Biology subjects

Whitehead, L.

Publications and source records attributed to Whitehead, L..

16 recordsLinked to original sources

bioRxiv2025

Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

Toxoplasma and other Apicomplexan parasites, switch between different developmental stages to persist in and transmit between hosts. Toxoplasma can alternate between systemic tachyzoites and encysted bradyzoite forms found in the CNS and muscle tissues. How parasites sense these tissue types and trigger differentiation remains largely unknown. We show that Toxoplasma differentiation is induced under glucose-limiting conditions and using a CRISPR screen identify parasite genes required for growth under these conditions. From [~]25 identified genes important for differentiation we show that lactate and glutamine metabolism is linked to differentiation and demonstrate the importance of an E3 ubiquitin ligase complex, orthologous to glucose induced degradation deficient (GID) complex in yeast and CTLH complex in humans. We show that TgGID likely regulates translational repression of a key transcription factor required for differentiation, BFD1, through its 3 utr. Overall, this work provides important new insight into how these divergent parasites sense different host cell niches and trigger stage conversion through a ubiquitination-dependent program.

microbiology↗

bioRxiv2025

What are all these soil fungi doing? Complex carbon use ability as a predictive trait for fungal community functions

Fungal communities in soil play important roles in decomposition processes and soil organic carbon cycling. These communities are tremendously diverse, making it challenging to assign relevant functions to individual species. Fungal communities may be differentiated at the level of functional guilds; beyond such broad classification we have little delimitation, especially in fungal taxa common in grassland and agricultural soils. To resolve the level of functional similarity in fungal communities and define traits predictive of soil carbon cycling, we characterized fungal isolates abundant in agricultural soils to test the hypotheses that (i) the majority of saprobic soil fungi have the ability to use complex carbon sources, (ii) differences in complex carbon use abilities correlate with fungal enzymatic profiles, following principles of the fungal economics spectrum and (iii) carbon use ability is a predictive trait for fungal community functions. Using specialized growth media, we isolated and characterized 105 isolates and developed a novel FungiResp approach that directly tests fungal activity on complex carbon sources. The largest amount of variance between isolates was explained by differential abilities to use cellulose and starch, with only few phylogenetically distinct fungal clades showing high respiratory activity on these biopolymers. A preference for bacterial necromass was another major distinction among taxa. These key traits correlated with soil fungal community shifts in response to carbon substrate availability. By contrast, enzymatic activity was a poor predictor of fungal carbon use ability, except for correlations in lignin use and laccase activity. The newly established functional trait of carbon use ability offers important insights into diverse fungal communities: Many taxa lack the ability to use complex carbon (on their own), while the most common enzymes analyzed in soil showed little correlation with fungal mineralization potential. The discovery of key functional traits is an important step towards predicting the significance of fungal community shifts for soil carbon cycling.

ecology↗

bioRxiv2023

Spatial analysis of Hofbauer cell transcriptome, distribution and morphology in placentas exposed to Plasmodium falciparum

Placental infection remains a significant health burden for mothers and their babies in low-income countries, especially in sub-Saharan Africa, where malaria transmission is intense. An increase in inflammatory biomarkers and poor vascularisation are characteristics of placentas infected with malaria. Hofbauer cells (HBCs) - placental villous macrophages of fetal origin - are one of the most abundant immune cells in the placenta. HBCs are thought to have roles in angiogenic processes and have been linked with the pathophysiology of several infections and inflammatory conditions during pregnancy, including malaria (caused by Plasmodium falciparum). However, there is limited in situ data on the transcriptional, proteomic or morphologic profile of these cells either during or following clearance of P. falciparum infection. We leveraged placental samples prospectively collected at delivery from 610 Malawian women enduring a high burden of malaria and other infections and nutritional deficiencies. We profiled placentas through spatial transcriptomic and proteomic platforms to discern in situ HBC features that could distinguish placentas with or without evidence of past malaria. In this cohort, past placental infection was common and was associated with lower birth weight babies (adjusted effect [95% confidence interval], -80.9 [-165.9, -3.7] g, P= 0.040). However, at term, HBC numbers, abundance, and transcriptional profiles from placentas with evidence of past infection were similar to those of placentas without malaria. HBCs may recover post-infection back to a basal state or may be replaced in the tissue over the course of pregnancy. Placentas with evidence of past malaria did show evidence of reduced fetal vessel development (mean area difference: -22.8% [-37.6, -7.9], P=0.003). Reduced vascular development following infection early in pregnancy may reflect disturbances to the normal vasculogenic and angiogenic processes, of which HBCs are an integral part.

