Trajectories and nuclear arrangement of PML bodies are influenced by A‐type lamin deficiency

Abstract

Background information Promyelocytic leukaemia (PML) bodies are specific nuclear structures with functional significance for acute promyelocytic leukaemia. In this study, we analysed the trajectories of PML bodies using single‐particle tracking. Results We observed that the recovery of PML protein after photobleaching was ATP dependent in both wild‐type (wt) and A‐type lamin‐deficient cells. The movement of PML bodies was faster and the nuclear area occupied by particular PML bodies was larger in A‐type lamin‐deficient fibroblasts compared with their wt counterparts. Moreover, dysfunction of the LMNA gene increased the frequency of mutual interactions between individual PML bodies and influenced the morphology of these domains at the ultrastructural level. As a consequence of A‐type lamin deficiency, PML protein accumulated in nuclear blebs and frequently appeared at the nuclear periphery. Conclusions We suggest that the physiological function of lamin A proteins is important for events that occur in the compartment of PML bodies. This observation was confirmed in other experimental models characterised by lamin changes, including apoptosis or the differentiation of mouse embryonic stem cells.

Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies

“…31 Differentiation of mouse ESCs was induced by alltrans retinoic acid according to previously published protocols. 32 We also used HeLa cells stably expressing GFP-colin (a generous gift from the laboratory of Prof Angus Lamond, University of Dundee 16 ). For detection of endogenous proteins in DNA lesions, we used HeLa cells stably expressing GFP-H2B to visualize nuclei.…”

“…The detailed methodological approach was described previously. 32 For Cajal body analysis, we analyzed the average areas enclosed by ellipses determined by the area occupied by CBs. The average enclosing ellipse is the ellipse with minimal area that enclosed all the positions of the CB centroids that appeared in time.…”

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

“…Accumulated protein mobility in the nucleus also can be addressed by additional biophysicsrelated computational methods, such as FRAP analysis of protein diffusion or computation of mean-square displacement (MSD). 18 However, diffusive motion analysis via MSD curve construction presumes a small size for the analyzed objects, and this presumption is not the case for large UBF1-positive nucleolar regions. Therefore, we could not apply this analysis for our data.…”

“…3A) were performed according to Stixov a et al (2012). 18 The anti-lamin B primary antibody (sc-6217, Santa Cruz) was used for these analyses. Total protein levels were measured using a mQuant spectrophotometer (BioTek).…”

“…The following methods can be applied for such analyses: 1] FRAP analysis of protein diffusion at DNA lesions, 2] advanced image processing algorithms to follow DNA repair foci trajectories, and 3] image analysis to describe morphology of protein-abundant DNA lesions. [15][16][17][18] Critical parameters also include the particle dynamics and fluorescence signal intensity of radiation-damaged chromatin, which can be monitored in 3 dimensions over time. Thus, the use of above mentioned experimental approaches can allow for the proper characterization of DNA lesion behavior in space and time.…”