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bioRxiv2022

Insights into the activation of Kinesin1 from the molecular characterisation of JIP3/4 binding to Kif5b

Whereas our understanding of kinesin auto-inhibition mechanisms is improving faster, important insights into kinesin activation mechanisms such as those controlled by cargo-motor adaptors are still missing. JIP3 and JIP4 are versatile motor-cargo adaptors for kinesin1 and dynein-dynactin motors enabling bi-directional transport on microtubules. JIP3 activates kinesin1 heavy chains, independently of kinesin1 light chains. In this report, we characterize the molecular details of the binding of the kinesin1 heavy chain, Kif5b to the motor-cargo adaptors, JIP3 and JIP4, using biophysical approaches. The definition of the exact binding site of Kif5b, as well as the specificity of interaction between JIP3 and JIP4 provide new insights into kinesin1 activation.

biophysics↗

bioRxiv2022

Cryo-EM reveals the membrane binding phenomenon of EspB, a virulence factor of the Mycobacterial Type VII secretion system

Mycobacterium tuberculosis utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. ESX-1 is one of the essential and well-studied secretion systems which transport various virulence factors, including EspB. EspB, a ~36 kDa secreted substrate, has been implicated to play vital role in protecting the bacteria from hostile environment within the host cell phagosome. It is also involved in bacterial pathogenesis and has been shown to bind phospholipids. Recently, two cryo-EM structures of EspB full-length and the secreted isoforms were resolved. Despite the availability of multiple high-resolution structures of EspB, the physiological relevance and mechanism of virulence of this secreted substrate remains poorly characterized. In this current work, we implemented cryo-EM-based structural studies, including various functional assays, TEM imaging, and biophysical approach to demonstrate the interaction of EspB with lipids and bio-membrane. Our findings also indicated that EspB may play a crucial role in binding to and rupturing host mitochondrial membrane. Through cryo-EM studies we were able to show the possible membrane-binding region of EspB. Our study sheds light on host-pathogen interactions and bacterial pathogenesis mediated by EspB.

biophysics↗

bioRxiv2022

Loss of the first β-strand of human prion protein generates an aggregation-competent partially "open" form

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). The pathway of PrP misfolding is still unclear, though previous data indicate the presence of a structural core in cellular PrP (PrPC), whose cooperative unfolding presents a substantial energy barrier on the path to prion formation. PrP is a GPI-anchored membrane protein, and a number of studies suggest that membrane interactions play an important role in the conversion of PrPC to its disease-associated form, including a transmembrane form of PrP in which a highly conserved region (residues 110 - 136) spans the ER membrane. Insertion of this region results in the detachment of the PrPC first {beta}-strand from the structural core. The effect of this removal on the structure, stability and self-association of the folded domain of PrPC is determined here through a biophysical characterisation of a truncated form of PrPC lacking this region. Whilst markedly destabilised, NMR chemical shifts show that the truncated protein exhibits tertiary structure characteristic of a fully folded protein and retains its native secondary structure elements, including the second strand of the PrP {beta}-sheet, but with altered conformational flexibility in the {beta}2-2 loop and first -helix. The latter is destabilised relative to the other helical regions of the protein, with markedly increased solvent exposure. This truncated form of PrP fibrilises more readily than the native form of the protein. These data suggest a stepwise mechanism, in which a destabilised "open" form of PrPC may be a key intermediate in the refolding to the fibrillar, pathogenic form of the protein.

biophysics↗

bioRxiv2022

Topological Gelation of Reconnecting Polymers

DNA recombination is a ubiquitous process that ensures genetic diversity. Contrary to textbook pictures, DNA recombination, as well as generic DNA translocations, occur in a confined and highly entangled environment. Inspired by this observation, here we investigate a solution of semiflexible polymer rings undergoing generic cutting and reconnection operations under spherical confinement. Our setup may be realised using engineered DNA in presence of recombinase proteins or by considering micelle-like components able to form living (or reversibly breakable) polymer rings. We find that in such systems there is a topological gelation transition, which can be triggered by increasing either the stiffness or concentration of the rings. Flexible or dilute polymers break into an ensemble of short, unlinked and segregated rings, whereas sufficiently stiff or dense polymers self-assemble into a network of long, linked and mixed loops, many of which are knotted. We predict the two phases should behave qualitatively differently in elution experiments monitoring the escape dynamics from a permeabilised container. Besides shedding some light on the biophysics and topology of genomes undergoing DNA reconnection in vivo, our findings could be leveraged in vitro to design polymeric complex fluids, e.g., DNA-based complex fluids or living polymer networks, with desired topologies.

