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First published online 19 September 2006
doi: 10.1242/jcs.03183

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Dynamics and anchoring of heterochromatic loci during development

¹ Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA
² Center for Automated Learning and Discovery, Carnegie Mellon University, Pittsburgh, PA 15213, USA

View larger version (21K): [in a new window]   Fig. 1. Distance measurements of bw-centromere signals to determine whether the CIDGFP dot closest to the mRFP dot is the centromere of the second chromosome. (A) Projected image of undifferentiated nuclei anterior to the morphogenetic furrow in the eye imaginal discs. The green dots are CIDGFP and the single purple dot represents the lacO repeats inserted in the bw region bound by mRFP-LacI. The background fluorescence of the unbound mRFP-LacI marks the nucleus. In interphase nuclei, the centromeres of the four paired chromosomes in Drosophila are typically observed as three or four dots. (B) Distance between the tagged locus and the closest CIDGFP dot was computed and divided by the radius of the nucleus. The distribution of distances is displayed as box plots. Box plots are calibrated representations of histograms wherein each horizontal line delimits the 10th, 25th, 50th (median), 75th and 90th percentiles. The numbers within the box plots are the number of nuclei included in the analysis. P-values (Mann-Whitney U-test) are shown above brackets for the respective sets. (C) The distribution is similar to that observed in an earlier study using FISH. Probes specific to the bw locus and the AACAC satellite repeats that make up a small subset of 2Rh were used (Thakar and Csink, 2005). (D) A histogram of mRFP-CIDGFP distances corrected by the radius.

View larger version (31K): [in a new window]   Fig. 2. bwD association is observed in differentiated embryonic neurons but not undifferentiated neuroblasts. (A) Immunofluorescence with antibody against ELAV, a neuron-specific protein, on embryonic neuroblasts cultured for either 5 hours (left) or 15 hours (right). Although the 5-hour cells do not show much signal for ELAV, those cultured for 15 hours do. The 15-hour cells also tend to form ganglionic clusters and extrude neuronal processes. (B) Single section from a series of z-stack images of embryonic neuroblasts expressing CIDGFP (green) and mRFP-LacI protein bound to the bw-region tagged with lacO repeats (purple). (C) Box plots displaying the 2D distance between the mRFP dot and the closest CIDGFP dot corrected by the nuclear radius from projected images. At least three to four coverslips were imaged with no more than 40 nuclei imaged per coverslip. Change in nuclear organization of the bwD locus is observed with a significant number of cells showing association between the bwD locus and centromere only after 15 hours of culturing. (D) Histograms of data shown in C.

View larger version (48K): [in a new window]   Fig. 3. Constraint of chromatin dynamics with differentiation restricts changes in nuclear organization. (A) An eye imaginal disc expressing GFP (pink) in differentiating cells present posterior to the morphogenetic furrow. There is a gradient of differentiation, with cells further away from the furrow having started differentiating earlier than those present closer to the furrow. A series of images of the posterior end of the disc were taken (green box). (B) A projected image representing the area demarcated by the green box is shown. Each image was divided into two halves, categorizing the nuclei into early differentiating and late-differentiating nuclei. (C) Box plots of distances between the mRFP signal from the tagged bwD locus and the closest CIDGFP dot was measured from projected images and corrected by the nuclear radius. No significant difference in the distribution was observed in the late-differentiating cells as compared with the early differentiating cells.

View larger version (27K): [in a new window]   Fig. 4. Association of bwD 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 bwD 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) bwD nuclei were further categorized into IU (distance between bwD and the closest CIDGFP dot was >1.0 µm at t=0) and IA (distance between bwD 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 bwD nuclei. (D) Histograms of step size (d) at t=15 seconds. The mean step size of bwD (IA) was found to be statistically significantly lower with a P-value of <0.001 (t-test), than either bw+ or bwD (IU). (E) To calculate the true radius of confinement for bw+ and IU bwD, 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 bwD tend to plateau at approximately t=21 minutes. As observed in A, the plateau height of the graph for IA bwD was significantly lower than that observed for bw+ and IU bwD, indicating that its movement is highly confined.

View larger version (52K): [in a new window]   Fig. 5. bwD can get closer to the centromere than bw+. (A) Histograms of all the 3D measurements from all the movies in this study with t= 30 seconds and 3 minutes suggest that the heterochromatic insertion in bwD allows it to come into close proximity (<1.0 µm) to centric heterochromatin. (B,C) Traces showing the distances between mRFP-CID at each time point in individual movies (each movie a different color trace). Movement of the tagged bw region in undifferentiated nuclei of the eye imaginal discs was followed for 1-2 hours. Images were taken every 3 minutes. Image analysis was done in 3D. Nuclei were divided into the three categories. The first category contains nuclei where the distance between CIDGFP and mRFP at t=0 was >2.0 µm, the second category contains nuclei where the distance was between 1.0 and 2.0 µm, and the third category contains nuclei where the distance was <1.0 µm. When the distance between the centromere and the bwD locus is >2.0 µm the fluctuation in distances between the two loci is more apparent (left panel), as compared with the change in distance when the relative distance between bwD and the closest centromere is <2.0 µm. (B) Traces from movies of wild-type nuclei. (C) Traces from movies of bwD nuclei. (D) Graph of the distance between the two closest CIDGFP dots in six nuclei. These centromeres do not experience any large-scale movements.

View larger version (21K): [in a new window]   Fig. 6. Microtubule destabilizers decrease the step size of centromeres, but do not perturb nuclear organization or chromatin dynamics of bwD. (A) Box plots showing the distribution of distances between bwD locus and the nearest CIDGFP signal corrected by the radius. No change was observed in nuclear organization upon treatment with nocodazole and colchicine, two chemicals that destabilize microtubules, as compared with the untreated control. (B) MSD plot (±1 s.e.m.) comparing the movement of bwD-CIDGFP in movies where images were taken at a single plane of focus every 3 seconds. A large change in chromatin dynamics was not observed for the bwD locus upon treatment. (C) MSD (±1 s.e.m.) plots comparing the movement of centromeres in the presence of microtubule-destabilizing agents. Although the diffusion coefficient for the movement of centromeres was comparable, a significant decrease in the radius of confinement was observed upon treatment. (D) Histograms of the distribution of step sizes of centromeres calculated at t=15 seconds. A significant decrease in step size of centromeres was observed in nuclei treated with microtubule destabilizers (P<0.0001).

View larger version (15K): [in a new window]   Fig. 7. Models of bwD behavior near the centromere. A constraint in movement is imposed when the distance between bwD-CIDGFP is between 1 and 2 µm. The distribution of the traces in Fig. 6 could be interpreted in two ways. (A) Constraint may be imposed wherever bwD contacts the surface of the heterochromatic compartment and the seemingly large variation in distance from the centromere could be because of the variable position of the centromere within the mass of heterochromatin. (B) bwD moves within the centric heterochromatin, varying its distance to the centromere. Possibly the endpoint of this random walk is a stable, close association with the centromere.