Spatial and Physical Organisation of the Mammalian Nucleus

The nucleus is a dynamic and highly adaptable structure, essential for eukaryotic cell survival and function. As the largest and stiffest organelle in the cell, the nucleus is especially sensitive to mechanical input. As a result, it is becoming increasingly evident that in addition to housing and protecting genomic material, the nucleus is capable of not only sensing but adapting and responding to its physical and biochemical environments. Considerable effort has been made in the past years to understand how mechanical cues can affect nuclear structure and nuclear processes. However, less is known regarding how the activation of cellular pathways and nanoscale organisation of nuclear proteins can affect local and overall nuclear mechanics and mechanotransduction. A link between nuclear activity and the mechanical properties of the nucleus also becomes more evident as chromatin arises as a major contributor to cell stiffness. The work presented in this thesis employed a multidisciplinary approach to study nuclear architecture and function – from large-scale nuclear adaptation to external stimuli and signalling pathways, to the nanoscale organisation of nuclear activity. As this thesis was written in manuscript format – as a collection of peer-reviewed publications or pre-print manuscripts submitted to different journals - my work is shown alongside that of others. For this reason, throughout the thesis, I use ‘we’ instead of ‘I’ when describing findings. For clarification on my individual contributions, I have detailed the work I performed for each manuscript at the beginning of each results chapter.

In a first instance, this thesis describes how chromatin is a major contributor to the viscoelastic response of the nucleus to mechanical strains. An important outcome of the work shown here was the understanding that chromatin mechanics are not homogeneous throughout the organelle. The work led by Lherbette and myself proposes that chromatin crosslinking, possibly by regulatory DNA-binding proteins, is important in defining the material properties and the mechanical response of the nucleus (Lherbette et al., 2017). This suggests that nuclear activity can directly impact the mechanical state of the organelle. To test this, I then investigated how DNA damage and activation of DNA repair signalling pathways affects nuclear stiffness. My work shows that, following cisplatin treatment, ATM kinase-dependent large-scale chromatin decondensation causes nuclear softening (dos Santos et al., 2021). This further supports our hypothesis, showing a clear link between biochemical processes and mechanical changes to the organelle. Furthermore, it highlights the importance of proteins that modulate nuclear processes, such as DNA repair factors, transcription regulators and proteins that regulate chromatin architecture. An example of a protein with important roles in transcription regulation and chromatin architecture is Myosin VI. This molecular motor is mostly known for cytoplasmic functions in cargo transport, endocytosis and cell adhesions. Interestingly, recent work has linked it to gene pairing events and RNA Polymerase II regulation. However, at the time of the work presented here, it was not yet clear how Myosin VI nuclear activation occurs or the molecular mechanism through which the protein performs it regulatory role in transcription. Here, we investigated how nuclear Myosin VI is activated and how this activity impacts RNA Polymerase II organisation and dynamics (Hari-Gupta et al., 2020).
We defined a general activation model for Myosin VI, whereby interactions with binding partners, such as the nuclear dot protein 52 (NDP52) or Disabled-2 (Dab2) release the protein from its auto-inhibited state and allow its dimerization and motor processivity (dos Santos et al., 2020; Fili et al., 2017). This motor activity of Myosin VI is essential for RNA Polymerase II clustering at transcriptional sites. In particular, the work presented here proposes that molecular anchoring of nuclear Myosin VI on actin filaments could be essential for increased RNA Polymerase II binding times at transcription initiation sites, leading to higher transcription efficiency.

As in Fili et al., we explored Myosin VI nuclear activity, we also uncovered novel nuclear roles for its binding partner NDP52 (Fili et al., 2017). NDP52 has been previously described in a cytoplasmic context, where, through interactions with Myosin VI and other autophagy adapters, it participates in the recognition and clearance of pathogens and damaged organelles. However, although NDP52 was first identified in the nucleus and shares high homology with a transcription coactivator (the Coiled-coil coactivator, CoCoA), until the study presented in this thesis, no clear functions had been attributed to the protein. My work indicates that NDP52 is involved in RNA Polymerase II transcription, through two possible mechanisms: either through direct interactions with transcription machinery and co-regulators, or through direct/indirect changes to chromatin structure (dos Santos et al., 2022).

Overall, this thesis describes different aspects of nuclear architecture, from overall organelle structure to the spatial distribution of enzymatic nuclear activity.