biophysics↗

bioRxiv2022

Design principles of 3D epigenetic memory systems

The epigenetic state of a cell is associated with patterns of chemical modifications of histones ("marks") across the genome, with different marks typical of active (euchromatic) and inactive (heterochromatic) genomic regions. These mark patterns can be stable over many cell generations--a form of epigenetic memory--despite their constant erosion due to replication and other processes. Enzymes that place histone marks are often stimulated by the same marks, as if "spreading" marks between neighboring histones. But this positive feedback may not be sufficient for stable memory, raising the question of what is. In this work, we show how 3D genome organization--in particular, the compartmental segregation of euchromatin and heterochromatin-- could serve to stabilize an epigenetic memory, as long as (1) there is a large density difference between the compartments, (2) the modifying enzymes can spread marks in 3D, and (3) the enzymes are limited in abundance relative to their histone substrates. We introduce a biophysical model stylizing chromatin and its dynamics through the cell cycle, in which enzymes spread self-attracting marks on a polymer. We find that marks localize sharply and stably to the denser compartment, but over several cell generations, the model generically exhibits uncontrolled spread or global loss of marks. Strikingly, imposing limitation of the modifying enzymes--a plausible but oft-neglected element--totally changes this picture, yielding an epigenetic memory system, stable for hundreds of cell generations. Our model predicts a rich phenomenology to compare to experiments, and reveals basic design principles of putative epigenetic memory systems relying on compartmentalized 3D genome structure for their function.

biophysics↗

bioRxiv2022

Technical insights into fluorescence lifetime microscopy of mechanosensitive Flipper probes

Measuring forces within living cells remains a technical challenge. We developed hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing and can report on membrane tension. Here, we describe technical optimization of the probe imaging, and diverse characterizations in various biological and in vitro systems. We provide a guideline to measure biophysical parameters of cellular membranes by FLIM microscopy with Flipper probes, providing evidences that flippers can report long range forces in cells, tissues and organsi.

biophysics↗

bioRxiv2022

Emergent chirality in active solid rotation of pancreas spheres

Collective cell dynamics play a crucial role in many developmental and physiological contexts. While two-dimensional (2D) cell migration has been widely studied, how three-dimensional (3D) geometry and topology interplay with collective cell behavior to determine dynamics and functions remains an open question. In this work, we elucidate the biophysical mechanism underlying rotation in spherical tissues, a phenomenon widely reported both in vivo and in vitro. Using murine pancreas-derived organoids as a model system, we find that epithelial spheres exhibit persistent rotation, rotational axis drift and rotation arrest. Using a 3D vertex model, we demonstrate how the interplay between traction force and polarity alignment can account for these distinct rotational dynamics. Furthermore, our analysis shows that the spherical tissue rotates as an active solid and exhibits spontaneous chiral symmetry breaking. Using a continuum model, we demonstrate how the types and location of topological defects in the polarity field underlie this symmetry breaking process. Altogether, our work shows that tissue chirality can arise via topological defects in the pattern of cell traction forces, with potential implications for left-right symmetry breaking processes in morphogenetic events.

biophysics↗

bioRxiv2022

Single-particle tracking and machine-learning classification reveals heterogeneous Piezo1 diffusion

The mechanically-activated ion channel PIEZO1 is critical to numerous physiological processes, and is activated by diverse mechanical cues. The channel is gated by membrane tension and has been found to be mobile in the plasma membrane. We employed single particle tracking (SPT) of endogenous, tdTomato-tagged PIEZO1 using Total Internal Reflection Fluorescence Microscopy in live cells. Application of SPT unveiled a surprising heterogeneity of diffusing PIEZO1 subpopulations, which we labeled "mobile" and "immobile". We sorted these trajectories into the two aforementioned categories using trajectory spread. To evaluate the effects of the plasma membrane composition on PIEZO1 diffusion, we manipulated membrane composition by depleting or supplementing cholesterol, or by adding margaric acid to stiffen the membrane. To examine effects of channel activation on PIEZO1 mobility, we treated cells with Yoda1, a PIEZO1 agonist, and GsMTx-4, a channel inhibitor. We collected thousands of trajectories for each condition, and found that cholesterol removal and Yoda1 incubation increased the channels propensity for mobility. Conversely, we found that GsMTx-4 incubation and cholesterol supplementation resulted in a lower chance of mobile trajectories, whereas margaric acid incubation did not have a significant effect on PIEZO1 mobility. The "mobile" trajectories were analyzed further by fitting the time-averaged mean-squared displacement as a function of lag time to a power-law model, revealing mobile PIEZO1 puncta exhibit anomalous subdiffusion. These studies illuminate the fundamental properties governing PIEZO1 diffusion in the plasma membrane and set the stage to determine how cellular processes and interactions may influence channel activity and mobility. SIGNIFICANCEPIEZO1 is a mechanically-activated ion channel that regulates a number of physiological processes. Here we examine a fundamental biophysical property of PIEZO1 - its movement in the plasma membrane. We find that the mobility of PIEZO1 is surprisingly heterogeneous, with some PIEZO1 puncta showing high mobility and some displaying very limited mobility. Cholesterol depletion from the plasma membrane increases PIEZO1 mobility while cholesterol supplementation decreases mobility. Yoda1 treatment increases PIEZO1 mobility whereas GsMTx-4 treatment decreases channel mobility.

