Figure 7.
DscI is a component of the mechanoresponsive CKs. (A and B) Western blot of co-immunoprecipitation studies of GFP-DscIA in cell lysates of indicated cell lines is shown. FT, flow-through; IQG2, IQGAP2. No antibody for IQGAP2 is available so cell lines expressing mCh-fused IQGAP2 and mouse anti-mCh antibody (Novus Biologicals, 25157SS) were used. In A, bands boxed in the same color (orange or blue) are from the same Western blot. (C) Results from FCCS analysis examining interactions between DscIA and MyoII in different cellular backgrounds are shown. The apparent, or in vivo, KD for unlinked mCherry and GFP (negative control) and fusion GFP-mCherry (positive control) was 5.0 and 1.1 µM, respectively. P values were calculated using the log-transformed data and the ANOVA followed by Fisher’s LSD test (*, P < 0.05; ***, P < 0.0001). Number of cells analyzed, n = 19–30 from two independent experiments. (D) FCCS analysis confirmed the positive interaction between CortI and IQGAP2 in cortI-complemented background (with an apparent KD = 1.2; Kothari et al., 2019b). Median in vivo KDs for negative (=3.9 and 3.8) and positive (=1.1 and 1.1) controls in cortI-complemented and cortI; dsc1 double mutant background were similar to that of the WT background (C). In the absence of dsc1, CortI and IQGAP2 interacted with an apparent KD of 2.0, which is significantly weaker than that measured in the presence of DscI. Number of cells analyzed, n = 18–28 from two independent experiments. (E) FCCS-detected interactions between CortI and MyoII in the WT background (with KD = 1.5). The affinity between CortI and MyoII was significantly weakened in dsc1 null mutants (with KD = 2.6). Positive and negative controls (shaded) in WT background are reproduced from C for direct comparison. Positive and negative controls were also measured in dsc1 background (in dsc1: KD for negative control = 5.2, KD for positive control = 1.3). All P values were calculated on the log-transformed data using an Kruskal-Wallis followed by the Wilcoxon test (*, P < 0.05; **, P < 0.001; ***, P < 0.0001). P values signifying the statistical differences in the interactions between the presence and absence of DscI are highlighted in red. Number of cells analyzed, n = 24–36 from two independent experiments. (F) FCCS data in this study show that DscI helps stabilize the associations between key proteins in the mechanoresponsive CKs. Combined with previous proteomic analysis and co-immunoprecipitation results, it is likely that these proteins, including DscI, function in the same complex, and the absence of one affects the stability of other interactions within the assembly. Source data are available for this figure: SourceData F7.

DscI is a component of the mechanoresponsive CKs. (A and B) Western blot of co-immunoprecipitation studies of GFP-DscIA in cell lysates of indicated cell lines is shown. FT, flow-through; IQG2, IQGAP2. No antibody for IQGAP2 is available so cell lines expressing mCh-fused IQGAP2 and mouse anti-mCh antibody (Novus Biologicals, 25157SS) were used. In A, bands boxed in the same color (orange or blue) are from the same Western blot. (C) Results from FCCS analysis examining interactions between DscIA and MyoII in different cellular backgrounds are shown. The apparent, or in vivo, KD for unlinked mCherry and GFP (negative control) and fusion GFP-mCherry (positive control) was 5.0 and 1.1 µM, respectively. P values were calculated using the log-transformed data and the ANOVA followed by Fisher’s LSD test (*, P < 0.05; ***, P < 0.0001). Number of cells analyzed, n = 19–30 from two independent experiments. (D) FCCS analysis confirmed the positive interaction between CortI and IQGAP2 in cortI-complemented background (with an apparent KD = 1.2; Kothari et al., 2019b). Median in vivo KDs for negative (=3.9 and 3.8) and positive (=1.1 and 1.1) controls in cortI-complemented and cortI; dsc1 double mutant background were similar to that of the WT background (C). In the absence of dsc1, CortI and IQGAP2 interacted with an apparent KD of 2.0, which is significantly weaker than that measured in the presence of DscI. Number of cells analyzed, n = 18–28 from two independent experiments. (E) FCCS-detected interactions between CortI and MyoII in the WT background (with KD = 1.5). The affinity between CortI and MyoII was significantly weakened in dsc1 null mutants (with KD = 2.6). Positive and negative controls (shaded) in WT background are reproduced from C for direct comparison. Positive and negative controls were also measured in dsc1 background (in dsc1: KD for negative control = 5.2, KD for positive control = 1.3). All P values were calculated on the log-transformed data using an Kruskal-Wallis followed by the Wilcoxon test (*, P < 0.05; **, P < 0.001; ***, P < 0.0001). P values signifying the statistical differences in the interactions between the presence and absence of DscI are highlighted in red. Number of cells analyzed, n = 24–36 from two independent experiments. (F) FCCS data in this study show that DscI helps stabilize the associations between key proteins in the mechanoresponsive CKs. Combined with previous proteomic analysis and co-immunoprecipitation results, it is likely that these proteins, including DscI, function in the same complex, and the absence of one affects the stability of other interactions within the assembly. Source data are available for this figure: SourceData F7.

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