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Fig. 4. Association of bw^D with the centromere greatly decreases the movement and radius of confinement of the locus. Data obtained from short time-lapse movies was used to calculate the diffusion coefficient and step size and to test for directionality of movement in bw^D and bw⁺ nuclei. (A) Movement of the bw locus was followed relative to the closest centromere marked by CIDGFP in undifferentiated cells of the eye imaginal discs. The average mean square change in distance <d²> over all possible t values (±1 s.e.m.) was plotted for each genotype. The 1.6 Mb insertion of heterochromatic repeats does not alter the dynamics of movement of the bw locus. The diffusion coefficients in Table 2 were calculated by taking the mean value for <d²> at t= 3 and 6 seconds from the data set shown here. (B) Histogram of the distribution of distances between mRFP-CIDGFP at t=0, from the data shown in A. Note that no bw⁺ nuclei were observed where the distance between mRFP-CIDGFP signal was less than 1.0 µm. (C) bw^D nuclei were further categorized into IU (distance between bw^D and the closest CIDGFP dot was >1.0 µm at t=0) and IA (distance between bw^D and the closest CIDGFP dot was <1.0 µm at t=0). The graph shows the mean square change in distance of the IA and IU bw^D nuclei. (D) Histograms of step size (d) at t=15 seconds. The mean step size of bw^D (IA) was found to be statistically significantly lower with a P-value of <0.001 (t-test), than either bw⁺ or bw^D (IU). (E) To calculate the true radius of confinement for bw⁺ and IU bw^D, chromatin movement was followed for a longer time (1-2 hours) with images taken every 3 minutes to obtain graphs where the plots for the average mean square change in distance <d²> over all possible t values reached a plateau. Image analysis was done in 3D. The plot of <d²> (±1 s.e.m.) in the longer movies for both bw⁺ and IU bw^D tend to plateau at approximately t=21 minutes. As observed in A, the plateau height of the graph for IA bw^D was significantly lower than that observed for bw⁺ and IU bw^D, indicating that its movement is highly confined.

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