Biology subjects

Kwok, S.

Publications and source records attributed to Kwok, S..

9 recordsLinked to original sources

bioRxiv2024

BMP signalling attenuates intercellular adhesion to drive mesenchyme migration during fin fold morphogenesis

The development of the vertebrate limb bud is a multifactorial process involving the coordinated activity of diverse signalling pathways and cell types. BMP signalling has been consistently shown to play an integral role during the patterning of mouse and chick limbs. However, the molecular mechanisms and cellular processes underlying its contribution to limb development are poorly understood. Using zebrafish embryos, we demonstrate a novel role of BMPs in driving median fin fold morphogenesis, an evolutionary related structure. Our findings indicated that a graded BMP signal is established in the emerging fin fold along the proximo-distal axis, with the fin mesenchymal cells being identified as the primary target of the signalling cue. Pharmacological and genetic ablation of BMP signalling in the fin fold inhibited the normal migration of the mesenchymal cells into the fin fold. The observed inhibition of migration was accompanied by the elongation in mesenchymal cell shape, which hinted at the possibility of altered physical forces acting on the cell population. We observed that loss of BMP signalling led to the cellular re-distribution and altered membrane dynamics of N-cadherin in the fin mesenchymal cells, which manifested as an increase in homotypic cell adhesion. This change in cell-adhesion serves as the mechanistic basis for the observed defect in migration. Furthermore, inability to detach due to loss of BMP signalling occurs independently of sphingosine-1-phosphate signalling that generates a gradient of mesenchyme adhesion to the underlying matrix. Overall, this study highlights the cellular and molecular mechanism underlying the role of BMPs in coordinating early fin bud morphogenesis.

developmental biology↗

bioRxiv2023

Parasite-Induced IFN-g Regulates Host Defense via CD115 and mTOR-Dependent Mechanism of Tissue-Resident Macrophage Death

Host resistance to a common protozoan parasite Toxoplasma gondii relies on a coordinated immune response involving multiple cell types, including macrophages. Embryonically seeded tissue-resident macrophages (TRMs) play a critical role in maintaining tissue homeostasis but their role in parasite clearance is poorly understood. In this study, we uncovered a crucial aspect of host defense against T. gondii mediated by TRMs. Through the use of neutralizing antibodies and conditional IFN-{gamma} receptor-deficient mice, we demonstrated that IFN-{gamma} directly mediated the elimination of TRMs. Mechanistically, IFN-{gamma} stimulation rendered macrophages unresponsive to macrophage colony-stimulating factor (M-CSF) and inactivated mTOR signaling by causing the shedding of CD115 (CSFR1), the receptor for M-CSF. Further experiments revealed the essential role of macrophage IFN-{gamma} responsiveness in host resistance to T. gondii. The elimination of peritoneal TRMs emerged as a host defense mechanism aimed at limiting the parasites reservoir. The identified mechanism, involving IFN-{gamma}-induced CD115 and mTOR-dependent cell death, provides insights into the adaptation of macrophage subsets during infection and highlights a crucial aspect of host defense against intracellular pathogens.

immunology↗

bioRxiv2023

Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

RNA replication is considered a crucial stage in the origins of life. However, both enzymatic and non-enzymatic RNA replication cycles are impeded by the "strand separation problem" (SSP), a form of product inhibition arising from the extraordinary stability of RNA duplexes and their rapid kinetics of reannealing. Here we show that RNA trinucleotide triphosphates (triplets) can overcome the SSP by binding to and kinetically trapping dissociated RNA strands in a single-stranded form, while simultaneously serving as substrates for RNA replication by a triplet polymerase ribozyme (TPR). This enabled exponential replication of both (+) and (-) strands of double-stranded RNAs by the TPR when driven by coupled pH and freeze-thaw cycles. We demonstrate replication of a fragment of the ribozyme itself, and open-ended amplification of random RNA sequence pools over >70 cycles, with emergence of partial, distributive TPR self-replication and triplet codon drift towards a primordial genetic code. One-sentence summaryRNA trinucleotide substrates together with simple physicochemical cycles enable RNA-catalysed replication of double-stranded RNA and partial, distributive self-replication of an RNA polymerase ribozyme.

