Biology subjects

Knott, G. W.

Publications and source records attributed to Knott, G. W..

8 recordsLinked to original sources

bioRxiv2025

A bioengineered human urothelial organoid model reveals the urine-urothelium interplay in tissue resilience and UPEC recurrence in urinary tract infections

Urine is a dynamic and highly variable biofluid. Urine-urothelium interactions are a critical yet underexplored factor in bladder homeostasis and urinary tract infections (UTIs). Here, we report on a human mini-bladder model that exposes a stratified urothelium to urine of defined composition, and incorporates micturition. Prolonged exposure to high-solute concentration urine weakens tight junctions, dysregulates immune responses, and reduces bladder tissue resilience. This increases susceptibility to colonization of the bladder by uropathogenic E. coli (UPEC) which reduces efficacy of antibiotic therapy. In high-solute concentration urine, Fosfomycin monotherapy - prescribed for uncomplicated UTIs, induces the formation of cell wall-deficient (CWD) UPEC in the urine (as observed in patients with recurrent UTIs) but also within deeper urothelial layers. Tissue-associated CWD UPEC directly contributed to recurrence. Our findings expand the conceptual role for CWD UPEC in UTIs, and demonstrate the power of the mini-bladder platform to capture urine-urothelial microenvironment dynamics that actively shape UTI pathogenesis and antibiotic tolerance.

bioengineering↗

bioRxiv2023

Membrane mechanics dictate axonal morphology and function

Axons are thought to be ultrathin membrane cables of a relatively uniform diameter, designed to conduct electrical signals, or action potentials. Here, we demonstrate that unmyelinated axons are not simple cylindrical tubes. Rather, axons have nanoscopic boutons repeatedly along their length interspersed with a thin cable with a diameter of [~]60 nm like pearls-on-a-string. These boutons are only [~]200 nm in diameter and do not have synaptic contacts or a cluster of synaptic vesicles, hence non-synaptic. Our in silico modeling suggests that axon pearling can be explained by the mechanical properties of the membrane including the bending modulus and tension. Consistent with modeling predictions, treatments that disrupt these parameters like hyper- or hypo-tonic solutions, cholesterol removal, and non-muscle myosin II inhibition all alter the degree of axon pearling, suggesting that axon morphology is indeed determined by the membrane mechanics. Intriguingly, neuronal activity modulates the cholesterol level of plasma membrane, leading to shrinkage of axon pearls. Consequently, the conduction velocity of action potentials becomes slower. These data reveal that biophysical forces dictate axon morphology and function and that modulation of membrane mechanics likely underlies plasticity of unmyelinated axons.

neuroscience↗

bioRxiv2021

NeuroMechFly, a neuromechanical model of adult Drosophila melanogaster

Animal behavior emerges from a seamless interaction between neural network dynamics, musculoskeletal properties, and the physical environment. Accessing and understanding the interplay between these intertwined elements requires the development of integrative and morphologically realistic neuromechanical simulations. Until now, there has been no such simulation framework for the widely studied model organism, Drosophila melanogaster. Here we present NeuroMech-Fly, a data-driven model of the adult female fly within a physics-based simulation environment. NeuroMechFly combines a series of independent computational modules including a biomechanical exoskeleton with articulating body parts-legs, halteres, wings, abdominal segments, head, proboscis, and antennae-muscle models, and neural network controllers. To enable illustrative use cases, we first define minimal leg degrees-of-freedom by analyzing real 3D kinematic measurements during real Drosophila walking and grooming. Then, we show how, by replaying these behaviors using NeuroMechFlys biomechanical exoskeleton in its physics-based simulation environment, one can predict otherwise unmeasured torques and contact reaction forces. Finally, we leverage NeuroMechFlys full neuromechanical capacity to discover neural networks and muscle parameters that enable locomotor gaits optimized for speed and stability. Thus, NeuroMechFly represents a powerful testbed for building an understanding of how behaviors emerge from interactions between complex neuromechanical systems and their physical surroundings.

neuroscience↗

bioRxiv2021

Dynamic persistence of intracellular bacterial communities of uropathogenic Escherichia coli in a human bladder-chip model of urinary tract infections

Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbours of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

microbiology↗

bioRxiv2020

Disentangling the sequence, cellular and ultrastructural determinants of Huntingtin nuclear and cytoplasmic inclusion formation

Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntingtons disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon1 of the Htt protein. Our findings provide novel insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve 1) polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; 2) recruitment and accumulation of remodeled or dysfunctional membranous organelles, and 3) impairment of the protein quality control and degradation machinery. We also show that nuclear and cytoplasmic Htt inclusions exhibit distinct biochemical compositions and ultrastructural properties, suggesting different mechanisms of aggregation and toxicity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/226977v2_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@295056org.highwire.dtl.DTLVardef@594664org.highwire.dtl.DTLVardef@11c5384org.highwire.dtl.DTLVardef@af0e0a_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic depictions and original electron micrographs of cytoplasmic inclusions formed by native (tag-free) mutant Huntingtin exon1 proteins (Httex1 72Q, left) and the corresponding GFP fusion protein (Httex1 72Q-GFP, right).

neuroscience↗

bioRxiv2020

Maturation of complex synaptic connections of layer 5 cortical axons in the posterior thalamic nucleus requires SNAP25

Synapses are able to form in the absence of neuronal activity, but how is their subsequent maturation affected in the absence of regulated vesicular release? We explored this question using 3D electron microscopy and immuno electron microscopy analyses in the large, complex synapses formed between cortical sensory efferent axons and dendrites in the posterior thalamic nucleus. Using a Snap25 conditional knockout we found that during the first two postnatal weeks the axonal boutons emerge and increase in the size similar to the control animals. However, by P18, when an adult-like architecture should normally be established, axons were significantly smaller with 3D reconstructions showing that each Snap25-cko bouton only forms a single synapse with the connecting dendritic shaft. No excrescences from the dendrites were formed, and none of the normally large glomerular axon endings were seen. These results show that activity mediated through regulated vesicular release from the presynaptic terminal is not necessary for the formation of synapses, but it is required for the maturation of the specialised synaptic structures between layer 5 corticothalamic projections in Po.

neuroscience↗

bioRxiv2020

Ultrastructural comparison of dendritic spine morphology preserved with cryo and chemical fixation

Previously we showed that cryo fixation of brain tissue gave a truer representation of brain ultrastructure in comparison with a standard chemical fixation method (Korogod et al 2005). Extracellular space matched physiological measurements, there were larger numbers of docked vesicles and less glial coverage of synapses and blood capillaries. Here, using the same preservation approaches we compared the morphology of dendritic spines. We show that the length of the spine and the volume of its head is unchanged, however, the spine neck width is thinner by more than 30 % after cryo fixation. In addition, the weak correlation between spine neck width and head volume seen after chemical fixation was not present in cryo-fixed spines. Our data suggest that spine neck geometry is independent of the spine head volume, with cryo fixation showing enhanced spine head compartmentalization and a higher predicted electrical resistance between spine head and parent dendrite.

neuroscience↗

bioRxiv2018

The architecture of the endoplasmic reticulum is regulated by the reversible lipid modification of the shaping protein CLIMP-63

The endoplasmic reticulum (ER) has a complex morphology generated and maintained by membrane-shaping proteins and membrane energy minimization, though not much is known about how it is regulated. The architecture of this intracellular organelle is balanced between large, thin sheets that are densely packed in the perinuclear region and a connected network of branched, elongated tubules that extend throughout the cytoplasm. Sheet formation is known to involve the cytoskeleton-linking membrane protein 63 (CLIMP-63), though its regulation and the depth of its involvement remain unknown. Here we show that the post-translational modification of CLIMP-63 by the palmitoyltransferase ZDHHC6 controls the relative distribution of CLIMP-63 between the ER and the plasma membrane. By combining data-driven mathematical modeling, predictions, and experimental validation, we found that the attachment of a medium chain fatty acid, so-called S-palmitoylation, to the unique CLIMP-63 cytoplasmic cysteine residue drastically reduces its turnover rate, and thereby controls its abundance. Light microscopy and focused ion beam electron microcopy further revealed that enhanced CLIMP-63 palmitoylation leads to strong ER-sheet proliferation. Altogether, we show that ZDHHC6-mediated S-palmitoylation regulates the cellular localization of CLIMP-63, the morphology of the ER, and the interconversion of ER structural elements in mammalian cells through its action on the CLIMP-63 protein.\n\nSignificance StatementEukaryotic cells subcompartmentalize their various functions into organelles, the shape of each being specific and necessary for its proper role. However, how these shapes are generated and controlled is poorly understood. The endoplasmic reticulum is the largest membrane-bound intracellular compartment, accounting for more than 50% of all cellular membranes. We found that the shape and quantity of its sheet-like structures are controlled by a specific protein, cytoskeleton-linking membrane protein 63, through the acquisition of a lipid chain attached by an enzyme called ZDHHC6. Thus, by modifying the ZDHHC6 amounts, a cell can control the shape of its ER. The modeling and prediction technique used herein also provides a method for studying the interconnected function of other post-translational modifications in organelles.

cell biology↗