Biology subjects

Verkade, P.

Publications and source records attributed to Verkade, P..

14 recordsLinked to original sources

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

ER-liquid condensate contacts sequester FAM134B/C and RhoA to govern cell morphology

ER-phagy receptors have elusive physiological functions beyond ER remodelling. Here, we use proximity biotinylation to identify their cytoplasmic interactomes. Secondary CRISPR/Cas9 screening reveals regulators of the prototypical FAM134B/C receptors, which include PRKAR1A, canonically known as a subunit of PKA. PRKAR1A directly binds an amphipathic helix in the otherwise disordered cytoplasmic domain of FAM134B. Super-resolution, FRAP and CLEM imaging reveal novel interorganellar contacts between liquid-like condensates of cAMP-bound PRKAR1A and the ER. Condensates promote clustering of ER-embedded FAM134B/C with LC3B, independently of regulation of PKA by PRKAR1A. Proteomics reveal that cytoplasmic RhoA interacts with FAM134B/C clusters and that sequestration of both these molecules occurs within lysosomes embedded within the proximal condensates. This results in reduced actomyosin contractility and ER-condensate interactions thusly determine cell morphology and cancer cell invasion modality. In summary, ER-condensate contacts mediated by FAM134B/C are novel cellular degradation hubs that coordinate ER and cellular remodelling.

cell biology↗

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↗

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

Five inhibitory receptors display distinct vesicular distributions in T cells

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

immunology↗

bioRxiv2023

TECPR1 provides E3-ligase like activity to the ATG5-ATG12 complex to conjugate LC3/ATG8 to damaged lysosomes

Autophagosomes deliver cytosolic material to lysosomes to provide amino acids during starvation and to degrade damaged proteins and organelles to maintain tissue homeostasis. Delivery to lysosomes requires LC3/ATG8 (LC3), the major membrane protein of the autophagosome, that uses adaptor proteins to capture cargo and recruits tethering and SNARE proteins to promote fusion with lysosomes. LC3 is also recruited to endo-lysosome compartments in response to increases in vacuolar pH to facilitate degradation of material entering cells by endocytosis. A series of ubiquitin-like reactions conjugate LC3 to amino groups exposed by phosphatidylethanolamine (PE) or phosphatidylserine (PS) in target membranes. The E1 and E2-ubiquitin-like activities of ATG7 and ATG3 use thioester bonds to transfer LC3 to the ATG5-ATG12 conjugate. At the same time binding of ATG16L1 to ATG5-ATG12 provides the E3 ubiquitin-ligase like activity necessary to conjugate LC3 to PE or PS. The ATG5-ATG12 conjugate can also bind TECPR1 (tectonin beta propeller repeat-containing protein) which shares an ATG5 interaction (AIR) region with ATG16L1 and can binds LC3 directly through a LC3 interaction region (LIR). In this study we have used cells lacking ATG16L1 to determine if TECPR1 can substitute for ATG16L1 during LC3 conjugation. The results show that ATG16L1-/-MEFS can conjugate LC3 to lysosomes damaged by chloroquine or LLOMes and conjugation is dependent on the ubiquitin-like enzymes ATG3, ATG5 and ATG7 upstream of ATG16L1. TECPR1, ATG5 and galectin 3 are recruited to LAMP positive damaged lysosomes in the absence of ATG16L1 suggesting that TECPR-1 recruits ATG5-ATG12 to conjugate LC3 to damaged lysosomes. This was confirmed when truncation of TECPR1 at the central PH domain required for lysosome binding prevented LC3 conjugation, and LC3 conjugation could be restored by full length TECPR1. Recruitment of TECPR1 to damaged lysosomes required the N-terminal LIR motif and was partially dependent on the central PH domain that binds PI4P exposed during lysosome repair. TECPR1 can therefore conjugate LC3 to damaged lysosomes independently of ATG16L1 by providing E3 ligase-like activity to ATG5-ATG12. Direct conjugation of LC3 by TECPR1 may contribute to the autophagosome tethering functions reported for TECPR1 by increasing recruitment of cargo receptors, tethering proteins and SNARE proteins required for fusion with lysosomes and protein degradation. TECPR1-dependent conjugation of LC3 may also facilitate lysosome repair pathways involving autophagic lysosomal reformation and transcriptional activation of autophagy by TFEB.

