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

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models. View Full-Tex

DNA damage alters nuclear mechanics through chromatin reorganization

AbstractDNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance

Myosin VI regulates the spatial organisation of mammalian transcription initiation.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression

Casos clínicos de paratuberculose em pequenos ruminantes no Distrito de Bragança

Dissertação de Mestrado Integrado em Medicina VeterináriaA paratuberculose, também denominada Doença de Johne, é uma doença de natureza infeciosa causada por Mycobacterium avium subespécie paratuberculosis (Map). Os ruminantes são as espécies maioritariamente afetadas, nos quais origina uma enterite crónica granulomatosa. O quadro clínico carateriza-se por um emagrecimento progressivo que pode ser fatal, acompanhado frequentemente de diarreia. O tratamento médico é raramente realizado, pois é dispendioso e pouco eficaz. Por essa razão, a vacinação é considerada o método de controlo mais eficaz. O objetivo deste trabalho consistiu em desenvolver e consolidar os conhecimentos sobre a paratuberculose através do acompanhamento de casos clínicos. Foram utilizados seis casos clínicos, dos quais dois já tinham um diagnóstico laboratorial positivo, nos quais apenas se realizou a vacinação, dois foram casos novos, em que se realizou a recolha de amostras e cujo diagnóstico de paratuberculose foi positivo, finalmente, os outros dois, foram casos em que havia a suspeita, no entanto a paratuberculose foi descartada após as análises laboratoriais. O médico veterinário desempenha um papel muito importante nas explorações. O seu auxílio na suspeita, diagnóstico e tratamento é essencial. É importante que haja uma observação atenta dos animais para detetar os sinais clínicos e em seguida tentar diagnosticar ou descartar a infeção por Map. Nem sempre, apesar do quadro clínico ser compatível, é diagnosticada a paratuberculose. Caso esta seja detetada, a vacinação é o meio de controlo maioritariamente usado. O efeito da vacinação apesar de difícil de avaliar é maioritariamente benéfico.Paratuberculosis, also known as Johne's disease, is an infectious disorder caused by Mycobacterium avium subspecies paratuberculosis (Map). Ruminants are most affected by this disease and as a result they develop a chronic granulomatous enteritis. The clinical presentation is characterized by a progressive weight loss, which can be fatal, often accompanied by diarrhea. Medical treatment is rarely used, because it is expensive and inneficient. For this reason, vaccination is considered the most efficient method of controlling this illness. The aim of this work was to develop and consolidate the knowledge about paratuberculosis, which was achieved by monitoring and describing clinical cases of this disease. Six cases were included in this dissertation, of which two had a previous positive diagnosis of paratuberculosis and only received vaccination, other two were new cases that were submitted to sampling and received a positive diagnosis, and finally, the last two cases were suspected to have this disorder, however the differential diagnosis of paratuberculosis was excluded after laboratory testing. Veterinary practioners play a very important role on farms. Their assistance in suspicion, diagnosis and treatment is essential. It is important to have a close observation of the animals to detect the clinical signs and then try to diagnose or rule out Map infection. The positive diagnosis for paratuberculosis isn’t always obtained, even in cases with a similar clinical presentation. If this disease is detected, vaccination is the main control method used. The effect of vaccination, despite being difficult to evaluate, is mostly beneficial

Binding partners regulate unfolding of myosin VI to activate the molecular motor

Myosin VI is the only minus-end actin motor and it is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single-molecule imaging to investigate the impact of binding partners upon myosin VI molecular organization and dynamics