Biology subjects

Hughes, K.

Publications and source records attributed to Hughes, K..

25 records · Page 2Linked to original sources

bioRxiv2019

Zygote morphogenesis but not the establishment of cell polarity in Plasmodium berghei is controlled by the small GTPase, RAB11A

Plasmodium species are apicomplexan parasites whose zoites are polarized cells with a marked apical organisation where the organelles associated with host cell invasion and colonization reside. Plasmodium gametes mate in the mosquito midgut to form the spherical and presumed apolar zygote that morphs during the following 24 hours into a polarized, elongated and motile zoite form, the ookinete. Endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and the small GTPase RAB11A is an important regulator of protein transport via recycling endosomes. PbRAB11A is essential in blood stage asexual of Plasmodium. Therefore, a promoter swap strategy was employed to down-regulate PbRAB11A expression in gametocytes and zygotes of the rodent malaria parasite, Plasmodium berghei which demonstrated the essential role of RAB11A in ookinete development. The approach revealed that lack of PbRAB11A had no effect on gamete production and fertility rates however, the zygote to ookinete transition was almost totally inhibited and transmission through the mosquito was prevented. Lack of PbRAB11A did not prevent meiosis and mitosis, nor the establishment of polarity as indicated by the correct formation and positioning of the Inner Membrane Complex (IMC) and apical complex. However, morphological maturation was prevented and parasites remained spherical and immotile and furthermore, they were impaired in the secretion and distribution of microneme cargo. The data are consistent with the previously proposed model of RAB11A endosome mediated delivery of plasma membrane in Toxoplasma gondii if not its role in IMC formation and implicate it in microneme function. Author SummaryAccording to the WHO there was estimated to be over 200 million cases of malaria in 2017 and nearly half a million deaths. The disease is caused by specific species of Plasmodium which are passed between human hosts by a mosquito vector. In order to transmit through the mosquito the single-celled parasite undergoes many developmental changes as it morphs from non-motile blood forms to become a polarised and motile ookinete in the mosquito midgut. Transport of proteins within the cell during these critical morphological transitions relies on specific endosome vesicles to correctly target proteins within the parasite. We investigated the role of the RAB11A protein which is known to be involved in endosomal vesicle targeting to generate cellular polarity in other organisms. Because RAB11A is also essential for parasite growth in the mammalian host we used a promoter swap system to specifically switch off RAB11A in the sexual transmission stages. In the absence of RAB11A parasites were unable to form elongated, motile ookinetes and were unable to pass through the mosquito. Interestingly the parasites were able to form some of the (polarising) structures specific to ookinetes however full morphological transformation did not occur and the parasites were not motile. We show that although proteins are still delivered to the parasite surface, secretion is impaired and that the mutant parasites are smaller despite obvious microtubule formation implying that there is a deficit in delivery of membrane to the surface.

cell biology↗

bioRxiv2019

Developmental stage-specific distribution of macrophages in mouse mammary gland

Mammary gland development begins in the embryo and continues throughout the reproductive life of female mammals. Tissue macrophages (M{phi}s), dependent on signals from the M{phi} colony stimulating factor 1 receptor (CSF1R), have been shown to regulate the generation, regression and regeneration of this organ, which is central for mammalian offspring survival. However, the distribution of M{phi}s in the pre- and post-natal mammary gland, as it undergoes distinct phases of development and regression, is unknown or has been inferred from immunostaining of thin tissue sections. Here, we used optical tissue clearing and 3-dimensional imaging of mammary tissue obtained from Csf1r-EGFP mice. Whilst tissue M{phi}s were observed at all developmental phases, their abundance, morphology, localization and association with luminal and basal epithelial cells exhibited stage-specific differences. Furthermore, sexual dimorphism was observed at E14.5, when the male mammary bud is severed from the overlying epidermis. These findings provide new insights into the localization and possible functions of heterogeneous tissue M{phi} populations in mammogenesis.

developmental biology↗

bioRxiv2019

Mammary mechanobiology: PIEZO1 mechanically-activated ion channels in lactation and involution

A mothers ability to produce a nutritionally-complete neonatal food source has provided a powerful evolutionary advantage to mammals. Milk production by secretory mammary epithelial cells is adaptive, its release is exquisitely timed and its own glandular stagnation with the permanent cessation of suckling triggers the programmed cell death and tissue remodeling that enables female mammals to nurse successive progeny. Both chemical and mechanical signals control epithelial expansion, function and remodeling. Despite this duality of input, however, the nature and function of mechanical forces in the mammary gland remain unknown. Here, we characterize the mammary force landscape and the capacity of luminal and basal epithelial cells to experience and exert force. We explore the molecular instruments for force-sensing in the mammary gland and the physiological requirement for PIEZO1 in lactation and involution. Our study supports the existence of a multifaceted system of chemical and mechanical sensing in the mammary gland, and a protective redundancy that ensures continued lactational competence and offspring survival.

