Biology subjects

Castle, E. L.

Publications and source records attributed to Castle, E. L..

5 recordsLinked to original sources

bioRxiv2023

Time- and lineage-resolved transcriptional profiling uncovers gene expression programs and clonal relationships that underlie human T lineage specification

T cells develop from multi-potent hematopoietic progenitors in the thymus and provide adaptive protection against pathogens and cancer. However, the emergence of human T cell-competent blood progenitors, and their subsequent specification to the T lineage, has been challenging to capture in real time. Here, we leveraged a pluripotent stem cell differentiation system to understand the transcriptional dynamics and cell fate restriction events that underlie this critical developmental process. Time-resolved single cell RNA sequencing revealed that cell-cycle exit, downregulation of the multipotent hematopoietic program, and upregulation of >90 lineage-associated transcription factors all occur within a highly co-ordinated and narrow developmental window. Computational gene-regulatory network inference elucidated the transcriptional logic of T lineage specification, uncovering an important role for YBX1. We mapped the differentiation cell fate hierarchy using transcribed lineage barcoding and mathematical trajectory inference and discovered that mast and myeloid potential bifurcate from each other early in haematopoiesis, upstream of T lineage restriction. Collectively, our analyses provide a quantitative, time-resolved model of human T cell specification with relevance for regenerative medicine and developmental immunology.

immunology↗

bioRxiv2021

DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8{beta}+, CD4-, CD3+, TCR{beta}+ T cells from PSCs.

bioengineering↗

bioRxiv2021

A viral protein utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies

Kaposis sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposis sarcoma (KS). We previously demonstrated that expression of the KSHV protein, Kaposin B (KapB), promoted inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). Processing bodies (PBs) modify gene expression by silencing or degrading labile messenger RNAs (mRNAs) including many transcripts that encode inflammatory or angiogenic proteins that are associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via the phosphorylation of the autophagy regulatory protein, Beclin 1. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin 1 phosphorylation or autophagic flux. Moreover, we reveal that PB disassembly mediated by KSHV or KapB depended on canonical autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection. Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of the inflammatory, endothelial cell cancer, Kaposis Sarcoma (KS). KSHV induces a pro-tumourigenic inflammatory environment which aids in the establishment and maintenance of the KS lesion. Processing bodies (PBs) are cellular structures that dampen inflammatory gene expression by suppression or decay of their cognate RNA molecules. We previously showed that the viral protein KapB caused PB disappearance during KSHV infection, identifying a new pathway used by KSHV to elicit inflammation. Now, we show that KSHV and KapB hijack the cellular degradative process of autophagy to promote PB disassembly and increase inflammatory gene expression. This places autophagy as central to the regulation of inflammation by KSHV and illustrates another remarkable strategy used by these viruses to create a tumourigenic microenvironment.

microbiology↗

bioRxiv2020

Human coronaviruses disassemble processing bodies

A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this report, no information was known about how human coronaviruses (hu CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold hu CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that N protein-mediated PB loss correlated with elevated RNA for PB-localized transcripts encoding TNF and IL-6. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV-1) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to enhanced proinflammatory cytokine production observed during severe SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Viral manipulation of a novel mechanoresponsive signalling axis disassembles processing bodies

Processing bodies (PBs) are ribonucleoprotein granules that suppress cytokine mRNA translation that are targeted for disassembly by many viruses. Kaposis sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposis sarcoma, and a PB-regulating virus. The virus encodes Kaposin B (KapB), which induces actin stress fibres (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB and mechanoresponsive PB disassembly also required YAP. Using the viral protein KapB, we identified a new consequence of the exposure of cells to mechanical forces that alter actin dynamics and activate YAP, namely the disassembly of PBs. ImportanceFor the first time, we demonstrate that processing bodies (PBs), cytoplasmic sites of RNA decay, are regulated by mechanical signaling events that alter actin dynamics and that this requires the mechanoresponsive transcription factor, YAP. Using the overexpression of a viral protein called KapB, known previously to mediate PB disassembly, we show that actin stress fibers (SFs) and the mechanoresponsive transcription factor, YAP, are required for PB loss. We also show that other established mechanical signals (shear stress or stiff extracellular matrix) that lead to the formation of SFs and activate YAP also cause PB disassembly. This is important because it means that KapB activates, from the inside out, a pathway that links cell shape to post-transcriptional gene regulation via cytoplasmic PBs.

cell biology↗