Biology subjects

McMillan, P.

Publications and source records attributed to McMillan, P..

7 recordsLinked to original sources

bioRxiv2026

Phase-Separated RNA Condensates Govern Cas13 Target Accessibility and Cleavage

RNA-guided CRISPR-Cas13 nucleases must efficiently locate and target specific transcripts amidst the millions of other RNA molecules that are spatially regulated in the cell. Yet the mechanisms by which Cas13 finds its targets within this crowded, compartmentalized environment remain elusive. Here, we show that diverse Cas13 orthologs assemble into distinct cytoplasmic granules in a crRNA-concentration dependent manner. These granules exhibit hallmark features of phase-separated condensates, displaying liquid-like viscoelasticity and dynamic molecular exchange with the cytoplasm, as demonstrated by FRAP analysis in both human and bacterial cells. Molecular profiling revealed that Cas13 co-localizes with polyadenylated RNAs and noncoding RNAs within condensates that display markers of canonical RNA granules. Biochemical purification coupled with RNA sequencing shows Cas13 associated with thousands of transcripts within condensates, likely mediated by electrostatic interactions with its positively charged surface. Notably, Cas13 retains catalytic activity within these condensates, efficiently cleaving co-localized targets, whereas RNA species excluded from the condensates remain largely protected from Cas13 cleavage. This data indicates that condensate-based spatial organization facilitates efficient sampling and binding of diverse RNA targets by concentrating Cas13 and its substrates within a confined, liquid-like compartment. Together, our findings uncover a conserved spatial mechanism regulating Cas13 activity across bacterial and mammalian cells, where localization within RNA dense granules governs Cas13 activity in cells.

molecular biology↗

bioRxiv2025

Exploring chemotaxis in spatio-temporal stoichiometric models of metabolism: Pseudomonas simiae as a case study

Summary/AbstractChemotaxis is the movement of cells or organisms in the direction of an increasing or decreasing gradient of a chemical, i.e. a chemoattractant or chemorepellent, respectively. In bacteria, this movement is typically manifested through the presence of bias in the random walk generated by run-and-tumble motility. Often, the chemoattractant is consumed by the organism, thus coupling the biophysical, directed motion with the biochemical, metabolic activity in the growing bacterial colony. In this chapter, we describe our approach to simulating chemotaxis, coupled with detailed metabolic activity, within the framework of our modeling software COMETS (Computation of Microbial Ecosystem in Time and Space). By combining genome-scale metabolic models, dynamic flux balance analysis and the newly implemented biophysical chemotaxis model, we built and qualitatively tested a chemotaxis module for COMETS. We applied this model to an automatically constructed genome-scale model of the plant commensal bacterium Pseudomonas simiae WCS417, which we hypothesized would perform chemotaxis on amino acids similar to its close relative Pseudomonas putida. COMETS simulations recapitulated the typical ring colony pattern, which is due to propagation at the colony front, indicative of bacteria growing on a chemoattractant-filled plate. By allowing for detailed examination of spatial distributions and time dynamics of the biomass, as well as concentration profiles of the metabolites involved in the growth and chemotaxis in bacterial colonies, the software method described here enables simulations of complex microbial patterns involving metabolism and directed motility. Beyond testing the appearance of the ring, future model validation could include comparisons of local growth rates and metabolic fluxes, including rates of metabolite uptake and secretion. The newly added COMETS chemotaxis capability integrates flux balance modeling with gradient-induced biomass propagation, paving the way for more realistic simulations of microbial ecosystem dynamics in complex environments, such as plant roots, where chemotaxis may play a fundamental role in microbiome-host dynamics.

systems biology↗

bioRxiv2025

Bringing the Ends together: Cryo-EM structures of mycobacterial Ku in complex with DNA define its role in NHEJ synapsis

Non-homologous end joining (NHEJ) is the sole pathway for repairing double-strand breaks in Mycobacterium tuberculosis during dormancy, relying on mycobacterial Ku (mKu) and ligase D, with mKu as the rate-limiting factor. Despite its essential role, the lack of structural information on prokaryotic Ku has hindered understanding of the molecular mechanisms underlying bacterial two-component NHEJ machinery. Here, we present the first cryo-EM structures of mKu in DNA-bound and higher-order super-complex forms, revealing a Ku-mediated DNA synapsis mechanism unique to prokaryotes. Integrating cryo-EM with hydrogen-deuterium exchange mass spectrometry, we define key mKu-mKu dimerization, DNA-binding, and synapsis interactions essential for efficient NHEJ, bridging structure with function. Structure-guided in silico mutagenesis, coupled with electrophoretic mobility shift assays, identifies residues essential for DNA binding and synaptic assembly, which are crucial for NHEJ. Forster resonance energy transfer confirms DNA-dependent mKu oligomerization in solution, while live-cell imaging captures its spatiotemporal dynamics during DSB repair. These findings provide fundamental insights into the architecture and function of prokaryotic NHEJ, positioning mKu as a potential therapeutic target against tuberculosis and offering a framework for understanding DNA repair across bacterial species.

biochemistry↗

bioRxiv2024

TMEM106B C-terminal fragments aggregate and drive neurodegenerative proteinopathy.

