The role of nuclear F-actin in chromatin organisation

Abstract

Re-establishment of nuclear structure and chromatin organisation after cell division is essential for genome regulation and cell function. However, the mechanisms underlying these events remain incompletely understood. Previous research identified the transient formation of filamentous actin (F-actin) that arises in the nucleus after mitosis, in several mammalian cell types. Given that the emergence of these filaments coincides with nuclear expansion and chromatin de-condensation, it was hypothesised that nuclear F-actin may drive these processes. Accordingly, this work describes the development of three assays to measure chromatin compaction and/or nuclear structure; through the combined use of fluorescence lifetime imaging (FLIM), electron microscopy (EM) and atomic force microscopy (AFM), a robust platform to detect changes in chromatin compaction was developed. These systems were then deployed to dissect the role of nuclear F-actin in nuclear re-organisation after cell division. AFM and live cell imaging were used to show that nuclear F-actin promotes nuclear expansion after cell division, a process which was impaired by the expression of a nuclear-targeted non-polymerisable actin mutant (NLS-actinR62D). Additionally, FLIM and quantitative EM showed that these actin mutant cells have defective chromatin de-condensation after mitosis, compared to wild-type cells. This increase in chromatin condensation was further confirmed by MNase assays, and the analysis of epigenetic markers associated with chromatin de-condensation. These results also suggest a potential role for nuclear F-actin in the regulation of chromatin re-modelling complexes, including Aurora B. In light of these results, the biological consequences of this increase in chromatin compaction was investigated under conditions, in which polymerisation of nuclear actin after cell division was inhibited (NLS-actinR62D). Using immunofluorescence, it was shown that transcription and progression through G1 was impaired in the daughter cells. Further, using flow cytometry and immunofluorescence to dissect the fate of these cells, defects in the timing of DNA replication were identified, as well as the pronounced accumulation of γH2AX foci in S phase. Finally, these defects were linked to the increase in chromatin compaction observed in G1. Collectively, these findings identify a novel role for nuclear F-actin, which contributes to the re-establishment of an interphase nucleus after cell division. This work has potential implications in the global regulation of chromatin structure throughout the cell cycle, and further emphasises the importance of post-mitotic genome organisation on healthy nuclear function and genome stability.

Date of Award	23 Jan 2019
Original language	English
Awarding Institution	University of Bristol
Supervisor	Stefan Roberts (Supervisor) & Paul Verkade (Supervisor)