Biology subjects

Lowe, J.

Publications and source records attributed to Lowe, J..

15 recordsLinked to original sources

bioRxiv2026

Bridging the lipid divide: archaeal ESCRT-III binds phosphoinositol and polarises the cytokinetic membrane

All cells remodel their membranes to divide. The highly conserved ESCRT-III system forms contractile polymers which, through direct interactions with membrane lipids, remodel membranes across the tree of life. In exploring how ESCRT-III divides the chemically and structurally unique archaeal membrane, we reveal that the homologue CdvB1 is required for the establishment of a distinct membrane domain within the division bridge of Sulfolobus acidocaldarius, associated with an accumulation of membrane-spanning inositol phosphate lipids. We show that CdvB1 associates with phosphoinositides in vitro and that this interaction aids cytokinesis in vivo. Together, we suggest that although eukaryotes inherited their membrane lipids from bacteria during eukaryogenesis, key features of the ESCRT-III:membrane interface that allow these polymers to bind, organise, and remodel eukaryotic membranes, may originate in archaea.

cell biology↗

bioRxiv2025

Platelet concentrate-derived extracellular vesicles promote adult hippocampal neurogenesis

Platelet-derived materials are emerging as promising, cell-free biotherapies for regenerative medicine. While platelet lysates have shown neuroprotective activity in preclinical models, the neurogenic potential of platelet concentrate-derived extracellular vesicles (pEVs) remains underexplored. Here, we evaluated the effects of human pEVs and a neuroprotective heat-treated human platelet lysate (HPPL) on adult hippocampal neurogenesis using both an ex vivo neurosphere assay and an in vivo intranasal administration model. pEVs selectively enhanced dentate gyrus (DG)-derived neurosphere growth, even in the absence of exogenous growth factors, and were internalized by neural precursors. In vivo, short-term pEV delivery increased EdU proliferating cells in the DG, while long-term administration (28 days) elevated the proportion of newborn mature neurons. By contrast, HPPL primarily promoted early neurogenesis by expanding immature DCX neurons. Quantitative proteomics of DG tissue after pEV treatment revealed 111 differentially expressed proteins, with enrichment in pathways related to oxidative phosphorylation, Notch4 signaling, myelination, and MHC class I-mediated antigen presentation. Downregulated proteins included cytoskeletal and translation-related regulators, suggesting a shift toward neuronal differentiation and circuit integration. Biophysical characterization confirmed the purity and vesicular nature of pEVs, with a defined protein cargo including immune modulators and ECM-interacting molecules such as CD44, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), and complement proteins. These findings identify allogeneic pEVs as multifunctional agents that modulate neural precursor cell fate and brain tissue remodeling through coordinated metabolic and immunoregulatory mechanisms. This work supports the translational potential of pEV-based therapeutics for promoting hippocampal neurogenesis and cognitive repair in neurodegenerative and age-related brain disorders.