molecular biology↗

bioRxiv2022

Single molecule co-occupancy of RNA-binding proteins with an evolved RNA deaminase

RNA-protein interactions broadly regulate gene expression. To understand RNA regulation, it is critical to measure RNA-protein interactions in cells. Current approaches to measure RNA-protein interactions often rely on crosslinking and shortread RNA sequencing, which has considerably advanced the understanding of gene expression but also suffers from some limitations. We present REMORA (RNA Encoded Molecular Recording in Adenosines), a new strategy to measure RNA-binding events on single RNA molecules in cells. In REMORA, adenosine deamination serves as a molecular record of RNA-protein interactions that are identified by mutations by sequencing. We performed RNA-based directed evolution to identify an RNA deaminase variant with high activity on arbitrary adenosine residues in RNA. We show that this RNA deaminase has high activity, low local sequence or structure bias, low background, and is generally applicable to diverse RNA-binding proteins. By combining our improved A-to-I RNA deaminase with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, our approach enables simultaneous recording of the locations two RNA binding proteins on single mRNA molecules. Orthogonal RNA molecular recording of two Pumilio family proteins, PUM1 and PUM2, reveals that PUM1 competes with PUM2 for some but not all Pumilio binding sites in cells, despite having the same in vitro binding preferences. Our work thus measures competition between RNA-binding proteins for RNA sites in cells, and our genetically encodable RNA deaminase enables single-molecule identification of RNA-protein interactions with cell type specificity.

molecular biology↗

bioRxiv2022

A Legionella toxin mimics tRNA and glycosylates the translation machinery to trigger a ribotoxic stress response.

Pathogens often secrete proteins or nucleic acids that mimic the structure and/or function of molecules expressed in their hosts. Molecular mimicry empowers pathogens to subvert critical host processes and establish infection. We report that the intracellular bacterium Legionella pneumophila secretes the toxin SidI (substrate of icm/dot transporter I), which possesses a transfer RNA (tRNA)-like shape and functions as a mannosyl transferase. The 3.1 [A] cryo-EM structure of SidI reveals an N-terminal domain that exhibits a characteristic inverted L-shape and charge distribution that is present in two other known protein mimics of tRNAs, the bacterial elongation factor EF-G and the mammalian release factor eRF1. In addition, we show that SidIs C-terminal domain adopts a glycosyl transferase B fold similar to a mannosyl transferase. This molecular coupling of the proteins fold and enzymatic function allows SidI to bind and glycosylate components of the host translation apparatus, including the ribosome, resulting in a robust block of protein synthesis that is comparable in potency to ricin, one of the most powerful toxins known. Additionally, we find that translational pausing activated by SidI elicits a stress response signature reminiscent of the ribotoxic stress response that is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAK and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus, and orchestrates the transcription of stress-inducible genes that promote cell death. Thus, using Legionella and its effectors as tools, we have unravelled the role of a ribosome-to-nuclear signalling pathway that regulates cell fate.

cell biology↗

bioRxiv2021

Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme and Establish Fin Morphology

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identified a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the Apical Ectodermal Ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

cell biology↗

bioRxiv2021

Spatio-temporal coordination at the maternal-fetal interface promotes trophoblast invasion and vascular remodeling in the first half of human pregnancy

Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels left with a thin, discontinuous smooth muscle layer and partially lined with EVTs. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodeling (SAR). However, it remains a matter of debate which immune and stromal cell types participate in these interactions and how this process evolves with respect to gestational age. Here, we used a multiomic approach that combined the strengths of spatial proteomics and transcriptomics to construct the first spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time of flight (MIBI-TOF) and a 37-plex antibody panel to analyze [~]500,000 cells and 588 spiral arteries within intact decidua from 66 patients between 6-20 weeks of gestation, integrating this with coregistered transcriptomic profiles. Gestational age substantially influenced the frequency of many maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, Galectin-9, and IDO-1 increasingly enriched and colocalized at later time points. In contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting unique monotonic and biphasic trends. Lastly, we developed an integrated model of SAR supporting an intravasation mechanism where invasion is accompanied by upregulation of pro-angiogenic, immunoregulatory EVT programs that promote interactions with vascular endothelium while avoiding activation of immune cells in circulating maternal blood. Taken together, these results support a coordinated model of decidualization in which increasing gestational age drives a transition in maternal decidua towards a tolerogenic niche conducive to locally regulated, EVT-dependent SAR.

