Biology subjects

Mantell, J.

Publications and source records attributed to Mantell, J..

12 recordsLinked to original sources

bioRxiv2026

Distinct roles for TANGO1S domains in maintaining ER-Golgi architecture

The endoplasmic reticulum (ER)-Golgi interface is a dynamic trafficking hub maintained in part by TANGO1, a scaffolding protein that coordinates proteins and membranes at ER exit sites (ERES). TANGO1 has two isoforms: TANGO1L, which has a lumenal SH3 domain, and TANGO1S, which lacks this domain but retains the transmembrane and cytoplasmic coiled coil (CC), TEER, and PRD domains common to both forms. We showed previously that loss of both isoforms disrupts ER - Golgi organization more severely than TANGO1L loss alone, indicating TANGO1S is functional and can compensate. Here we dissect the role of each TANGO1 cytoplasmic domain in maintaining secretory pathway organisation by expressing TANGO1S domain-deletion mutants in TANGO1L-/S- knockout cells. We show that TANGO1 loss causes cis-Golgi vesiculation that cannot be rescued by TANGO1S. TANGO1S re-expression also failed to rescue COPII and Sec16 recruitment in this cell line. Meanwhile, the TEER domain is essential for the organisation of the ER, and the TEER, CC2 and PRD domain are required for a defined ERGIC. This study represents an advance towards a domain-level resolution of TANGO1S function.

cell biology↗

bioRxiv2025

Cryo-FIB Lift-out and Electron Tomography Workflow for Bacteria-Nanopillar Interface Imaging Under Native Conditions: Investigating Dragonfly Inspired Bactericidal Titanium Surfaces

Dragonfly- and cicada-wing-inspired titanium nanostructured surfaces exhibit promising bactericidal properties. However, direct visualisation of the bacteria-material interface under hydrated conditions remains limited, restricting experimental interrogation how bacteria interact with nanostructured surfaces. This limitation reflects a long-standing methodological gap, as visualisation of bacteria-nanostructure interfaces has largely relied upon indirect or dehydrated imaging approaches. Cryo-electron tomography (cryo-ET) enables three-dimensional visualisation of cellular ultrastructure in a native hydrated environment, but imaging bacteria attached to metallic nanostructures by cryo-ET requires preparation of thin electron-transparent lamellae. Obtaining such lamellae from vitrified bacteria on titanium substrates is technically challenging because cryo-focused ion beam (cryo-FIB) milling must simultaneously section soft biological material and hard metal whilst bacteria embedded in vitreous ice are not directly visible. Here, we establish a correlative cryogenic workflow enabling targeted extraction and cryo-ET imaging of defined bacterium-nanopillar interfaces. Individual bacteria interacting with titanium nanopillars are identified by correlative cryo-fluorescence microscopy, followed by targeted cryo-FIB lift-out, transfer to a receiver grid, and thinning into electron-transparent lamellae. The data presented demonstrate that bacteria-nanostructure interfaces can be targeted, extracted, and structurally analysed in situ under fully hydrated conditions. This workflow provides a methodological framework for future cryo-ET studies of bacteria-nanotopography interactions.

microbiology↗

bioRxiv2025

Synaptic high-frequency jumping synchronises vision to high-speed behaviour

During high-speed behaviour, animals must synchronise perception and action despite rapid environmental and self-generated motion. How neural systems achieve such precision remains unclear. Here we show how the housefly (Musca domestica) maintains visual accuracy during fast motion. Using intracellular and photomechanical recordings during saccade-like stimulation, we traced information flow from photoreceptors to large monopolar cells (LMCs). Visual neurons achieved record-high information sampling ([~]2,500 bits{middle dot}s-1) and synaptic transmission ([~]4,100 bits{middle dot}s-1), far exceeding previous estimates. We identify a previously unknown mechanism - synaptic high-frequency jumping - in which photoreceptor-LMC synapses dynamically shift transmission toward higher frequencies during saccades, extending visual bandwidth to [~]1,000 Hz, effectively eliminating synaptic delays, and quadrupling classical flicker-fusion limits ([~]230 Hz). Behavioural experiments show flies respond synchronously within [~]13-20 ms, even before photoreceptor responses peak. A biophysically realistic model reveals how photomechanical-stochastic-refractory quantal sampling co-adapts with saccadic behaviour: through self-motion, flies efficiently translate image motion into temporally-precise, predictive high-speed vision.