biophysics↗

bioRxiv2022

Structure and assembly of an extremely long bacteriophage tail tube

Tail tube assembly is an essential step in the assembly of long-tailed bacteriophages. Limited structural and biophysical information has impeded an understanding of assembly and stability of their long, flexible tail tubes. The hyperthermophilic phage P74-26 is particularly intriguing as it has the longest tail of any known virus (nearly 1 m) and is the most stable known phage. Here, we present the structure of the P74-26 tail tube and introduce an in vitro system for studying the kinetics of tube assembly. Our high resolution cryo-EM structure provides insight into how the P74-26 phage achieves its flexibility and thermostability through assembly of flexible loops into neighboring rings through tight "ball-and-socket"-like interactions. Guided by this structure, and in combination with mutational, light scattering, and molecular dynamics simulations data, we propose a model for the assembly of conserved tube-like structures across phage and other entities possessing Tail Tube-like proteins. Our model proposes that formation of a full ring licenses the adoption of a tube elongation-competent conformation among the flexible loops and their corresponding sockets, which is further stabilized by an adjacent ring. Tail assembly is controlled by the cooperative interaction of dynamic intra- and inter-ring contacts. Given the structural conservation among tail tube proteins and tail-like structures, our model can explain the mechanism of high-fidelity assembly of long, stable tubes.

biophysics↗

bioRxiv2022

The maximum solubility product marks the threshold for condensation of multivalent biomolecules

Clustering of weakly interacting multivalent biomolecules underlies the formation of membraneless compartments known as condensates. As opposed to single component (homotypic) systems, the concentration dependence of multi-component (heterotypic) condensate formation is not well understood. We previously proposed the solubility product (SP), the product of monomer concentrations in the dilute phase, as a tool for understanding the concentration dependence of multi-component systems. In the current study, we further explore the limits of the SP concept using spatial Langevin dynamics and rule-based stochastic simulations. We show, for a variety of idealized molecular structures, how the maximum SP coincides with the onset of the phase transition, i.e., the formation of large clusters. We reveal the importance of intra-cluster binding in steering the free and cluster phase molecular distributions. We also show how structural features of biomolecules shape the solubility product profiles. The interplay of flexibility, length and steric hindrance of linker regions controls the phase transition threshold. Remarkably, when solubility products are normalized to non-dimensional variables and plotted against the concentration scaled to the threshold for phase transition, the curves all coincide independent of the structural features of the binding partners. Similar coincidence is observed for the normalized clustering vs. concentration plots. Overall, the principles derived from these systematic models will help guide and interpret in vitro and in vivo experiments on the biophysics of biomolecular condensates. Significance StatementBiomolecular condensates are macroscopic intracellular structures that are composed of weakly interacting macromolecules. Because their composition can be complex, there are no simple rules for how condensates form as a function of the concentrations of their individual components. In this work, we show how the solubility product (SP), the product of monomer concentrations in the dilute phase, might serve as a tool for predicting the concentration dependence for condensation of multi-component systems. Specifically, Langevin dynamics simulations of the clustering of a series of multivalent binding partners reveals how the maximum SP is always attained at the same concentration as the appearance of large clusters. Experimental application of the SP concept should help rationalize the cellular formation of biomolecular condensates.

biophysics↗

bioRxiv2022

Mechanosensitive channel-based optical membrane tension biosensor

Plasma membrane tension functions as a global physical organizer of cellular activities. Technical limitations of current membrane tension measurement techniques have hampered in-depth investigation of cellular membrane biophysics and the role of plasma membrane tension in regulating cellular processes. Here, we develop an optical membrane tension biosensor by repurposing an E. coli mechanosensitive channel via insertion of circularly permuted GFP (cpGFP), which undergoes a large conformational rearrangement associated with channel activation and thus fluorescence intensity changes under increased membrane tension.