neuroscience↗

bioRxiv2022

Divisome core complex in bacterial cell division revealed by cryo-EM

Cell division, or cytokinesis is a fundamental process of life and, in most bacteria, is driven by peptidoglycan synthesis at the septum1. It is catalysed by the divisome, a multi-protein complex with more than 20 components that spans the cell envelope in bacteria harbouring a cell wall2. Central to the divisome is the peptidoglycan-synthesising protein complex FtsWI, with the transglycosylase (TG) FtsW polymerising glycan strands from its substrate Lipid II3,4, and the transpeptidase (TP) FtsI crosslinking peptide stems, thus forming a covalent mesh between glycan strands5,6 (Fig. 1a). Septal peptidoglycan synthesis occurs after activation of the divisome glycosyltransferase-transpeptidase pair FtsWI3, in particular through an interaction with the heterotrimer FtsQBL7. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/517367v1_fig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1242fe6org.highwire.dtl.DTLVardef@c4d2c2org.highwire.dtl.DTLVardef@1e8eb0forg.highwire.dtl.DTLVardef@a02fff_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1:C_FLOATNO Biochemical and structural characterisation of the core divisome complex FtsWIQBL from P. aeruginosa. a) Septal peptidoglycan synthesis by FtsWIQBL during Gram-negative bacterial cell division. The transglycosylase FtsW (red), and transpeptidase FtsI (blue) bind the non-enzymatic subcomplex FtsQBL (green, violet and yellow, respectively). The complex contains 14 transmembrane helices - ten from FtsW and one each from FtsIQLB. The transglycosylase FtsW catalyses the polymerisation of GlcNAc-MurNAc disaccharides from Lipid II. The transpeptidase FtsI crosslinks the peptides from the nascent chain to adjacent peptides in the peptidoglycan layer between residues three and four. OM: outer membrane, IM: inner membrane, GlcNAc: N-acetylglucosamine, MurNAc: N-acetylmuramic acid, CC: coiled coil, TM: transmembrane. b) SDS-PAGE of the co-purified PaFtsWIQBL complex after size-exclusion chromatography. c) Western blot showing glycan strand ladders synthesised by divisome core complexes from Lipid II, demonstrating transglycosylase activity. The negative control does not contain any FtsWIQBL (lane 1). WT P. aeruginosa and E. coli FtsWIQBL complexes (lanes 2 and 4) are active transglycosylases, while the P. aeruginosa putative active site mutant FtsWD275AIQBL (lane 3) is inactive. d) Three representative 2D classes of our PaFtsWIQBL cryo-EM data. e) Left panel: side-view of the PaFtsWIQBL cryo-EM density at an overall resolution of 3.7 [A]. Protein colours are the same as those in a). Residual density from the detergent micelle is visible around the transmembrane domain in grey. Right panel: model of PaFtsWIQBL, rotated by 120{degrees} with respect to the density on the left-hand side. The putative FtsW active site residue D275 is indicated, as is the FtsI active site residue S294. The FtsW loop 219-233 and FtsI loop 45-50 are shown as a dotted line as they were too flexible to build. FtsQ and FtsQ were not resolved and are not shown. f) Top view of the periplasmic domain, showing interactions between FtsI, FtsL, FtsB and FtsQ. C_FIG Here, we present the cryo-EM structure of the catalytic divisome core complex FtsWIQBL from Pseudomonas aeruginosa at 3.7 [A] resolution. The structure reveals the intricate details of the periplasmic interfaces within FtsWIQBL, including the positioning of FtsI by the coiled coil of FtsBL, as well as a transmembrane domain containing FtsWIBL but not FtsQ. With our structure we are able to provide molecular mechanisms of a multitude of known mutations that interfere with divisome activation and regulation. Finally, we reveal a large conformational switch between presumably inactive and active states of the FtsWI core enzymes. Our work is foundational for further structural, biochemical and genetic studies elucidating the molecular mechanisms of bacterial cell division. Since the divisome peptidoglycan synthase is essential for cell division in most bacteria, and is absent in eukaryotic cells entirely, it is a key target of important antibiotics and antibiotic development8, and we suggest that our structure will help to accelerate these efforts.

molecular biology↗

bioRxiv2021

Characterisation of the Ubiquitin-ESCRT pathway in Asgard archaea sheds new light on origins of membrane trafficking in eukaryotes

The ESCRT machinery performs a critical role in membrane remodelling events in all eukaryotic cells, including in membrane trafficking, membrane repair, cytokinetic abscission, in viral egress, and in the generation of extracellular vesicles. While the machinery is complex in modern day eukaryotes, where it comprises dozens of proteins, the system has simpler and more ancient origins. Indeed, homologues of ESCRT-III and the Vps4 ATPase, the proteins that execute the final membrane scission reaction, play analogous roles in cytokinesis and potentially in extracellular vesicle formation in TACK archaea where ESCRT-I and II homologues seem to be absent. Here, we explore the phylogeny, structure, and biochemistry of homologues of the ESCRT machinery and the associated ubiquitylation system found in genome assemblies of the recently discovered Asgard archaea. In these closest living prokaryotic relatives of eukaryotes, we provide evidence for the ESCRT-I and II sub-complexes being involved in the ubiquitin-directed recruitment of ESCRT-III,_as it is in eukaryotes. This analysis suggests a pre-eukaryotic origin for the Ub-coupled ESCRT system and a likely path of ESCRT evolution via a series of gene duplication and diversification events.

cell biology↗

bioRxiv2021

Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the worlds population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

immunology↗

bioRxiv2020

Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP

The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 [A]. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

molecular biology↗

bioRxiv2020

Scc2 and Scc3 promote distinct modes of topological association between cohesin and DNA

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are "clamped" in a sub-compartment created by Scc2s association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

biochemistry↗

bioRxiv2020

A Novel Performance Metric for Multiclass Subject Invariant Brain Computer Interfaces with Imbalanced Classes