Genetic variation in the lysosomal and transmembrane protein 106B (TMEM106B) modifies risk for a diverse range of neurodegenerative disorders, especially frontotemporal lobar degeneration (FTLD) with progranulin (PGRN) haplo-insufficiency, although the molecular mechanisms involved are not yet understood. Through advances in cryo-electron microscopy (cryo-EM), homotypic aggregates of the C-Terminal domain of TMEM106B (TMEM CT) were discovered as a previously unidentified cytosolic proteinopathy in the brains of FTLD, Alzheimers disease, progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB) patients. While it remains unknown what role TMEM CT aggregation plays in neuronal loss, its presence across a range of aging related dementia disorders indicates involvement in multi-proteinopathy driven neurodegeneration. To determine the TMEM CT aggregation propensity and neurodegenerative potential, we characterized a novel transgenic C. elegans model expressing the human TMEM CT fragment constituting the fibrillar core seen in FTLD cases. We found that pan-neuronal expression of human TMEM CT in C. elegans causes neuronal dysfunction as evidenced by behavioral analysis. Cytosolic aggregation of TMEM CT proteins accompanied the behavioral dysfunction driving neurodegeneration, as illustrated by loss of GABAergic neurons. To investigate the molecular mechanisms driving TMEM106B proteinopathy, we explored the impact of PGRN loss on the neurodegenerative effect of TMEM CT expression. To this end, we generated TMEM CT expressing C. elegans with loss of pgrn-1, the C. elegans ortholog of human PGRN. Neither full nor partial loss of pgrn-1 altered the motor phenotype of our TMEM CT model suggesting TMEM CT aggregation occurs downstream of PGRN loss of function. We also tested the ability of genetic suppressors of tauopathy to rescue TMEM CT pathology. We found that genetic knockout of spop-1, sut-2, and sut-6 resulted in weak to no rescue of proteinopathy phenotypes, indicating that the mechanistic drivers of TMEM106B proteinopathy may be distinct from tauopathy. Taken together, our data demonstrate that TMEM CT aggregation can kill neurons. Further, expression of TMEM CT in C. elegans neurons provides a useful model for the functional characterization of TMEM106B proteinopathy in neurodegenerative disease.

neuroscience↗

bioRxiv2021

Identification and characterisation of splicing regulators in Toxoplasma gondii

The splicing of mRNA constitutes a major source of co- and post-transcriptional regulation in metazoans. In particular, members of the serine/arginine (SR) protein family are essential splicing factors that are implicated in the regulation of gene expression and RNA metabolism. However, very little is known about these proteins in apicomplexans, a phylum that includes some of the most important global parasites. In this study, we investigated the suite of three uncharacterised SR proteins in Toxoplasma gondii and show that all three are found localised to nuclear speckles. We show, by genetic ablation, that TgSR1 is particularly important for T. gondii growth. Using RNA-seq, we also characterised the global gene expression and splicing regulation of these proteins. We find that the SR proteins regulate several types of alternative splicing of distinct but overlapping subsets of transcripts, as well as impacting transcript abundance. Most of the alternative splicing events are non-productive intron retention events that do not appear to affect transcript abundance. The splicing sites of the impacted transcripts are enriched in characteristic SR binding motifs. We also identified and conditionally knocked down two putative kinases of SR proteins. The kinases are localised to nuclear speckles and are essential to parasite survival. Their perturbation resulted in widespread changes to splicing, but the affected transcripts did not mirror the patterns seen in knockouts of individual SRs, suggesting an absence of a simple relationship between SRs and these putative kinase regulators. Overall, this study reveals a complex system of splicing factors and kinases that post-transcriptionally regulate gene expression in T. gondii.

cell biology↗

bioRxiv2019

The Plasmodium falciparum protein VCAP1 controls Maurer’s cleft morphology, knob architecture and PfEMP1 trafficking

The malaria parasite, Plasmodium falciparum, traffics the virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurers clefts. We developed a method for efficient enrichment of Maurers clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterised or poorly characterised Maurers cleft proteins. We generated transfectants expressing GFP-fusions of 7 proteins and confirmed their Maurers cleft location. Using co-immunoprecipitation and mass spectrometry we have generated a protein interaction map of proteins at the Maurers clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurers clefts. We focus on a putative PfEMP1 loading complex that includes the newly characterised virulence complex assembly protein 1 (VCAP1). Disruption of VCAP1 causes Maurers cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression and loss of the PfEMP1 directed adhesion. {Delta}VCAP1 parasite lines have a growth advantage compared to wildtype parasites; and the infected RBCs are more deformable and more osmotically fragile.\n\nImportanceThe trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite infected RBCs is central to severe adhesion related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurers clefts controlling PfEMP1 loading and unloading. This work adds significantly to our understanding of virulence protein trafficking and will provide crucial knowledge that will be required to determine the mechanisms underpinning parasite driven host cell remodelling, parasite survival within the host and virulence mechanisms.

microbiology↗

bioRxiv2018

Stromal fibroblast activation and inflammation in frozen shoulder

IntroductionFrozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement. Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the stromal compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder,\n\nMethodsShoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Stromal activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1{beta} upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR.\n\nResultsImmunohistochemistry demonstrated increased expression of stromal activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1{beta}) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM).\n\nConclusionsThese results show that stromal fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated stromal fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

immunology↗