cell biology↗

bioRxiv2019

Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

Uridylate insertion/deletion RNA editing in Trypanosoma brucei is a complex system that is not found in humans, so there is interest in targeting this system for drug development. This systeme uses hundreds of small non-coding guide RNAs (gRNAs) to modify the mitochondrial mRNA transcriptome. This process occurs in holo-editosomes that assemble several macromolecular trans factors around mRNA: the RNA-free RNA editing core complex (RECC) and auxiliary RNPs. Yet, the regulatory mechanisms of editing remain obscure. The enzymatic accessory RNP complex, termed the REH2C, includes mRNA substrates and products, the multi-domain 240 kDa RNA Editing Helicase 2 (REH2) and an intriguing 8-zinc finger protein termed REH2-Associated Factor 1 (H2F1). Both proteins are essential in editing. REH2 is a member of the DExH/RHA subfamily of RNA helicases with a conserved C-terminus that includes a regulatory OB-fold domain. In trypanosomes, H2F1 recruits REH2 to the editing apparatus, and H2F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 and H2F1 for the assembly of REH2C, the stability of REH2, and the RNA-mediated association of REH2C with other editing trans factors. We identified functional OB-fold amino acids in eukaryotic DExH/RHA helicases that are conserved in REH2 and impact the assembly and interactions of REH2C. H2F1 upregulation stabilized the large REH2 polypeptide in vivo. Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property of H2F1 but not its interactions with other examined editing components. Thus, most if not all fingers may contribute to REH2 stabilization. Finally, a recombinant REH2 (240 kDa) established that the full-length protein is a bona fide RNA helicase with ATP-dependent unwinding activity. REH2 is the only DExH/RHA-type helicase in kinetoplastid holo-editosomes.

molecular biology↗

bioRxiv2018

Dexi disruption depletes gut microbial metabolites and accelerates autoimmune diabetes

Non-coding genetic variants in the CLEC16A gene on human chromosome 16p13.13 are associated with risk of autoimmune diseases, including type 1 diabetes and multiple sclerosis. In this region, we previously identified DEXI, a candidate causal gene of unknown function, which alters the risk of type 1 diabetes, where the T1D predisposing allele is associated with lower DEXI expression. Here, we demonstrate by CRISPR mutagenesis in vivo and deep phenotyping that disrupted Dexi expression accelerates diabetes in the non-obese diabetic (NOD) mouse, a spontaneous model of autoimmune pancreatic beta-cell destruction. Mutant mice have increased serum IgM and IgA concentrations compared to wild-type NOD mice, as well as changes in both the gut microbiome and molecular metabolites associated with microbial metabolism. These findings suggest that the mechanism by which DEXI alters diabetes risk involves the composition and function of the microbiome and its impact on host metabolites. Such metabolites, including short chain fatty acids such as butyrate, have been shown to alter the activity of the immune cells involved in beta-cell destruction and susceptibility of the beta cells to autoimmune attack.\n\nOne Sentence Summary: Disruption of the Dexi gene leads to accelerated diabetes in the non-obese diabetic (NOD) mouse, accompanied by changes in serum immunoglobulins, gut microbiome and microbial metabolites.

genetics↗

bioRxiv2018

Intracellular calcium dysregulation precedes muscle decline in C. elegans modeling Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by loss of the protein dystrophin. In humans, DMD has early onset, causes developmental delays, muscle necrosis, loss of ambulation, and early death. Current animal models have been challenged by their inability to model the early onset and severity of the disease. Thus it remains unresolved if increased sarcoplasmic calcium observed in dystrophic muscles follows or leads the mechanical insults caused by the muscles disrupted contractile machinery. This knowledge has important applications for patients, as potential physiotherapeutic treatments may either help or exacerbate symptoms, depending on how dystrophic muscles differ from healthy ones. Recently we showed how burrowing dystrophic (dys-1) C. elegans recapitulate many salient phenotypes of DMD, including loss of mobility and muscle necrosis. Here we report dys-1 worms display early pathogenesis, including dysregulated sarcoplasmic calcium, and increased lethality. Sarcoplasmic calcium dysregulation in dys-1 worms precedes overt structural phenotypes (e.g. mitochondrial, and contractile machinery damage) and can be mitigated by silencing calmodulin expression. To learn how dystrophic musculature responds to altered physical activity, we cultivated dys-1 animals in environments requiring high amplitude, or high frequency of muscle exertion during locomotion. We find that several muscular parameters (such as size) improve with increased activity. However, longevity in dystrophic animals was negatively associated with muscular exertion, regardless of the duration of the effort. The high degree of phenotypic conservation between dystrophic worms and humans provides a unique opportunity to gain insights into the etiology of the disease, as well as the initial assessment of potential treatment strategies.\n\nSIGNIFICANCEDuchenne muscular dystrophy is a degenerative disease affecting tens of thousands of people in the US alone. Much remains unknown about the disease, including the chain of events that links the loss of dystrophin to muscle death, or the extent to which exercise might be able to protect degenerating muscles. We used the nematode C. elegans to show that sarcoplasmic calcium dysregulation takes place in dystrophic muscles long before other overt signs of damage manifest. When placed in assays that altered muscular activity by increasing either contraction frequency or amplitude, we observed several metrics associated with muscular repair increase. However, no treatment positively affected the life expectancy of dystrophic animals.

molecular biology↗

bioRxiv2018

An examination of the evolve-and-resequence method using Drosophila simulans

We develop a set of analytical and simulation tools for Evolve-and-Resequence (E&R) experiments and apply them to a new study of rapid evolution in Drosophila simulans. Likelihood based test statistics applied to pooled population sequencing data suggest parallel evolution of 138 polymorphisms (SNPs) across the genome. This number is reduced by orders of magnitude from previous studies (thousands or tens of thousands), owing to differences in both experimental design and statistical analysis. Whole genome simulations calibrated from several Drosophila genetic datasets support the contention that the observed genome-wide response could be generated by as few as 30 loci under strong directional selection, with a corresponding hitch-hiking effect. Finally, the SNPs that showed strong parallel evolution in the novel laboratory environment exhibit an (initial) allele frequency spectrum indicative of balancing in nature. These loci also exhibit elevated differentiation among natural populations of D. simulans.

evolutionary biology↗