Biology subjects

Smith, C. W.

Publications and source records attributed to Smith, C. W..

7 recordsLinked to original sources

bioRxiv2023

Damage-induced pyroptosis drives endogenous thymic regeneration viainduction of Foxn1 by purinergic receptor activation

Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression of Foxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTP{gamma}S promotes rapid regeneration of the thymus in vivo following acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration. SUMMARYO_LIThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration. C_LIO_LIDamage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes. C_LIO_LIElevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage. C_LIO_LIExtracellular ATP release promotes Foxn1 expression in cTECs via activation of P2Y2 C_LIO_LITherapeutic targeting of the P2Y2 receptor promotes thymic regeneration. C_LI

immunology↗

bioRxiv2020

Attenuation of homeostatic signaling from apoptotic thymocytes triggers a global regenerative response in the thymus

The molecular triggers of organotypic tissue repair are unknown. The thymus, which is the primary site of T cell development, is a model of tissue damage and regeneration as it is particularly sensitive to insult, but also has a remarkable capacity for repair. However, acute and profound damage, such as that caused by common cytoreductive therapies or age-related decline, lead to involution of the thymus and prolonged T cell deficiency, precipitating life-threatening infections and malignant relapse. Consequently, there is an unmet need to boost thymic function and enhance T cell immunity. Here, we demonstrate an innate trigger of the reparative response in the thymus, centered on the attenuation of signaling directly downstream of apoptotic cell detection as thymocytes are depleted after acute damage. We found that the intracellular pattern recognition receptor NOD2, via induction of microRNA-29c, suppressed the induction of the regenerative factors IL-23 and BMP4, from thymic dendritic cells (DCs) and endothelial cells (ECs), respectively. During steady-state, when a high proportion of thymocytes are undergoing apoptosis (as a consequence of selection events during T cell development), this suppressive pathway is constitutively activated by the detection of exposed phosphatidylserine on apoptotic thymocytes by cell surface TAM receptors on DCs and ECs, with subsequent downstream activation of the Rho GTPase Rac1. However, after damage, when profound cell depletion occurs across the thymus, the TAM-Rac1-NOD2-miR29c pathway is abrogated, therefore triggering the increase in IL-23 and BMP4 levels. Importantly, this pathway could be modulated pharmacologically by inhibiting Rac1 GTPase activation with the small molecule inhibitor EHT1864, leading to increased thymic function and T cell recovery after acute damage. In conclusion, our work not only represents a novel regenerative strategy for restoring immune competence in patients whose thymic function has been compromised due to cytoreductive conditioning, infection, or age; but also, identifies a mechanism by which tissue regenerative responses are triggered.

immunology↗

bioRxiv2020

An adaptable analysis workflow for characterization of platelet spreading and morphology

The assessment of platelet spreading through light microscopy, and the subsequent quantification of parameters such as surface area and circularity, is a key assay for many platelet biologists. Here we present an analysis workflow which robustly segments individual platelets to facilitate the analysis of large numbers of cells while minimising user bias. Image segmentation is performed by interactive learning and touching platelets are separated with an efficient semi-automated protocol. We also use machine learning methods to robustly automate the classification of platelets into different subtypes. These adaptable and reproducible workflows are made freely available and are implemented using the open source software KNIME and ilastik.

bioinformatics↗

bioRxiv2019

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation, loss of which results in severe macrothrombocytopenia and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

biochemistry↗

bioRxiv2019

Identification of RBPMS as a smooth muscle master splicing regulator via proximity of its gene with super-enhancers

Alternative splicing (AS) programs are primarily controlled by regulatory RNA binding proteins (RBPs). It has been proposed that a small number of master splicing regulators might control cell-specific splicing networks and that these RBPs could be identified by proximity of their genes to transcriptional super-enhancers. Using this approach we identified RBPMS as a critical splicing regulator in differentiated vascular smooth muscle cells (SMCs). RBPMS is highly down-regulated during phenotypic switching of SMCs from a contractile to a motile and proliferative phenotype and is responsible for 20% of the AS changes during this transition. RBPMS directly regulates AS of numerous components of the actin cytoskeleton and focal adhesion machineries whose activity is critical for SMC function in both phenotypes. RBPMS also regulates splicing of other splicing, post-transcriptional and transcription regulators including the key SMC transcription factor Myocardin, thereby matching many of the criteria of a master regulator of AS in SMCs.

molecular biology↗

bioRxiv2018

Lamin A/C controls nuclear matrin-3 levels and localization, but not alternative splicing of cassette exons

Disruptions in connections between the nuclear lamina and nuclear matrix occur in myopathic disorders. However, the biological significance of nuclear lamina - nuclear matrix coupling still remains largely undetermined. Previously it has been demonstrated that the nuclear matrix protein, matrin-3, binds to lamin A/C and this interaction is disrupted in laminopathies resulting in enhanced separation between the lamina and matrix. Matrin-3 has recently been identified as a core regulator of alternative splicing, whereas the involvement of lamin A/C in splicing still remains controversial. In this study, we demonstrate that lamin A/C is not only required for maintaining the nuclear organization of matrin-3, but also of other splicing activators and small nuclear ribonucleoproteins (snRNP) components. Interestingly, mis-localization of these splicing components did not appear to significantly disrupt alternative splicing events of cassette exons regulated by matrin-3. Thus, the lamin A/C-matrin3 interaction is unlikely to be involved in controlling alternative splicing but could be important in coordinating other nuclear activities. Interestingly, matrin-3 knock-down results in misshapen nuclei suggesting its interaction with lamin A/C maybe important in maintaining nuclear structural integrity.

cell biology↗

bioRxiv2018

Heteromeric RNP assembly at LINEs controls lineage-specific RNA processing

It is challenging for RNA processing machineries to select exons within long intronic regions. We find that intronic LINE repeat sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs). This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites in LINEs. Both RBPs repress splicing and 3 end processing within and around LINEs, as demonstrated in cultured human cells and mouse brain. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are confined to deep intronic regions. These RBPs insulate both LINEs and surrounding regions from RNA processing. Upon evolutionary divergence, gradual loss of insulation diversifies the roles of LINEs. Older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for assembly of repressive RBPs, and contribute to evolution of new, lineage-specific transcripts in mammals.

genomics↗