biophysics↗

bioRxiv2022

PIEZO1 regulates leader cell formation and cellular coordination during collective keratinocyte migration

The collective migration of keratinocytes during wound healing requires both the generation and transmission of mechanical forces for individual cellular locomotion and the coordination of movement across cells. Leader cells along the wound edge transmit mechanical and biochemical cues to ensuing follower cells, ensuring their coordinated direction of migration across multiple cells. Despite the observed importance of mechanical cues in leader cell formation and in controlling coordinated directionality of cell migration, the underlying biophysical mechanisms remain elusive. The mechanically-activated ion channel PIEZO1 was recently identified to play an inhibitory role during the reepithelialization of wounds. Here, through an integrative experimental and mathematical modeling approach, we elucidate PIEZO1s contributions to collective migration. Time-lapse microscopy reveals that PIEZO1 activity inhibits leader cell formation at the wound edge. To probe the relationship between PIEZO1 activity, leader cell formation and inhibition of reepithelialization, we developed an integrative 2D continuum model of wound closure that links observations at the single cell and collective cell migration scales. Through numerical simulations and subsequent experimental validation, we found that coordinated directionality plays a key role during wound closure and is inhibited by upregulated PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits coordinated directionality between cells during collective migration. Author summaryDuring the healing of a wound, cells called keratinocytes that make up the outer layer of the skin migrate collectively to close the wound gap. The mechanically activated ion channel PIEZO1 was previously found to inhibit wound closure. Here, through a combined modeling and experimental approach, we investigate the role of PIEZO1 in regulating collective migration. Specialized cells called leader cells, which typically form along the wound edge, are important for guiding the migration of neighboring cells. These leader cells dictate the coordinated directionality, or the cohesiveness of the migration direction between neighboring cells, through the transmission of mechanical and biochemical cues. We find that PIEZO1 activity inhibits the formation of these leader cells and, as a result, inhibits cell coordinated directionality causing the collective movement of cells to become disorganized and less effective in closing the wound. Our findings shed light on the complex mechanisms underlying collective migration, providing valuable insight into how mechanical cues affect the movement of cells during wound closure.

biophysics↗

bioRxiv2022

An experimental demonstration of ensemble epistasis in the lac repressor

Epistatic, non-additive, interactions between mutations reveal the functional architecture of living systems, strongly shape evolution, and present a difficult challenge for bioengineers. Interpreting and modeling epistasis requires knowledge of the mechanisms that bring it about. We recently argued that "ensemble epistasis" could be a generic mechanism for epistasis between mutations introduced into a single macromolecule. Because proteins exist as ensembles of interconverting conformations, a mutation could induce epistasis by subtly altering ensemble composition and thus the effects of subsequent mutations. Here we show experimentally that the thermodynamic ensemble does indeed yield high magnitude epistasis in the lac repressor. We observed two- and three-way epistasis in DNA binding, with magnitudes as large or larger than the individual effects of mutations. This biophysical effect propagated to substantial epistasis in gene expression in vivo. As predicted in previous theoretical work, IPTG concentration tunes the magnitude of ensemble epistasis. Further, our observations could all be captured with a rigorous mathematical model of the lac repressor ensemble. Given that conformational ensembles are unavoidable features of macromolecules, we expect this is a ubiquitous and underappreciated cause of intramolecular epistasis.

biophysics↗

bioRxiv2022

Thumb-domain dynamics modulate the functional repertoire of DNA-Polymerase IV (DinB)

In order to cope with the risk of stress-induced mutagenesis, cells in all kingdoms of life employ Y-family DNA polymerases to resolve resulting DNA lesions and thus maintaining the integrity of the genome. In Escherichia coli (E. coli) the DNA polymerase IV, or DinB, plays this crucial role in coping with these type of mutations via the so-called translesion DNA synthesis. Despite the availability of several high-resolution crystal structures important aspects of the functional repertoire of DinB remain elusive. In this study, we use advanced solution NMR spectroscopy methods in combination with biophysical characterization to elucidate the crucial role of the Thumb domain within DinBs functional cycle. We find that the inherent dynamics of this domain guide the recognition of double-stranded (ds) DNA buried within the interior of the DinB domain arrangement and trigger allosteric signals through the DinB protein. Subsequently, we characterized the RNA polymerase interaction with DinB, revealing an extended outside surface of DinB and thus not mutually excluding the DNA interaction. Altogether the obtained results lead to a refined model of the functional repertoire of DinB within the translesion DNA synthesis pathway.

biophysics↗

bioRxiv2022

Simulation of Gilbert Theory for Self-Association in Sedimentation Velocity Experiments: A Guide to Evaluate Best Fitting Models

