulting in differences in the absolute values of the fluorescence intensity. The arrowhead points to the region of higher Rac activity at the leading edge of the RLC-D,D cell, across from the rear. Representative cells are shown. Bar, 20 µm. (E) FRET quantification. Data are the mean ± SD (error bars) of the difference between FRET indices at the front and rear (RLC-D,D) and rear/sides (RLC-WT) from three independent experiments (RLC-D,D, n = 40; RLC-WT, n = 34). P represents significance using a Student’s t test. (F) CHO.K1 cells were transfected with wild-type RLC or RLC-D,D coupled to GFP (top) or mCherry (bottom) and mCherry-βPIX (top) or FLAG-DOCK180 (bottom), plated on fibronectin, fixed, stained with an anti-FLAG antibody (bottom), and imaged using confocal microscopy (FV300). Arrowheads point to clusters of βPIX and DOCK180, respectively, whereas arrows denote the RLC-D,D–decorated rears. Representative morphologies are shown. Bars: (A–C) 10 µm; (D and F) 20 µm.

RLC-D,D restricts Rac activation and GEF localization to the front of polarized cells. (A) CHO.K1 cells were transfected with constitutively active Rac1 (myc-V12Rac1, top) or Tiam1 (HA-Tiam1 C1199, bottom) and RLC-D,D–GFP, plated onto fibronectin-coated coverslips (2 µg/ml, 60 min), stained for myc or HA, and imaged using a confocal microscope (FV300; Olympus). Representative morphologies are shown. (A, bottom) Axis ratio as defined in Fig. 2. Data are the mean ± SD of three independent experiments (error bars; n > 200). (B) CHO.K1 cells were transfected with RLC-D,D–mCherry (magenta) and mVenus-tagged PA-Rac (green), plated onto 5 µg/ml fibronectin for 15–30 min (initial phases of spreading and polarization), and photoactivated in the rear; e.g., region of RLC bundles (marked with a box). Photoactivation and imaging were done with a confocal microscope (FV1000; Olympus). Protrusion was observed by differential interference contrast to define the edge of the cell (bottom, white line). Image on the right depicts a color overlay before (green) and after (magenta) photoactivation (5 min) using mVenus fluorescence. Inset shows the detail of a fragment of the photoactivated area that displays the most robust protrusion. A representative experiment is shown. (C) CHO.K1 cells were transfected with RLC-D,D–mCherry (green) and PA-Rac (magenta), plated onto fibronectin as in Fig. 6 B, and photoactivated globally. Representative examples are on the left. (C, right) Data are the mean ± SEM (error bars) of the polarity axis scored in three independent experiments. P represents significance using a Student’s t test. (D) CHO.K1 cells were cotransfected with RLC wild type (top left) or RLC-D,D (bottom left) coupled to mCherry and the Raichu-Rac FRET sensor (right). Localized activation of Rac was visualized in a confocal microscope (FV1000) by imaging the intensity ratio image of YFP and CFP, which represents FRET efficiency. Note the lower FRET index at the rear as defined by the RLC-D,D bundles (arrows). FRET values outside of the cell contour were set to zero for representation. Note that the scales are different because of differences in expression levels and imaging parameters, resulting in differences in the absolute values of the fluorescence intensity. The arrowhead points to the region of higher Rac activity at the leading edge of the RLC-D,D cell, across from the rear. Representative cells are shown. Bar, 20 µm. (E) FRET quantification. Data are the mean ± SD (error bars) of the difference between FRET indices at the front and rear (RLC-D,D) and rear/sides (RLC-WT) from three independent experiments (RLC-D,D, n = 40; RLC-WT, n = 34). P represents significance using a Student’s t test. (F) CHO.K1 cells were transfected with wild-type RLC or RLC-D,D coupled to GFP (top) or mCherry (bottom) and mCherry-βPIX (top) or FLAG-DOCK180 (bottom), plated on fibronectin, fixed, stained with an anti-FLAG antibody (bottom), and imaged using confocal microscopy (FV300). Arrowheads point to clusters of βPIX and DOCK180, respectively, whereas arrows denote the RLC-D,D–decorated rears. Representative morphologies are shown. Bars: (A–C) 10 µm; (D and F) 20 µm.

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