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Mullin, N.

Publications and source records attributed to Mullin, N..

7 recordsLinked to original sources
bioRxiv2026

Protein architectures of the bacterial spore envelope - common principles of assembly

Bacterial spores are among the most durable of cell forms, protected by robust envelopes of layered protein assemblies. Remarkably, these envelopes often share similar architectures across distantly related bacteria despite extensive divergence in their molecular components. How evolution has converged on such similar highly ordered and resilient cellular structures is relatively unexplored. Here, we combine targeted mutagenesis, cryo-electron microscopy, atomic force microscopy and structure prediction to determine the organisation and assembly of the outer spore envelope of Clostridium sporogenes, a genetically tractable surrogate for Group I Clostridium botulinum. We reveal a hierarchy of protein structures, including a semi-permeable two-dimensional crystalline exosporium; CsxA forms the exosporium scaffold with an outer "hairy nap" partially composed of BclA. We observe a previously uncharacterised multilayered three-dimensional crystalline parasporal assembly within the interspace between exosporium and coat (CsxC). These distinct structures are built from related cysteine-rich SPOCS (SpoVID-CotE-SipL)-domain proteins that self-assemble into highly ordered lattices. Such crystalline organisation, combined with high symmetry, can provide a template for local enhancement of cysteine concentration, favouring cooperative disulphide cross-linking and the formation of exceptionally stable supramolecular structures. Unexpectedly, the three-dimensional CsxC structure grows through screw dislocations, a mechanism familiar from inorganic and synthetic crystal growth but rarely demonstrated in native biological assemblies, with the exception of some biomineralisation processes. Our observations now suggest that this classical crystal-growth mechanism can be exploited in both mineralised and proteinaceous biological materials. Related SPOCS-domain proteins are implicated in spore-envelope assembly across diverse Clostridia, where they have diversified to act as both structural components and morphogenetic organisers. Remarkably, distantly related Bacilli construct similarly highly symmetric crystalline, cysteine-rich and disulphide-stabilised spore layers using proteins with very different protein folds. Thus, crystallisation and cooperative disulphide formation appear to represent a convergent physicochemical strategy for building diverse self-assembling proteins into exceptionally robust cellular assemblies. Together these findings provide the first molecular framework for understanding the organisation and assembly of the Clostridium spore envelope and reveal previously unrecognised principles governing the evolution of protective proteinaceous structures across the Bacillota (Firmicutes).

microbiology↗
bioRxiv2021

Atomic force microscopy reveals the mechanical properties of breast cancer bone metastases

Mechanically dependent processes are essential in cancer metastases. However, reliable mechanical characterisation of metastatic cancer remains challenging whilst maintaining the tissue complexity and an intact sample. Using atomic force microscopy, we quantified the micro-mechanical properties of relatively intact metastatic breast tumours and their surrounding bone microenvironment isolated from mice, and compared with other breast cancer models both ex vivo and in vitro. A unique mechanical distribution of extremely low elastic modulus and viscosity was identified on metastatic tumours, which were significantly more compliant than both 2D in vitro cultured cancer cells and subcutaneous tumour explants. The presence of mechanically distinct metastatic tumour did not result in alterations of the mechanical properties of the surrounding microenvironment at meso-scale distances (> 200 {micro}m). These findings demonstrate the utility of atomic force microscopy in studies of complex tissues and provide new insights into the mechanical properties of cancer metastases in bone.

cancer biology↗
bioRxiv2020

TET1 interacts directly with NANOG via independent regions containing hydrophobic and aromatic residues

The DNA demethylase TET1 is highly expressed in embryonic stem cells. Knockout experiments indicate that TET1 is important for lineage commitment, and paradoxically, also for reprogramming to naïve pluripotency. TET1 binds to promoters through a CXXC domain which recognises unmethylated CpG dinucleotides. TET1 also binds to enhancers, presumably via interactions with partner proteins. The transcription factor NANOG interacts with TET1 and is predominantly localised at enhancers in ESCs. Therefore, NANOG may contribute to TET1 biological activity in pluripotent cells. However, the regions of TET1 involved in protein-protein interactions are mostly unknown. Here, we characterise the physical interaction between TET1 and NANOG using embryonic stem cells and bacterial expression systems. TET1 and NANOG interact through multiple binding sites that act independently. Critically, mutating conserved hydrophobic and aromatic residues within TET1 and NANOG abolishes the interaction. Comparative ChIP-seq analysis identifies genomic loci bound by both TET1 and NANOG, that correspond predominantly to pluripotency enhancers. Importantly, around half of NANOG transcriptional target genes are associated with TET1-NANOG co-bound sites. These results indicate a mechanism by which TET1 protein is targeted to specific sites of action at enhancers by direct interaction with a transcription factor.HighlightsNANOG and TET1 have regulatory roles in maintaining and reprogramming pluripotencyTET1 and NANOG interact via multiple independent binding regionsTET1 and NANOG interactions are mediated by aromatic and hydrophobic residuesTET1 residues that bind NANOG are highly conserved in mammalsCo-localisation of TET1 and NANOG on chromatin is enriched at NANOG target genesCompeting Interest StatementThe authors have declared no competing interest.AbbreviationsDSBHdouble stranded beta helix domainESCembryonic stem cellOGTO-linked N-acetylglucosamine transferaseTETTen-eleven-translocationWRtryptophan-repeatView Full Text