neuroscience↗

bioRxiv2025

Molecular mechanism for cargo-mediated initiation of kinesin-1 activation

Kinesin-1 is a dynamic heterotetrameric assembly of two heavy and two light chains (KHC and KLC) that mediates microtubule-based intracellular transport of many different cargoes. The complex adopts a compact, autoinhibited state that is activated by cargo-adaptor proteins containing specific short linear peptide motifs (SLiMs). These motifs interact with the tetratricopeptide repeat (TPR) domains of the KLCs. The mechanism coupling SLiM recognition to activation-associated conformational changes in the complex is unknown. Here we combine protein design, computational modelling, biophysical analysis, and electron microscopy to examine the structural and mechanistic consequences of SLiM binding to the KLC-TPR domain within the complete heterotetrameric holoenzyme. We show that coiled coil 1 (CC1) of the KHC docks KLC TPR domains in the autoinhibited complex, forming the shoulder feature observed in electron microscopy. Disrupting this interaction or binding an activating SLiM dislocates the TPR shoulder, freeing the motor domains and promoting transition between its closed, inactive, and open states. Opening the kinesin-1 complex facilitates binding to the microtubule-associated kinesin-1 cofactor, MAP7. Therefore, cargo-mediated dislocation of the TPR shoulder serves as a key initial step in kinesin-1 activation, allosterically linking cargo binding to motor dynamics.

biochemistry↗

bioRxiv2024

Maturation and conformational switching of a de novo designed phase-separating polypeptide

Cellular compartments formed by biomolecular condensation are a widespread feature of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels, through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arise in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using the droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely {beta} conformations, analogous to models of pathological phase separation.

synthetic biology↗

bioRxiv2023

CryoET reveals actin filaments within platelet microtubules

Crosstalk between the actin and microtubule cytoskeletons is essential for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

cell biology↗

bioRxiv2023

Allosteric regulation of a molecular motor through de novo protein design

Many enzymes are allosterically regulated. Our ability to manipulate these structural changes is limited. Here we install an allosteric switch into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed auto-inhibited states. The equilibrium between these centres on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modelled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities. One Sentence SummaryDe novo peptide and protein design are used to engineer an allosteric switch into kinesin-1 motors in vitro and directly in cells.

synthetic biology↗

bioRxiv2022

Discovery and biosynthetic assessment of Streptomyces ortus sp nov. isolated from a deep-sea sponge

The deep sea is known to host novel bacteria with the potential to produce a diverse array of undiscovered natural products. Understanding these bacteria is thus of broad interest in ecology and could also underpin applied drug discovery, specifically in the area of antimicrobials. Here, we isolate a new strain of Streptomyces from the tissue of the deep-sea sponge Polymastia corticata collected at a depth of 1869 m from the Gramberg seamount in the Atlantic Ocean. This strain, which was given the initial designation A15ISP2-DRY2T, has a genome size of 9.29 Mb with a GC content of 70.83%. Phylogenomics determined that A15ISP2-DRY2T represents a novel species within the genus Streptomyces as part of the Streptomyces aurantiacus clade. The biosynthetic potential of A15ISP2-DRY2T was assessed relative to other members of the aurantiacus clade via comparative gene cluster family (GCF) analysis. This revealed a clear congruent relationship between phylogeny and GCF content. A15ISP2-DRY2T contains six unique GCFs absent elsewhere in the clade. Culture-based assays were used to demonstrate the antibacterial activity of A15ISP2-DRY2T against two drug-resistant human pathogens. We thus determine A15ISP2-DRY2T to be a novel bacterial species with considerable biosynthetic potential and propose the systematic name Streptomyces ortus sp. nov. Impact StatementThe Streptomyces genus has contributed more to our antibiotic arsenal than any other group of bacteria or fungi. Despite decades of exploration, global analysis has suggested they still possess more undiscovered biosynthetic diversity than any other bacterial group. Isolating novel species of Streptomyces is therefore a priority for antibiotic discovery. Here we isolate a novel strain from a deep-sea sponge and use comparative cluster analysis to identify six biosynthetic clusters unique to our deep-sea strain. This work demonstrates the utility of continuing to isolate novel Streptomyces strains for antibiotic discovery and, for the first time, we used species tree-gene cluster tree reconciliation to assess the contribution of vertical evolution on the biosynthetic gene cluster content of Streptomyces.

genomics↗

bioRxiv2022

Molecular architecture of the autoinhibited kinesin-1 lambda particle

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the alpha-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1, in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drive kinesin-1 activation. Summary statementIntegration of computational structure prediction with electron microscopy reveals the coiled-coil architecture of the autoinhibited compact conformer of the microtubule motor, kinesin-1.

cell biology↗

bioRxiv2021

Molecular mechanism for kinesin-1 direct membrane recognition

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxy-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical and cell biology approaches we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a newly defined subset of long kinesin light chain paralogues and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids and is important for kinesin-1 dependent lysosome positioning, a canonical activity that until now has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a new protein-lipid coincidence detection framework for kinesin-1 mediated organelle transport.

cell biology↗

bioRxiv2020

Bacterial flagella disrupt host cell membranes and interact with cytoskeletal components

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of ligands and mechanisms of binding are still debated. In this study, the molecular basis of E. coli O157:H7 and S. Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. SEM revealed flagella disappearing into cellular surfaces and TEM of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6), and S. Typhimurium (P1 & P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerisation rates.

microbiology↗

bioRxiv2019

In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organisation to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small-molecule induced, microtubule-based cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in-situ study of microtubule structure and contents.

cell biology↗