Biology subjects

Baker, M. A.

Publications and source records attributed to Baker, M. A..

29 records · Page 2Linked to original sources

bioRxiv2022

The identification of a gene expression signature of primordial follicle activation in mouse pregranulosa cells

Primordial follicles are quiescent ovarian structures comprised of a single oocyte surrounded by a layer of somatic supporting pregranulosa cells. Primordial follicle activation is the first step towards oocyte maturation and, ultimately, ovulation. As the number of quiescent primordial follicles is finite, their rate of activation is a critical parameter of the duration of the female reproductive lifespan. Activation status is established by the presence of cuboidal and proliferative granulosa cells in primary follicles, rather than squamous and quiescent pregranulosa cells in primordial follicles. Here, using a continuous Entropy Sort Feature Weighting approach on single-cell RNA sequencing data, we identify a distinct transcriptomic signature of activating pregranulosa cells in neonatal wildtype mice. This signature contains several genes previously linked with mature granulosa cells as well several novel candidates: Slc18a2, Tnni3, Fam13a and Myo1e. We confirm expression of Slc18a2 and TNNI3 in the granulosa cells of activating follicles in embryonic, neonatal and adult mouse ovaries. Perturbation of cell cycle inhibitor p27kip1 in Cdkn1b-/- mice results in complete activation of all primordial follicles during this neonatal period. Contrary to previous reports on this established mouse model, we find substantial transcriptomic changes in embryonic Cdkn1b-/- ovaries. Upon loss of cell-cycle inhibition, we find increased expression of our signature of pregranulosa cell activation, particularly that of cardiac troponin I (Tnni3). We conclude that pregranulosa cells engage a distinct transcriptional programme prior to cell-cycle dependent primordial follicle activation.

developmental biology↗

bioRxiv2022

Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility

The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H+ powered motors), or PomA and PomB (in Na+ powered motors). In this study we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed ten ancestral sequences of MotA and found four of them were motile in combination with contemporary E. coli MotB and in combination with our previously published functional ancestral MotBs. Sequence comparison between wild-type (WT) E. coli MotA and MotA-ASRs revealed 30 critical residues across multiple domains of MotA that were conserved among all motile stator units. These conserved residues included pore-facing, cytoplasm-facing and MotA-MotA intermolecular facing sites. Overall, this work demonstrates the role of ASR in assessing conserved variable residues in a subunit of a molecular complex.

microbiology↗

bioRxiv2022

Computer-aided diagnosis of reflectance confocal images to differentiate between lentigo maligna (LM) and atypical intraepidermal melanocytic proliferation (AIMP)

Lentigo maligna (LM), a form of melanoma in situ that predominantly affects sun-exposed areas such as the face, has an ill-defined clinical border and has a high rate of recurrence. Atypical Intraepidermal Melanocytic Proliferation (AIMP) is a term used to describe the melanocytic proliferation of an uncertain malignant potential. Clinically and histologically, AIMP can be difficult to distinguish from LM, and indeed AIMP may in some cases progress to LM. Reflectance Confocal Microscopy (RCM) is often used to investigate these lesions non-invasively, however, RCM is often not readily available nor is the associated expertise for RCM image interpretation. Here, we demonstrate machine learning architectures that can correctly classify lesions between LM and AIMP on stacks of RCM images. Overall, our methods showcase the potential for computer-aided diagnosis in dermatology, which in conjunction with the remote acquisition, can expand the range of diagnostic tools in the community.

bioinformatics↗

bioRxiv2022

Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Intracellular compartments are functional units that support the metabolic processes within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer customised complex emulsion droplets as a multicompartment artificial cell chassis, using multiphase microfluidics and acoustic levitation. Such levitated constructs provide free-standing, dynamic, definable droplet networks for the encapsulation and organisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and large-conductance mechanosensitive channel (MscL) and their activation. The assembly of droplet networks is three-dimensionally patterned with fluidic inputs configurations determining droplet contents and connectivity. Whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. In addition, a mechanosensitive channel, MscL, can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact control of membrane protein function. Collectively, this will expand our capability to program and operate increasingly sophisticated artificial cells as life-like materials.

synthetic biology↗

bioRxiv2021

Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free swimming bacteria and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations, and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types, and in pathogenic and non-pathogenic strains. ImportanceHere we characterised two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 M across multiple strains, from non-pathogenic E. coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (EHEC/UPEC) and lastly in sodium-powered Vibrio alginolyticus. Broad anti-motility compounds such as these are important tools in our efforts control virulence of pathogens in health and agricultural settings.