biochemistry↗
bioRxiv2020

Characterisation of interactions between the pluripotency transcription factors Nanog, Oct4 and Sox2

The pluripotency transcription factors (TFs) Nanog, Sox2, and Oct4 are at the centre of the gene regulatory network that controls cell identity in embryonic stem (ES) cells. However, the mechanisms by which these factors control cell fate, and their interactions with one another are not fully understood. Here we combine biophysical and novel biochemical assays to assess how these factors interact with each other quantitatively. A confocal microscopy method to detect binding of a target protein to a fluorescently labelled partner (coimmunoprecipitated bead imaging microscopy [CBIM]) is presented and used to demonstrate homotypic binding of Nanog and heterotypic binding between Nanog and Sox2 and between Nanog and Oct4. Using fluorescence correlation spectroscopy we show that in solution, Nanog but not Oct4 or Sox2 can form homomultimers. However, both Sox2 and Oct4 can form heterotypic multimers with Nanog in a manner that depends on the presence of tryptophan residues within the Nanog tryptophan repeat. Fluorescence Cross Correlation Spectroscopy shows the affinity of Nanog for dimer formation is in the order Sox2 > Nanog > Oct4. Importantly, live cell analysis demonstrates the existence of Nanog homomultimers in vivo. Together these findings extend understanding of the molecular interactions occurring between these central mediators of the pluripotency gene regulatory network at the single-molecule level.

biochemistry↗
bioRxiv2020

Mechanical Heterogeneity in the Bone Microenvironment as Characterised by Atomic Force Microscopy

Bones are structurally heterogeneous organs with diverse functions that undergo mechanical stimuli across multiple length scales. Mechanical characterisation of the bone microenvironment is important for understanding how bones function in health and disease. Here we describe the mechanical architecture of cortical bone, the growth plate, metaphysis and marrow in fresh murine bones, probed using atomic force microscopy in physiological buffer. Both elastic and viscoelastic properties are found to be highly heterogeneous with moduli ranging over 3 to 5 orders of magnitude, both within and across regions. All regions include extremely soft areas, with moduli of a few Pascal and viscosities as low as tens Pa*s. Aging impacts the viscoelasticity of the bone marrow strongly but has limited effect on the other regions studied. Our approach provides the opportunity to explore the mechanical properties of complex tissues at the length scale relevant to cellular processes and how these impact on aging and disease. SIGNIFICANCEThe mechanical properties of biological materials at cellular scale are involved in guiding cell fate. However, there is a critical gap in our knowledge of such properties in complex tissues. The physiochemical environment surrounding the cells in in-vitro studies differs significantly from that found in vivo. Existing mechanical characterisation of real tissues are largely limited to properties at larger scales, structurally simple (e.g. epithelial monolayers) or non-intact (e.g. through fixation) tissues. In this paper, we address this critical gap and present the micro-mechanical properties of the relatively intact bone microenvironment. The measured Youngs moduli and viscosity provide a sound guidance in bioengineering designs. The striking heterogeneity at supracellular scale reveals the potential contribution of the mechanical properties in guiding cell behaviour.

biophysics↗
bioRxiv2020

Architecture and self-assembly of the Clostridium sporogenes/botulinum spore surface illustrate a general protective strategy across spore formers

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives have similar spore architectures incorporating protective proteinaceous envelopes. We reveal in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other Clostridial relatives, forms a hexagonally symmetric molecular filter. A cysteine-rich protein, CsxA, when expressed in E. coli, self-assembles into a highly thermally stable structure identical to native exosporium. Like exosporium, CsxA arrays require harsh reducing conditions for disassembly. We conclude that in vivo, CsxA self-organises into a highly resilient, disulphide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar in Bacillus spores, despite lack of protein homology. In both cases, intracellular disulphide formation is favoured by the high lattice symmetry. We propose that cysteine-rich proteins identified in distantly related spore formers may adopt a similar strategy for intracellular assembly of robust protective structures.

microbiology↗
bioRxiv2017

Distinct SoxB1 networks are required for naïve and primed pluripotency

Deletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naive pluripotency.

cell biology↗