Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation (2020)
Figure 5

Figure 5. HSPC differentiation is associated with microtubule-dependent heterochromatin redistribution from the nucleus periphery.

A) Spatial distribution of H3K9me3 in CD38-, CD33+ and CD19+ cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.

B) Spatial distribution of H3K9me3 at the indicated time points of differentiation of CD38- cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.

C) For each cell, the raw DAPI image at the equatorial Z stack is used to extract the nucleus contour and measure H3K9Me3 intensity (in arbitrary units [a.u]) along the contour (lower row).

D) H3K9me3 loss from the nucleus periphery occurs locally in nuclear invaginations. Line scans of H3K9me3 signal along nucleus contour of 2 representative cells are presented in the upper panel for each time point. Asterisks indicate the local loss of H3K9me3. The corresponding variations in H3K9Me3 intensity and curvature are plotted in blue in red, respectively. Negative curvature values correspond to local nuclear invaginations.

E) Local curvature of the nucleus envelope as a function of H3K9Me3 local intensity at the indicated time points of differentiation. Positive and negative curvatures are shown in grey and red, respectively. The absence of curvature is highlighted as a dashed line. During differentiation, negatively curved domains increase in number and size and display low H3K9me3 intensities.

F) H3K9me3 redistribution during differentiation. H3K9me3 signal dispersion at the nucleus periphery is low in CD38- (n=137, 3 donors) indicating that H3K9me3 is homogenously distributed at the nucleus periphery. Upon differentiation, H3K9me3 signal dispersion remains homogenous after 24 hours in culture (n=115, 3 donors), but progressively becomes heterogeneous (48 h: n=139, 3 donors) to attain a level at 72 h (n= 148, 3 donors) similar to that in freshly collected CD33+ cells (n=124, 3 donors), indicative of H3K9me3 redistribution from the nucleus periphery. Bars correspond to mean ± SD; ***:p<0.001; **:p<0.01; n.s: non significant; Mann Whitney test.

G) Microtubule perturbations impair H3K9me3 redistribution during HSPC differentiation. Microtubule-modifying agents were added to culture medium between 48 and 72 hours. Cells were fixed and analyzed after 72 hours of culture. For each indicated treatment, one representative nucleus is presented. Inverted images of equatorial Z stacks of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.

H) H3K9me3 signal dispersion significantly increases in non-treated cells between 48h (n=139/3 donors) and 72h (n=148/3 donors) of culture. At 72h, the dispersion is similar in non-treated and blebbistatin-treated cells (n=124/ 3 donors). By contrast, H3K9me3 signal dispersion is significantly reduced compared with non-treated cells after treatment with nocodazole and blebbistatin (n=55, 2donors), taxol (n= 109, 3 donors) and ciliobrevin-D (n=144, 3donors) to a level attained by non-treated cells at 48 hours, demonstrating that microtubules affect H3K9me3 redistribution during HSPC differentiation. Bars correspond to mean ± SD; ****:p<0.0001; n.s: non significant; Mann Whitney test.