Biology subjects

Richardson, J.

Publications and source records attributed to Richardson, J..

27 records · Page 2Linked to original sources

bioRxiv2021

Single-domain antibodies for targeting, detection and in vivo imaging of human CD4+ cells

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ T cells in regulating immunological processes, we generated novel single-domain antibodies (nanobodies, Nbs) that specifically recognize human CD4. After in depth analysis of their binding properties, recognized epitopes, and effects on T cell proliferation, activation and cytokine release, we selected CD4 Nbs that did not interfere with crucial T cell processes in vitro and converted them into immune tracers for non-invasive molecular imaging. By optical imaging, we demonstrate the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, time-resolved immune positron emission tomography (immunoPET) of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb in CD4+ T cell-rich tissues. We propose that the CD4 Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

immunology↗

bioRxiv2021

Notch controls the cell cycle to define leader versus follower identities during collective cell migration.

Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, as cancer. How cell cycle progression affects migration, and vice-versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish Trunk Neural Crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. We propose that migratory behaviours are defined through the interaction of Notch signalling and cell cycle progression.

developmental biology↗

bioRxiv2021

MBNL1 and RBFOX1 co-regulate alternative splicing events transcriptome-wide through a conserved buffering mechanism

Alternative splicing (AS) is controlled by cis-regulatory elements recognized by networks of trans-acting factors. Here we investigate modes and mechanisms of AS co-regulation by MBNL1 and RBFOX1, two RNA binding proteins (RBPs) critical for developmental AS transitions. We generated two cell models that express each RBP under separate inducible promoters. Transcriptome-wide categorization of the impacts of RBFOX1 expression on MBNL1 splicing revealed a common co-regulatory mode through which RBFOX1 buffers MBNL1 dose-dependent splicing regulation by reducing the total range of exon inclusion or exclusion. Minigene mutational analysis and in vitro binding experiments suggest that this buffering mechanism occurs through a shared cis-regulatory element previously unidentified as critical for MBNL1-dependent activity. Overall, our studies define a conserved co-regulatory mechanism through which RBFOX1 and MBNL1 can fine-tune and provide redundancy for AS outcomes. These studies indicate overlapping use of RNA motifs with potential implications for when activity of RBPs is disrupted.

biochemistry↗

bioRxiv2020

Making the invisible enemy visible

During the COVID-19 pandemic, structural biologists rushed to solve the structures of the 28 proteins encoded by the SARS-CoV-2 genome in order to understand the viral life cycle and enable structure-based drug design. In addition to the 204 previously solved structures from SARS-CoV-1, 548 structures covering 16 of the SARS-CoV-2 viral proteins have been released in a span of only 6 months. These structural models serve as the basis for research to understand how the virus hijacks human cells, for structure-based drug design, and to aid in the development of vaccines. However, errors often occur in even the most careful structure determination - and may be even more common among these structures, which were solved quickly and under immense pressure. The Coronavirus Structural Task Force has responded to this challenge by rapidly categorizing, evaluating and reviewing all of these experimental protein structures in order to help downstream users and original authors. In addition, the Task Force provided improved models for key structures online, which have been used by Folding@Home, OpenPandemics, the EU JEDI COVID-19 challenge and others.

molecular biology↗

bioRxiv2020

A conserved cell division protein directly regulates FtsZ dynamics in filamentous and unicellular actinobacteria

Bacterial cell division is driven by the polymerization of the GTPase FtsZ into a contractile structure, the so-called Z-ring. This essential process involves proteins that modulate FtsZ dynamics and hence the overall Z-ring architecture. Actinobacteria, like Streptomyces and Mycobacterium lack known key FtsZ-regulators. Here we report the identification of SepH, a conserved actinobacterial protein that directly regulates FtsZ dynamics. We show that SepH is crucially involved in cell division in Streptomyces and that it binds FtsZ via a conserved helix-turn-helix motif, stimulating the assembly of FtsZ protofilaments. Comparative in vitro studies using the SepH homolog from Mycobacterium further reveal that SepH can also bundle FtsZ protofilaments, indicating an additional Z-ring stabilizing function in vivo. We propose that SepH plays a crucial role at the onset of cytokinesis in actinobacteria by promoting the rapid assembly of FtsZ filaments into division-competent Z-rings that can go on to mediate septum synthesis.

microbiology↗

bioRxiv2020

Repeat length increases disease penetrance and severity in C9orf72 ALS/FTD BAC transgenic mice

C9orf72 ALS/FTD patients show remarkable clinical heterogeneity, but the complex biology of the repeat expansion mutation has limited our understanding of the disease. BAC transgenic mice were used to better understand the molecular mechanisms and repeat length effects of C9orf72 ALS/FTD. Genetic analyses of these mice demonstrate that the BAC transgene and not integration site effects cause ALS/FTD phenotypes. Transcriptomic changes in cell proliferation, inflammation and neuronal pathways are found late in disease and alternative splicing changes provide early molecular markers that worsen with disease progression. Isogenic sublines of mice with 800, 500 or 50 G4C2 repeats generated from the single-copy C9-500 line show longer repeats result in earlier onset, increased disease penetrance, and increased levels of RNA foci and RAN aggregates. These data demonstrate G4C2 repeat length is an important driver of disease and identify alternative splicing changes as early biomarkers of C9orf72 ALS/FTD.

neuroscience↗

bioRxiv2019

Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection.