systems biology↗

bioRxiv2020

ILC1-derived IFN-gamma mediates cDC1-dependent host resistance against Toxoplasma gondii

Host resistance against intracellular pathogens requires a rapid IFN-{gamma} mediated immune response. We reveal that T-bet-dependent production of IFN-{gamma} is essential for the maintenance of inflammatory DCs at the site of infection with a common protozoan parasite, Toxoplasma gondii. A detailed analysis of the cellular sources for T-bet-dependent IFN-{gamma} identified that ILC1s, but not NK or TH1 cells, were involved in the regulation of inflammatory DCs via IFN-{gamma}. Mechanistically, we established that T-bet dependent ILC1-derived IFN-{gamma} is critical for the induction of IRF8, an essential transcription factor for cDC1s. Failure to upregulate IRF8 in DCs resulted in acute susceptibility to T. gondii infection. Our data identifies that ILC1-derived IFN-{gamma} is indispensable for host resistance against intracellular infection via maintaining IRF8+ inflammatory DCs at the site of infection. Author SummaryMounting a robust type I innate immune response is essential for resistance against numerous intracellular pathogens. The type I immune response is characterized by the production of IFN-{gamma}, a central cytokine required for multiple non-redundant effector functions against bacterial, viral, and parasitic pathogens. Previous work has shown that group 1 innate lymphocyte cells (ILC1s) together with NK and CD4+ T cells play an indispensable IFN-{gamma} mediated protective role against Toxoplasma gondii infection; yet, the pathway of how IFN-{gamma} produced by ILC1s defend against T. gondii remains unknown. In this work we identified that early production of IFN-{gamma} by ILC1 is essential for maintaining dendritic cells (DCs) during infection. Mechanistically, we reveal that ILC1-derived IFN-{gamma} is indispensable for inducing the transcription factor IRF8 that is critical for sustaining inflammatory DCs. Finally, we demonstrate that IRF8+ DCs are critical for parasite elimination.

immunology↗

bioRxiv2019

Investigating mechanisms of polarized light sensitivity in the small white butterfly Pieris rapae

There is an ever increasing number of arthropod taxa shown to have polarization sensitivity throughout their compound eyes. However, the mechanisms underlying arthropod perception of polarized reflections from objects such as plants are not well understood. The small white butterfly, Pieris rapae, has been demonstrated to exploit foliar polarized reflections, specifically the degree of linear polarization (DoLP), to recognize host plants. The well-described visual system of P. rapae includes several photoreceptor types (red, green, blue) that are sensitive to polarized light. Yet, the mechanism underlying the behavioral responses of P. rapae to stimuli with different DoLPs remains unknown. To investigate potential mechanisms, we designed several two-choice behavioral bioassays, displaying plant images on paired LCD monitors which allowed for independent control of polarization, color and intensity. We found that shifts in image intensity had a similar effect on P. rapae preferences for stimuli dissimilar in DoLP and dissimilar in color, suggesting DoLP differences are perceived as color. When a DoLP choice was offered between plant images manipulated in a manner to minimizing the response of blue, red, or blue and red photoreceptors, P. rapae shifted its preference for DoLP, suggesting a role for red, green and blue polarization-sensitive photoreceptors. Modeling of P. rapae photoreceptor responses to test stimuli suggests that differential DoLP is not perceived solely as a color difference. Our combined results suggest that P. rapae females process and interpret polarization reflections in a way different from that described for other polarization-sensitive taxa.

animal behavior and cognition↗