Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly

Abstract

Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re-establish a functional nucleus. Time-lapse, FRAP and FRET analyses in human cells showed that barrier-to-autointegration factor (BAF), a DNA-binding protein, assembled first at the distinct `core' region of the telophase chromosome and formed an immobile complex by directly binding with other core-localizing NE proteins, such as lamin A and emerin. Correlative light and electron microscopy after live cell imaging, further showed that BAF formed an electron-dense structure on the chromosome surface of the core, close to spindle microtubules (MTs) prior to the attachment of precursor NE membranes, suggesting that MTs may mediate core assembly of BAF. Disruption of the spindle MTs consistently abolished BAF accumulation at the core. In addition, RNAi of BAF eliminated the core assembly of lamin A and emerin, caused abnormal cytoplasmic accumulation of precursor nuclear membranes and resulted in a significant delay of NE assembly. These results suggest that the MT-mediated BAF accumulation at the core facilitates NE assembly at the end of mitosis.

Lamin A precursor induces barrier-to-autointegration factor nuclear localization

“…18 Moreover, in living cells, BAF localization can be restricted to specific locations including telomeres and the "core region" of decondensing chromosomes. 30 The influence of BAF on chromatin organization has been experimentally demonstrated. In particular, it has been reported that BAF addition during nuclear assembly may have either positive or negative effects on chromatin condensation.…”

An epichromatin epitope: Persistence in the cell cycle and conservation in evolution

“…Another location that appears unreactive to the antiepichromatin antibody is the mitotic chromosome "core", a region facing the spindle pole, possessing centromeres, binding to the spindle microtubules, exhibiting proteins such as LAP2α and BAF and excluding LBR, LAP2β and lamin B. 23,24 Examples of mitotic chromosome cores are presented in Figure 5A and Supplementary Figure 1. Note especially that CREST antibody stains centromere regions of the cores, while anti-LBR and PL2-6 primarily react with the telophase chromosome "periphery".…”

How chromosomes unite

“…It will be interesting to learn how BAF 'shields' are dismantled, and how BAF contributes to chromosome re-engagement with lamins and nuclear-membrane proteins 6,14 , because these molecular connections are essential for customizing 3D genome organization in specific tissues and organs 15 . Further research might move beyond cell division, perhaps investigating the possibility of targeting BAF for anticancer therapies 16,17 , or detailing the mechanisms whereby BAF influences genome integrity, gene regulation and virus infection.…”