There is a long tradition in the Biophysics community of using simulations as a means to understand macromolecular behavior in various physicochemical methods. This allows a rigorous means to interpret observations in terms of fundamental principles, including chemical equilibrium, reaction kinetics, transport processes and thermodynamics. Here we simulate data for the Gilbert Theory for self-association, a fundamental analytical ultracentrifuge (AUC) technique to understand the shape of sedimentation velocity reaction boundaries that involve reversible monomer-Nmer interactions. Simulating monomer-dimer through monomer-hexamer systems as a function of concentration about the equilibrium constant allows a visual means to differentiate reaction stoichiometry by determining end points and inflexion positions. Including intermediates (eg A1-A2-A3-A4-A5-A6) in the simulations reveals the smoothing of the reaction boundary and the removal of sharp inflexions between monomers and polymers. The addition of cooperativity restores sharp boundaries or peaks to the observation and allows more discrimination in the selection of possible fitting models. Thermodynamic nonideality adds additional features when applied across wide ranges of concentration that might be appropriate for high concentration therapeutic monoclonal antibody (mAb) solutions. This presentation serves as a tutorial for using modern AUC analysis software like SEDANAL for selecting potential fitting models.

biophysics↗

bioRxiv2022

Tau amyloid polymorphism is shaped by local structural propensities of its protein sequence

Different tauopathies are characterized by specific amyloid filament folds that are conserved between patients. Disease-specific tau filament folds probably reflect the specific pathological contexts leading to their formation including isoforms or post-translational modifications. Little is known, however, as to whether and how intrinsic conformational tendencies of the tau sequence itself contribute to its polymorphism. Using cryo-EM structure determination we find that a short amyloidogenic C-terminal peptide consisting of residues 350-362 of the tau repeat domain adopts the same polymorphic conformations in isolation as it does in the context of major disease-associated protofilament folds. Biophysical characterisation and molecular modelling show that the amyloid conformations adopted by this peptide constitute core structural motifs stabilizing distinct disease-associated tau filament folds. In accordance this segment also contributes to the efficient propagation of human AD tau seeds in tau reporter cells while it is irrelevant to heparin-induced recombinant seeds. Our findings suggest that tau 350-362 is key to the propagation of disease-associated tau polymorphs and that the conformational preferences of this segment predispose to the topological diversity observed in tau filament folds.

biophysics↗

bioRxiv2022

Role of 14-3-3 proteins in human cardiac sodium channel Nav1.5 regulation

Background14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. AimsIn this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. Methods and ResultsAmongst the seven 14-3-3 isoforms, only 14-3-3{eta} (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3{eta} overexpression or inhibition with difopein, and 14-3-3{eta} did not affect physical interaction between Nav1.5 - subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. ConclusionsOur findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201. SummaryThis work shows that only 14-3-3{eta}, exhibits weak/transient interaction with Nav1.5, and does not modify its total protein expression, plasmalemmal trafficking, and basal biophysical properties of the whole-cell current. Furthermore, inhibition of endogenous 14-3-3/ligand interactions with difopein does not affect the dimerization of Nav1.5. Therefore, 14-3-3 proteins are suggested to be dispensable for the Nav1.5 regulation in a heterologous expression system.

biophysics↗

bioRxiv2022

Cell remote mechanics regulates large-spatial matrix modeling with dynamic simulations

Tissues are often isotropic and heterogeneous organizations, which developmental processes are coordinated by cells and extracellular matrix modeling. Cells have the capability of modeling matrix in distance, however, the biophysical mechanism is largely unknown. We investigated underlying mechanism of large collagen I (COL) fibrillary modeling by cell mechanics with designed arrays of cell clusters. By incorporating dynamic contractions, Molecular Dynamics simulations yielded highly matching isotropic outcomes with observed COL clustering in experiments from variable geometrical arrays without spatial limitation. Further designed single polygons from triangles to hexagons resulted in predicted structural assembly which showed maintained spatial balance. Cell cytoskeletal integrity (actin filaments, microtubules), actomyosin contractions, and endoplasmic reticulum calcium channels were essential for remote fiber inductions, while membrane mechanosensitive integrin and Piezo showed coordinative role in regulating the fiber assembly. The study provides new insights on cell mechanics-induced isotropic matrix modeling with dynamic large-spatial scales and the associated cellular mechanism. The assembled biomechanical scaffolds with pre-designs may lead to applications in micro-tissue engineering. This work implicates heterogeneous tissue structures maybe partially derived from isotropic cell mechanics.

biophysics↗