Tick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-eastern Asia. TBEV tropism is critical for neuropathogenesis, yet, little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis. We showed that infection of neuronal/glial cultures derived from human fetal neural progenitor cells (hNPCs) mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death and astrogliosis. We also showed that these cells had conserved their capacity to build an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures in either human neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV. Our results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They offer a new in vitro model to further study TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells.\n\nAuthor summaryTick-borne encephalitis virus (TBEV), a neurotropic Flavivirus that is responsible for encephalitis in humans, is of growing concern in Europe. Indeed, over the last two decades the number of reported cases has continuously increased and the virus has spread into new geographical areas. Whereas it is well established that neurons are the main target of TBEV in the human brain, the mechanisms that underlie this preferential tropism have not yet been elucidated. Here, we used neuronal/glial cells derived from human fetal neural progenitors to establish and characterize a new in vitro pathological model that mimics major hallmarks of TBEV infection in vivo; namely, neuronal tropism, neuronal death and astrogliosis. Using this highly relevant model, we showed that human neurons and astrocytes were both capable of developing an innate immune response against TBEV, but with dissimilar magnitudes that correlated with differential susceptibility to TBEV. Our results thus revealed that TBEV tropism for subsets of human brain cells is likely to depend on cell-type specific innate immunity. This improves our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells and may help guide development of future therapies.

microbiology↗

bioRxiv2019

The immunomodulatory effect of IrSPI, a tick salivary gland serine protease inhibitor involved in Ixodes ricinus tick feeding

Ticks are strict hematophagous arthropods and are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Moreover, they are second only to mosquitoes as vectors of human pathogens. Hard tick feeding is a slow process--taking up to several days for repletion prior to detachment--and necessitates extended control over the host response. The success of the feeding process depends upon injection of saliva by tick, which not only controls host haemostasis and wound healing, but also subverts the host immune response to avoid tick rejection during this long-lasting process. In turn, the manipulation of the host immune response creates a favourable niche for the survival and propagation of diverse tick-borne pathogens transmitted during feeding. Here, we report on the molecular and biochemical features and functions of IrSPI, an Ixodes ricinus salivary serine protease inhibitor involved in blood meal acquisition. Our results show that IrSPI harbours the typical conformational fold of Kunitz type I serine protease inhibitors and that it functionally inhibits the elastase and, to a lesser extent, chymotrypsin. We also show that IrSPI is injected into the host during feeding. Crucially, we found that IrSPI has no impact on tissue factor pathway-induced coagulation, fibrinolysis, apoptosis, or angiogenesis, but a strong effect on immune cells. IrSPI affects antigen-presenting macrophages by hampering IL-5 production. In addition, IrSPI represses proliferation of mitogen-stimulated CD4+ cells. The inhibition of T cell proliferation was associated with marked reductions in pro-inflammatory cytokine secretion. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in successful I. ricinus tick feeding.\n\nAuthor summaryTicks are the most important vector influencing both human and animal health in Europe, where Ixodes ricinus is the most abundant tick species. Ticks feed on animal or human blood for an extended period, during which their saliva allows both feeding and pathogen transmission by interfering with native host responses. A better understanding of tick-host-pathogen interactions is central to the discovery of improved control methods. Within this context, we previously identified IrSPI as an I. ricinus salivary molecule implicated in both tick feeding and bacterial transmission. This serine protease inhibitor was almost characterised as an elastase inhibitor, and here, we show IrSPI overexpression in several tick organs--especially salivary glands--during blood feeding. We demonstrate that IrSPI is injected into the host through saliva, and despite having no impact on endothelial cell angiogenesis or apoptosis during blood feeding, we report an immunomodulatory role, whereby CD4+ T lymphocyte proliferation is repressed and where the cytokine secretion pattern of both splenocytes and macrophages is modified. Our study provides new insights into the complex armament developed by ticks to overcome the host response, and uncovers a potential vaccine target for disruption of feeding processes and pathogen transmission.

animal behavior and cognition↗

bioRxiv2017

The Odd One Out: Aabidopsis Reticulon 20 Has A Role In Lipid Biosynthesis

The family of reticulon proteins has been shown to be involved in a variety of functions in eukaryotic cells including tubulation of the endoplasmic reticulum (ER), formation of cell plates and primary plasmodesmata. Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the reticulons sitting in the membrane in a W-topology. Here we report on a subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. We show that reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue reticulon 19 labels the whole ER. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in the roots indicating a role in lipid biosynthesis or regulation. A third homologue in this family - 3BETAHSD/D1- is localised to ER exit sites resulting in an intriguing location difference for the three proteins.

plant biology↗