Figure 6. UNC-45a knockout (KO) cells... | Rockefeller University Press

Figure 6.

$Figure 6. UNC-45a knockout (KO) cells display defects in formation of bipolar NM-IIA filaments and stacks. (A) SIM images of representative WT and UNC-45a knockout cells in which actin filaments were visualized by phalloidin. NM-IIA motor domains and tails (rod) were visualized by antibodies against RLC and NM-IIA heavy chain C terminus, respectively. Bars, 5 µm. Magnified regions (corresponding to the orange boxes) display characteristic NM-IIA filament distributions in WT and UNC-45a knockout cells. Bars, 1 µm. High-magnification images of individual filaments (corresponding to the white boxes in the magnified regions) display examples of 1, unipolar structure; 2, bipolar filament; 3, stack of bipolar filaments. Bars, 100 nm. (B) Distributions of different NM-IIA filament populations were quantified from the SIM data, registering all NM-IIA filaments within 2 µm toward the cell body from the first-detected filament at the leading edge. Data obtained on relative percentages of NM-IIA unipolar structures and bipolar filaments were then multiplied by the respective NM-IIA protein levels (WT, 1; knockout, 0. 43; Figs. 4 A and S2 B). n = 261 and 211 NM-IIA filaments analyzed from seven WT and seven UNC-45a knockout cells, respectively. Data are represented as means ± SEM (Student’s t test). (C) The fluorescence intensities of bipolar NM-IIA filaments were manually measured from SIM images of WT and UNC-45a knockout cells expressing NM-IIA with N-terminal mEmerald and C-terminal mApple fusions (Fig. S3 D). For the analysis, 16 bipolar filaments were quantified from five control cells, and 22 bipolar filaments were quantified six UNC-45a knockout cells. Data are represented as fluorescence intensity profiles of the motor and rod domains along the line scan length (=x axis); means ± SEM. (D) The fluorescence intensities of unipolar NM-IIA structures were manually measured from mEmerald–NM-IIA–mApple-expressing cells as shown in C. n = 17 and 32 unipolar structures analyzed from five control and six UNC-45a knockout cells, respectively. Data are represented as fluorescence intensity profiles of the NM-IIA motors and rods along the line scan length (=x axis); mean ± SEM. (E) Distributions of NM-IIA molecules in a density gradient. Identical amounts (100 µg) of total cell lysates from WT and UNC-45a knockout cells were subjected to density gradient (0–50%), and a total 11 fractions were collected with increasing density, followed by SDS-PAGE and WB with NM-IIA heavy chain antibody. Tubulin was probed for a fractionation control.$

UNC-45a knockout (KO) cells display defects in formation of bipolar NM-IIA filaments and stacks. (A) SIM images of representative WT and UNC-45a knockout cells in which actin filaments were visualized by phalloidin. NM-IIA motor domains and tails (rod) were visualized by antibodies against RLC and NM-IIA heavy chain C terminus, respectively. Bars, 5 µm. Magnified regions (corresponding to the orange boxes) display characteristic NM-IIA filament distributions in WT and UNC-45a knockout cells. Bars, 1 µm. High-magnification images of individual filaments (corresponding to the white boxes in the magnified regions) display examples of 1, unipolar structure; 2, bipolar filament; 3, stack of bipolar filaments. Bars, 100 nm. (B) Distributions of different NM-IIA filament populations were quantified from the SIM data, registering all NM-IIA filaments within 2 µm toward the cell body from the first-detected filament at the leading edge. Data obtained on relative percentages of NM-IIA unipolar structures and bipolar filaments were then multiplied by the respective NM-IIA protein levels (WT, 1; knockout, 0. 43; Figs. 4 A and S2 B). n = 261 and 211 NM-IIA filaments analyzed from seven WT and seven UNC-45a knockout cells, respectively. Data are represented as means ± SEM (Student’s t test). (C) The fluorescence intensities of bipolar NM-IIA filaments were manually measured from SIM images of WT and UNC-45a knockout cells expressing NM-IIA with N-terminal mEmerald and C-terminal mApple fusions (Fig. S3 D). For the analysis, 16 bipolar filaments were quantified from five control cells, and 22 bipolar filaments were quantified six UNC-45a knockout cells. Data are represented as fluorescence intensity profiles of the motor and rod domains along the line scan length (=x axis); means ± SEM. (D) The fluorescence intensities of unipolar NM-IIA structures were manually measured from mEmerald–NM-IIA–mApple-expressing cells as shown in C. n = 17 and 32 unipolar structures analyzed from five control and six UNC-45a knockout cells, respectively. Data are represented as fluorescence intensity profiles of the NM-IIA motors and rods along the line scan length (=x axis); mean ± SEM. (E) Distributions of NM-IIA molecules in a density gradient. Identical amounts (100 µg) of total cell lysates from WT and UNC-45a knockout cells were subjected to density gradient (0–50%), and a total 11 fractions were collected with increasing density, followed by SDS-PAGE and WB with NM-IIA heavy chain antibody. Tubulin was probed for a fractionation control.