immunology↗

bioRxiv2023

An immunohistochemical atlas of necroptotic pathway expression

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo. HighlightsO_LI13 automated immunohistochemistry protocols for detecting the necroptotic pathway C_LIO_LINecroptotic pathway expression is confined to fast-cycling immune barriers C_LIO_LINecroptotic pathway expression changes at sites of immunoinflammatory challenge C_LIO_LIImmunodetection of necrosomes in IBD patients is a putative new diagnostic tool C_LI

cell biology↗

bioRxiv2023

Divergent roles for caspase-8 and MLKL in high-fat diet induced obesity and NAFLD in mice

Cell death frequently occurs in the pathogenesis of obesity and non-alcoholic fatty liver disease (NAFLD). However, the exact contribution of core cell death machinery to disease manifestations remains ill-defined. Here, we show via the direct comparison of mice genetically deficient in apoptotic caspase-8 in myeloid cells, or the essential necroptotic regulators, Receptor-interacting protein kinase-3 (RIPK3) and Mixed lineage kinase domain-like (MLKL), that RIPK3-caspase-8 signaling regulates macrophage inflammatory responses and drives adipose tissue inflammation and NAFLD upon high-fat diet feeding. In contrast, MLKL, divergent to RIPK3, contributes to both obesity and NAFLD in a manner largely independent of inflammation. We also uncover that MLKL regulates the expression of molecules involved in lipid uptake, transport and metabolism and, congruent with this, we discover a shift in the hepatic lipidome upon MLKL deletion. Collectively, these findings highlight MLKL as an attractive therapeutic target to combat the growing obesity pandemic and metabolic disease.

immunology↗

bioRxiv2022

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Rapid advances in tissue clearing protocols have begun to outpace the capabilities of existing microscope objectives: High-resolution imaging inside cm-sized cleared samples is often not possible as it requires multi-immersion objectives with high numerical aperture (NA > 0.7), long working distance (WD > 10 mm) and a large field-of-view (FOV > 1 mm). Here, we introduce a novel mirror-based optical design, the "Schmidt objective", which meets all these criteria despite containing only two optical elements. It consists of a spherical mirror in contact with the immersion medium and an aspherical correction plate. We showcase a multi-photon variant of a Schmidt objective that reaches NA 1.08 at an refractive index of 1.56 and demonstrate its versatility by imaging fixed samples in a wide range of immersion media ranging from air and water to BABB, DBE, and ECI. In addition, we demonstrate in vivo imaging by recording neuronal activity in larval zebrafish.

neuroscience↗

bioRxiv2022

SOCS2 regulation of growth hormone signaling requires a canonical interaction with phosphotyrosine

Suppressor Of Cytokine Signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function.

cell biology↗

bioRxiv2021

The minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers

Minor splicing is a second splicing system required for the correct expression of [~]700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38. Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3. We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

cancer biology↗

bioRxiv2021

Elys deficiency constrains Kras-driven tumour burden by amplifying oncogenic stress

The nucleoporin ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using a zebrafish model of hepatocellular carcinoma, in which the expression of an inducible mutant kras transgene (krasG12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly shrinks tumour burden, while non-hyperplastic tissues are unaffected. We demonstrate that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of TP53-dependent and independent mechanisms of cell death and cell cycle arrest. This selective vulnerability of cancer cells to mild disruption of Elys function uncovers a novel synthetic lethal interaction between ahctf1 and kras mutations that could be exploited therapeutically. Heterozygous expression of both ahctf1 and ranbp2, or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocked krasG12V-driven hepatocyte hyperplasia, revealing promising avenues for combinatorial treatments.

cancer biology↗

bioRxiv2020

Epigenomic reprogramming of repetitive noncoding RNAs and IFN-stimulated genes by mutant KRAS

RAS genes are the most frequently mutated oncogenes in cancer. However, the effects of oncogenic RAS signaling on the noncoding transcriptome are unclear. We analyzed the transcriptomes of human airway epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We found that oncogenic KRAS upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements. These repetitive noncoding RNAs exhibit differential RNA editing in single cells, are released in extracellular vesicles, and are known targets of KRAB zinc-finger proteins, which are broadly down-regulated in mutant KRAS cells and lung adenocarcinomas. Moreover, mutant KRAS induces IFN-stimulated genes through both epigenetic and RNA-based mechanisms. Our results reveal that mutant KRAS remodels the noncoding transcriptome through epigenomic reprogramming, expanding the scope of genomic elements regulated by this fundamental signaling pathway and revealing how mutant KRAS induces an intrinsic IFN-stimulated gene signature often seen in ADAR-dependent cancers.