cell biology↗

bioRxiv2023

Self-assembly of nanofilaments in cyanobacteria for protein co-localization

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts but, so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that overexpression of a modified bacterial microcompartment shell protein, PduA*, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly only had a minor impact on cellular metabolism. Finally, we targeted the fluorescent reporter mCitrine to the nanofilaments using an encapsulation peptide that natively interacts with PduA. To our knowledge, this is the first study to apply bacterial microcompartment based nanotechnology in cyanobacteria. The establishment of nanofilaments in cyanobacterial cells is an important step towards cellular organization of heterologous pathways and the establishment of cyanobacteria as next generation hosts.

synthetic biology↗

bioRxiv2022

The Free Fatty Acid-Binding Pocket is a Conserved Hallmark in Pathogenic β-Coronavirus Spike Proteins from SARS-CoV to Omicron

As COVID-19 persists, severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor-binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA-binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic {beta}-coronaviruses ({beta}-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2 and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation while the open, infectious conformation is LA-free. Electron tomography of SARS-CoV-2 infected cells reveals that LA-treatment inhibits viral replication, resulting in fewer, deformed virions. Our results establish FFA-binding as a hallmark of pathogenic {beta}-CoV infection and replication, highlighting potential antiviral strategies. One-Sentence SummaryFree fatty acid-binding is conserved in pathogenic {beta}-coronavirus S proteins and suppresses viral infection and replication.

biochemistry↗

bioRxiv2021

Maintenance of Complex I and respiratory super-complexes by NDUF-11 is essential for respiratory function, mitochondrial structure and health in C. elegans

Mitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identified B0491.5 (NDUF-11) as the C. elegans homologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing expression by RNAi allowed development to the adult stage, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, resulting in excessive detrimental ROS production. Meanwhile, electron cryo-tomography highlight aberrant cristae morphology and widening of the inter-membrane space and cristae junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development highlights the importance of complex I/ super-complex maintenance. Their perturbation by this, or other means, is likely to be the cause of metabolic stress and disease.

molecular biology↗

bioRxiv2020

De Novo Designed Peptide and Protein Hairpins that Self-assemble into Sheets and Nanoparticles.

The design and assembly of peptide based materials has advanced considerably, leading to a variety of fibrous, sheet and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here we present a de novo peptide system that forms nanoparticles or sheets depending on the strategic placement of a disulfide pin between two elements of secondary structure that drive self-assembly. Specifically, we join homodimerizing and homotrimerizing de novo coiled-coil -helices with a flexible linker to generate a series of linear peptides. The helices are pinned back-to-back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling and advanced microscopy show that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.

synthetic 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

Membrane insertion of chromogranin B for granule maturation in regulated secretion

Regulated secretion serves responses to specific stimuli in eukaryotes. An anion conductance was found essential for maturation and acidification of secretory granules four decades ago, but its genetic identity was unknown. We now demonstrate that chromogranin B (CHGB), an obligate granule protein, constitutes the long-sought anion channel. High-pressure freezing immuno-electron microscopy and biochemical assays showed native CHGB in close proximity to secretory granule membranes, and its membrane-bound and soluble forms both reconstituted Cl- channels. Release of secretory granules delivered CHGB clusters to plasma membranes, which dominate whole-cell anion conductance. Intragranular pH measurements and cargo maturation assays found that CHGB channels supported proinsulin - insulin conversion and dopamine-loading in neuroendocrine cells. {beta}-cells from Chgb-/- mice exhibited significant granule deacidification, accounting for hyperproinsulinemia, altered glucose-tolerance response and lower dopamine concentration in chromaffin granules in these animals. Membrane insertion of well-conserved CHGB is thus indispensable for granule maturation in exocrine, endocrine and neuronal cells. HighlightsO_LINative CHGB is amphipathic and distributes in the lumen and membranes of secretory granules with contrastingly different destinies and functions. C_LIO_LINative CHGB, once delivered to cell surface via granule exocytosis, dominates anion conductance in plasma membranes. C_LIO_LICHGB channels facilitate granule acidification and cargo maturation in cultured and primary neuroendocrine cells. C_LIO_LICHGB channels from bovine, rat and mouse cells all serve the long-missing, intra-organellar anion shunt pathway in the secretory granules for regulated secretion. C_LI

cell biology↗

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↗