microbiology↗

bioRxiv2021

Spontaneous adaptation of ion selectivity in a bacterial flagellar motor

Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants which rescue function. The Bacterial Flagellar Motor (BFM) is an excellent candidate - an ancient and highly conserved molecular complex for propulsion which navigates bacteria towards favourable environments. In most species, rotation is powered by H+ or Na+ ion transit through the torque-generating stator subunit of the motor complex. The ion that drives the rotor has changed over evolutionary timescales but the molecular basis of this selectivity remains unknown. Here we used CRISPR engineering to replace the native Escherichia coli H+-powered stator with Na+-powered stator genes and report the rapid and spontaneous reversion of our edit in a low sodium environment. We followed the evolution of the stators during their reversion to H+-powered motility and used whole genome and transcriptome sequencing to identify both flagellar- and non-flagellar-associated genes involved in the cells adaptation. Our transplant of an unfit protein and the cells rapid response to this edit demonstrates the adaptability of the stator subunit and highlights the hierarchical modularity of the flagellar motor.

microbiology↗

bioRxiv2020

Separation and enrichment of sodium-motile bacteria using cost-effective microfluidics.

Many motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through membrane protein complexes known as stators, with the next most common ionic power source being sodium. Sodium-powered stators can be studied through the use synthetic chimeric stators that combine parts of sodium- and proton-powered stator proteins. The most well studied example is the use of the sodium powered PomA-PotB chimeric stator unit in the naturally proton-powered E. coli. Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria while varying the ionic composition of the media and thus the sodium-motive-force available to drive this chimeric flagellar motor. We measured separation efficiencies at varying ionic concentrations and confirmed using fluorescence that our device delivered eight-fold enrichment of the motile proportion of a mixed population. Furthermore, our results showed that we could select bacteria from reservoirs where sodium was not initially present. Overall, this technique can be used to implement selection of highly-motile fractions from mixed liquid cultures, with applications in directed evolution to investigate the adaptation of motility in bacterial ecosystems.

microbiology↗

bioRxiv2020

Optimised assembly of DNA-lipid nanostructures

Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol-modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA-lipid binding while allowing controlled switching by strand-displacement are not known. Here we characterised the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA-lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl2, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded. For larger DNA origami tiles, 4 - 8 cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid-binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand-displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.

biophysics↗

bioRxiv2020

Sialylation of Asparagine 612 inhibits Aconitase activity during mouse sperm capacitation; A possible mechanism for the switch from oxidative phosphorylation to glycolysis

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS), we found 142 non-reductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2) was the only protein to show an increase in Sia content during capacitation. Interestingly, while the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, the increase of sialylation in the ACO2 (N612) also resulted in a decrease of the activity of this TCA cycle enzyme. The latter was confirmed by N612D recombinant protein with both His and GFP tag, in which the N612D mutant had no activity compared to WT when protein. Computer modelling show that N612 sits atop the catalytic site of ACO2. The introduction of sialic acid causes a large confirmation change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

developmental biology↗

bioRxiv2018

Mutations close to the peptidoglycan-binding region of the stators of the bacterial flagellar motor influence phenamil resistance

The bacterial flagellar motor (BFM) is a molecular complex which powers the rotation of the filament that propels swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus/Eschrichia Coli chimeric stator PotB. We then selected for motors that exhibited resistance to the sodium-channel inhibitor phenamil. We found that single mutations that inferred resistance to phenamil occurred at two sites: 1) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus, and 2) near the peptidoglycan (PG) binding region that corresponds to the C-terminal region of the MotB stator from E. coli. We corroborated our swim plate observations with single cell rotation assays to confirm that individual cells could drive rotation of flagellar motors in the presence of up to 100 M phenamil. Our results demonstrate that it is not only the pore region of the stator that moderates the effect of motility in the presence of ion-channel blockers. We hypothesise that mutations in the PG region can allow motors to function in the presence of phenamil by allowing multiple semi-functioning stators to persist on the motor and drive flagellar rotation.

microbiology↗

bioRxiv2018

Fluorescence Microscopy of Piezo1 in Droplet Hydrogel Bilayers

Mechanosensitive ion channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, mechanosensitive ion channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis (Xerocytosis) and lymphatic dysplasia. Here we utilize an established in-vitro artificial bilayer system to interrogate single Piezo1 channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 ion channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertion and activation of hPiezo1-GFP in bilayers of varying composition. Furthermore, we compare the Piezo1 bilayer reconstitution with measurements of insertion and activation of KcsA channels to reproduce the channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of ion channel gating while visualising the channel proteins using fluorescence.

molecular biology↗