genomics↗

bioRxiv2020

Real-Time Multi-Angle Projection Imaging of Biological Dynamics

We introduce a cost-effective and easily implemented scan unit which enables any camera-based microscope to perform projection imaging from diverse viewing angles. We demonstrate this capability on Lattice Light-Sheet and Oblique Plane Microscopy by rapidly delivering projection images with an uncompromised lateral resolution and high optical contrast. By imaging the sample from one or multiple perspectives, our method enables visualization of rapid biological processes, real time stereoscopic imaging as well as three-dimensional particle localization throughout a cellular volume from just two images. Furthermore, because our projection imaging technique provides intuitive three-dimensional renderings in real-time, it improves microscope usability, allows users to more-readily optimize instrument performance and identify biological phenomena of interest on-the-fly, while also reducing data overhead by a factor of >100. We leverage our rapid projection method to image cancer cell morpho-dynamics and calcium signaling in cultured neurons, to perform three-dimensional localization of genetically encoded nanoparticles, as well as to image orthogonal views of an embryonic Zebrafish heart simultaneously.

biophysics↗

bioRxiv2020

A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.

cell biology↗

bioRxiv2020

BCL-XL is essential for the protection from secondary anemia caused by radiation-induced fatal kidney damage

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis, but less is known about the roles of these proteins in adults, including in the response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL-deficiency in erythroid cells. Unexpectedly, the combination of total-body {gamma}-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combinations with DNA damage-inducing drugs for cancer therapy. SummaryThe inducible loss of BCL-XL in all cells of adult mice causes primary anemia due to apoptosis of erythroid and megakaryocytic cell populations. In contrast {gamma}-radiation plus loss of BCL-XL in all cells except hematopoietic cells causes secondary anemia resulting from kidney damage.

pharmacology and toxicology↗

bioRxiv2019

Vasoactive intestinal peptide confers anticipatory mucosal immunity by regulating ILC3 activity

ILC3-mediated IL-22 cytokine production is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we show that group 3 ILC (ILC3) constitutive function is not constant across the day but instead oscilliates between active and resting phases. Coordinate responsiveness of ILC3 in the intestine depended on food-induced expression of the neuronal hormone vasoactive intestinal peptide (VIP). Intestinal ILC3 expressed high levels of the G protein-coupled receptor, VIPR2, and activation via enteric neuronal VIP markedly enhanced IL-22 production and conferred gut protection. Conversely, deficiency of VIPR2 signalling led to impaired production of IL-22 by ILC3 and increased susceptibility to inflammatory gut disease. As such, intrinsic cellular rhythms synergise with the cyclic patterns of food intake to drive IL-22 thereby syncronizing intestinal epithelial protection via the ILC3 VIP-VIPR2 pathway.

immunology↗

bioRxiv2019

Toxoplasma gondii bradyzoites induce transcriptional changes to host cells and prevent IFNγ-mediated cell death

Toxoplasma gondii, the causative agent of toxoplasmosis, lies dormant for life and is a reservoir for disease reactivation, causing blindness, encephalitis and congenital birth defects. Acute-stage tachyzoites extensively manipulate their host cell by exporting a repertoire of proteins across the parasitophorous vacuolar membrane (PVM). This interferes with the hosts transcriptional program, allowing for persistence during immune attack. It is unknown how bradyzoites persist and what role host manipulation plays in latency. Here we show that bradyzoite-containing host cells have a unique transcriptional landscape when compared to tachyzoite infection. We demonstrate that many of these changes are dependent parasite protein export. Furthermore, we show that bradyzoite effector proteins protect host cells from IFN{gamma}-mediated cell death, thus highlighting the functional importance of host manipulation. Together, our work provides the first understanding of how Toxoplasma sets up latency to persist in its host.

microbiology↗

bioRxiv2017

Structurally conserved primate lncRNAs are transiently expressed during human cortical differentiation and influence cell type specific genes

The cerebral cortex has expanded in size and complexity in primates, yet the underlying molecular mechanisms are obscure. We generated cortical organoids from human, chimpanzee, orangutan, and rhesus pluripotent stem cells and sequenced their transcriptomes at weekly time points for comparative analysis. We used transcript structure and expression conservation to discover thousands of expressed long non-coding RNAs (lncRNAs). Of 2,975 human, multi-exonic lncRNAs, 2,143 were structurally conserved to chimpanzee, 1,731 to orangutan, and 1,290 to rhesus. 386 human lncRNAs were transiently expressed (TrEx) and a similar expression pattern was often observed in great apes (46%) and rhesus (31%). Many TrEx lncRNAs were associated with neuroepithelium, radial glia, or Cajal-Retzius cells by single cell RNA-sequencing. 3/8 tested by ectopic expression showed [≥]2-fold effects on neural genes. This rich resource of primate expression data in early cortical development provides a framework for identifying new, potentially functional lncRNAs.

neuroscience↗