Figure 4.
BRCA2 and RAD51 display similar diffusive behavior in live cells that is disrupted by BRC3 overexpression. (A, top) Oblique illumination images of live cells expressing BRCA2-GFP, RAD51-GFP, and RAD54-GFP. Slowly diffusing particles were detected for both BRCA2 and RAD51. RAD54 was detected only as immobile clusters. Bars, 5 µm. (A, bottom) 2D histograms for all three proteins display the distribution of residence times in the mobile (particle jumps > 200 nm) and bound state (particle jumps < 200 nm) of all detected tracks. The insets illustrate representative tracks for characteristic different mobility behaviors and are also shown in Fig. 2 (B and C). The relative frequencies for tracks with the indicated times spent in the bound and mobile state are represented by the colors defined on the right. The number of acquired tracks was 4126 for BRCA2-GFP, 791 for RAD51-GFP, and 250 for RAD54-GFP, from at least four fields and 16 nuclei per sample. (B) Fluorescence images of cells transfected with BRC3 peptide expression vector and/or an RFP expression vector. Upon expression of BRC3-ΔFK peptide, which is deficient for RAD51 binding, slowly diffusing and transiently binding RAD51-GFP particles (green color channel) can be detected in Rad51GFP/WT cell nuclei. Upon BRC3 peptide expression, nuclei of most transfected cells now show bright, uniform RAD51-GFP fluorescence, indicating that BRCA2–RAD51 interactions are disrupted and RAD51 mobility is enhanced. BRC3 expression did not alter the mobile behavior of BRCA2-GFP. Bars, 5 µm. (C) Quantification of true detections (red circles) as in Fig. 1 B, in RFP-positive and -negative cells expressing BRC3, BRC3-ΔFK, or control transfection. Because of the relatively high RAD51 concentration (compared with BRCA2-GFP), and therefore enhanced fluorescence level, only relevant detections in the σ range of 1–3 were analyzed. Additionally, the exponential function was set off by 5 AU because the RFP+ BRC3 dataset did not have any detections to use for deriving a cutoff curve. Example data representative of individual video stacks are displayed. (D) Number of detections per condition (5–7 nuclei, number of initial detections: 5,000–12,000). The error bar represents the mean with the 95% confidence interval. Thus, BRC3-expressing cells have significantly fewer RAD51 detections than cells subjected to control transfection or BRC3-ΔFK expression.