Dynamic organization of chromosomes in the mammalian cell nucleus

摘要: Uncovering the motifs of a higher order nuclear architecture and its implications on function has raised increasing interest in past decade. The nucleus eukaryotes is considered to display highly dynamic interaction DNA protein factors. There an emerging view that there are hierarchical levels gene regulation, reaching from epigenetic modifications at DNA- histone level functional topology, context which gene-activating -repressing processes influence expression profile individual cell beyond sequence information DNA. The present work focuses analysis aspects living cells. As prerequisite, vivo replication labeling strategy was developed, enabled simultaneous visualization early mid-to-late replicating chromatin as well single chromosome territories basis labeling/segregation approach. presented scratch protocol combines high efficiency with reduced “damaging” effects can be successfully applied number adherently growing lines, including primary human fibroblasts. In addition, live observation system developed facilitates time-lapse confocal (4D) microscopy over elongated time periods made it possible follow complete cycle or more. To address long-range movements (CTs) during entire interphase, fluorescence small CTs performed HeLa cells stably expressing H2B-GFP. This achieved by fluorescent nucleotides. Labeled were cultivated for several cycles until labeled chromatids had segregated. Such followed time-scales up 20 hours covering major parts cycle. Positional changes intensity gravity centers µm observed G1, thereafter, positions remained within range ~1 till end G2. conclusion, CT arrangements constrained mid G1 late G2 / prophase, whereas neighborhoods occurred one next. More extended might play role when “home in” establish non-random radial arrangement. To analyze next, nuclei photobleached maintaining contiguous zone unbleached pole. preserved onset prophase segments often become located distant sites metaphase plates. Accordingly, patterns daughter differed significantly mother nucleus, indicating not mitosis. variability clonal growth further confirmed 3D-FISH experiments. A series experiments more preliminary character looked lamina constraining determining selectively interacting chromatin. Simultaneous immunodetection lamin B two-color neuroblastoma revealed specific attachment compartment only along periphery but also inside invaginations lamina. 4D-live C-GFP CHO simultaneously concomitant foci attached invaginations. Moreover, essay employed uses injection dominant negative A mutant (ΔNLA) cause reversible disruption Initial results point distortion pattern preferential respective partially disrupted (as compared pore complex). Finally, model positioning mammalian depending shape.