[Finding suitable common feature sets for use in multiclass subject independent brain-computer interface (BCI) classifiers is problematic due to characteristically large inter-subject variation of electroencephalographic signatures. We propose a wrapper search method using a one versus the rest discrete output classifier. Obtaining and evaluating the quality of feature sets requires the development of appropriate classifier metrics. A one versus the rest classifier must be evaluated by a scalar performance metric that provides feedback for the feature search algorithm. However, the one versus the rest discrete classifier is prone to settling into degenerate states for difficult discrimination problems. The chance of occurrence of degeneracy increases with the number of classes, number of subjects and imbalance between the number of samples in the majority and minority classes. This paper proposes a scalar Quality (Q)-factor to compensate for classifier degeneracy and to improve the convergence of the wrapper search. The Q-factor, calculated from the ratio of sensitivity to specificity of the confusion matrix, is applied as a penalty to the accuracy (1-error rate). This method is successfully applied to a multiclass subject independent BCI using 10 untrained subjects performing 4 motor tasks in conjunction with the Sequential Floating Forward Selection feature search algorithm and Support Vector Machine classifiers.]

neuroscience↗

bioRxiv2019

Systematic identification of factors bound to isolated metaphase ESC chromosomes reveals a role for chromatin repressors in compaction

Epigenetic information is transmitted from mother to daughter cells through mitosis. To identify trans-acting factors and cis-acting elements that might be important for conveying epigenetic memory through cell division, we isolated native (unfixed) chromosomes from metaphase-arrested cells using flow cytometry and performed LC-MS/MS to determine the repertoire of chromosome-bound proteins. Quantitative proteomic comparisons between metaphase-arrested cell lysates and chromosome-sorted samples revealed a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers (such as PRC2 and DNA methyl-transferases) and proteins governing chromosome architecture. We showed that deletion of PRC2, DNMT1/3a/3b or Mecp2 provoked an increase in the size of individual mitotic chromosomes consistent with de-condensation, as did experimental cleavage of cohesin complexes. These data provide a comprehensive inventory of chromosome-bound factors in pluripotent stem cells at mitosis and reveal an unexpected role for chromatin repressor complexes in preserving mitotic chromosome compaction.

cell biology↗

bioRxiv2019

Cryo-EM structure of the MinCD copolymeric filament from Pseudomonas aeruginosa at 3.1 A resolution

Positioning of the division site in many bacterial species relies on the MinCDE system, which prevents the cytokinetic Z-ring from assembling anywhere but the mid-cell, through an oscillatory diffusion-reaction mechanism. MinD dimers bind to membranes and, via their partner MinC, inhibit the polymerisation of cell division protein FtsZ into the Z-ring. MinC and MinD form polymeric assemblies in solution and on cell membranes. Here, we report the high-resolution cryo-EM structure of the copolymeric filaments of Pseudomonas aeruginosa MinCD. The filaments consist of three protofilaments made of alternating MinC and MinD dimers. The MinCD protofilaments are almost completely straight and assemble as single protofilaments on lipid membranes, which we also visualised by cryo-EM.

molecular biology↗

bioRxiv2018

A folded conformation of MukBEF and Cohesin

Structural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, where they have key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through topological entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: proteobacterial MukBEF and eukaryotic cohesin. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head/kleisin module, situated at opposite ends of the arms. Our findings favor SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.

molecular biology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2018

Structural analysis of the interaction between the bacterial cell division proteins FtsQ and FtsB

Most bacteria and archaea use similar proteins within their cell division machinery, which uses the tubulin homologue FtsZ as its central organiser. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. In biochemical assays we show that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the {beta} domain of FtsQ shows that only residues 64-87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ:FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ:FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalising view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.\n\nImportanceCells in most bacteria and archaea divide through a cell division process that is characterised through its filamentous organiser, FtsZ protein. FtsZ forms a ring structure at the division site and starts the recruitment of 10-20 downstream proteins that together form an elusive multi-protein complex termed divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

microbiology↗

bioRxiv2017

MreB Filaments Create Rod Shape By Aligning Along Principal Membrane Curvature.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion and increased glycan crosslinking. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

microbiology↗

bioRxiv2016

A polymerisation-associated conformational switch in FtsZ that enables treadmilling

Bacterial cell division in many organisms involves a constricting cytokinetic ring that is orchestrated by the tubulin-like protein FtsZ. FtsZ forms dynamic filaments close to the membrane at the site of division that have recently been shown to treadmill around the division ring, guiding septal wall synthesis.\n\nHere, using X-ray crystallography of Staphylococcus aureus SaFtsZ we reveal how an FtsZ can adopt two functionally distinct conformations, open and closed. The open form is found in SaFtsZ filaments formed in crystals and also in soluble filaments of E. coli FtsZ as deduced by cryoEM. The closed form is found within several crystal forms of two non-polymerising SaFtsZ mutants and corresponds to many previous FtsZ structures from other organisms.\n\nWe argue that FtsZ undergoes a polymerisation-associated conformational switch. We show that such a switch provides explanations for both how treadmilling may occur within a single-stranded filament, and why filament assembly is cooperative.

microbiology↗