Regulation of Nuclear Mechanics and the Impact on DNA Damage.

In eukaryotic cells, the nucleus houses the genomic material of the cell. The physical properties of the nucleus and its ability to sense external mechanical cues are tightly linked to the regulation of cellular events, such as gene expression. Nuclear mechanics and morphology are altered in many diseases such as cancer and premature ageing syndromes. Therefore, it is important to understand how different components contribute to nuclear processes, organisation and mechanics, and how they are misregulated in disease. Although, over the years, studies have focused on the nuclear lamina-a mesh of intermediate filament proteins residing between the chromatin and the nuclear membrane-there is growing evidence that chromatin structure and factors that regulate chromatin organisation are essential contributors to the physical properties of the nucleus. Here, we review the main structural components that contribute to the mechanical properties of the nucleus, with particular emphasis on chromatin structure. We also provide an example of how nuclear stiffness can both impact and be affected by cellular processes such as DNA damage and repair.

DNA damage alters nuclear mechanics through chromatin reorganization.

DNA 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.

Competition between two high- and low-affinity protein-binding sites in myosin VI controls its cellular function.

Myosin VI is involved in many cellular processes ranging from endocytosis to transcription. This multifunctional potential is achieved through alternative isoform splicing and through interactions of myosin VI with a diverse network of binding partners. However, the interplay between these two modes of regulation remains unexplored. To this end, we compared two different binding partners and their interactions with myosin VI by exploring the kinetic properties of recombinant proteins and their distribution in mammalian cells using fluorescence imaging. We found that selectivity for these binding partners is achieved through a high-affinity motif and a low-affinity motif within myosin VI. These two motifs allow competition among partners for myosin VI. Exploring how this competition affects the activity of nuclear myosin VI, we demonstrate the impact of a concentration-driven interaction with the low-affinity binding partner DAB2, finding that this interaction blocks the ability of nuclear myosin VI to bind DNA and its transcriptional activity in vitro We conclude that loss of DAB2, a tumor suppressor, may enhance myosin VI-mediated transcription. We propose that the frequent loss of specific myosin VI partner proteins during the onset of cancer leads to a higher level of nuclear myosin VI activity.

NDP52 activates nuclear myosin VI to enhance RNA polymerase II transcription.

Myosin VI (MVI) has been found to be overexpressed in ovarian, breast and prostate cancers. Moreover, it has been shown to play a role in regulating cell proliferation and migration, and to interact with RNA Polymerase II (RNAPII). Here, we find that backfolding of MVI regulates its ability to bind DNA and that a putative transcription co-activator NDP52 relieves the auto-inhibition of MVI to enable DNA binding. Additionally, we show that the MVI-NDP52 complex binds RNAPII, which is critical for transcription, and that depletion of NDP52 or MVI reduces steady-state mRNA levels. Lastly, we demonstrate that MVI directly interacts with nuclear receptors to drive expression of target genes, thereby suggesting a link to cell proliferation and migration. Overall, we suggest MVI may function as an auxiliary motor to drive transcription.

Atomic Force Microscopy micro-rheology reveals large structural inhomogeneities in single cell-nuclei.

During growth, differentiation and migration of cells, the nucleus changes size and shape, while encountering forces generated by the cell itself and its environment. Although there is increasing evidence that such mechanical signals are employed to control gene expression, it remains unclear how mechanical forces are transduced through the nucleus. To this end, we have measured the compliance of nuclei by applying oscillatory strains between 1 and 700 Hz to individual nuclei of multiple mammalian cell-lines that were compressed between two plates. The quantitative response varied with more than one order of magnitude and scaled with the size of the nucleus. Surprisingly, the qualitative behaviour was conserved among different cell-lines: all nuclei showed a softer and more viscous response towards the periphery, suggesting a reduced degree of crosslinking of the chromatin. This may be an important feature to regulate transcription via mechano-transduction in this most active and dynamic region of the nucleus.

Oligomerization domains in the glycan-binding receptors DC-SIGN and DC-SIGNR: Sequence variation and stability differences.

Human dendritic cell-specific intercellular adhesion molecule-1 grabbing nonintegrin, DC-SIGN, and the sinusoidal endothelial cell receptor DC-SIGNR or L-SIGN, are closely related sugar-binding receptors. DC-SIGN acts both as a pathogen-binding endocytic receptor and as a cell adhesion molecule, while DC-SIGNR has only the pathogen-binding function. In addition to differences in the sugar-binding properties of the carbohydrate-recognition domains in the two receptors, there are sequence differences in the adjacent neck domains, which are coiled-coil tetramerization domains comprised largely of 23-amino acid repeat units. A series of model polypeptides consisting of uniform repeat units have been characterized by gel filtration, differential scanning calorimetry and circular dichroism. The results demonstrate that two features characterize repeat units which form more stable tetramers: a leucine reside in the first position of the heptad pattern of hydrophobic residues that pack on the inside of the coiled coil and an arginine residue on the surface of the coiled coil that forms a salt bridge with a glutamic acid residue in the same polypeptide chain. In DC-SIGNR from all primates, very stable repeat units predominate, so the carbohydrate-recognition domains must be held relatively closely together. In contrast, stable repeat units are found only near the membrane in DC-SIGN. The presence of residues that disrupt tetramer formation in repeat units near the carbohydrate-recognition domains of DC-SIGN would allow these domains to splay further apart. Thus, the neck domains of DC-SIGN and DC-SIGNR can contribute to the different functions of these receptors by presenting the sugar-binding sites in different contexts.

Organization of the extracellular portion of the macrophage galactose receptor: a trimeric cluster of simple binding sites for N-acetylgalactosamine.

The properties of the human macrophage galactose receptor have been investigated. Specificity for N-acetylgalactosamine (GalNAc) residues with exposed 3- and 4-hydroxyl groups explains virtually all of the results obtained from a recently expanded array of synthetic glycans and is consistent with a model for the structure of the binding site. This simple interaction is sufficient to explain the ability of the receptor to bind to tumor-cell glycans bearing Tn and sialyl-Tn antigens, but not to more elaborate O-linked glycans that predominate on normal cells. This specificity also allows for binding of parasite glycans and screening of an array of bacterial outer membrane oligosaccharides confirms that the receptor binds to a subset of these structures with appropriately exposed GalNAc residues. A key feature of the receptor is the clustering of binding sites in the extracellular portion of the protein, which retains the trimeric structure observed in the cell membrane. Chemical crosslinking, gel filtration, circular dichroism analysis and differential scanning calorimetry demonstrate that this trimeric structure of the receptor is stabilized by an α-helical coiled coil that extends from the surface of the membrane to the globular carbohydrate-recognition domains. The helical neck domains form independent trimerization domains. Taken together, these results indicate that the macrophage galactose receptor shares many of the features of serum mannose-binding protein, in which clusters of monosaccharide-binding sites serve as detectors for a simple epitope that is not common on endogenous cell surface glycans but that is abundant on the surfaces of tumor cells and certain pathogens.