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bioRxiv2017

Intraflagellar transport proteins undergo nonaxonemal staged hindrance between the recruiting distal appendages and the cilium

Primary cilia play a vital role in cellular sensing and signaling [1]. An essential component of ciliogenesis is intraflagellar transport (IFT), which first requires IFT-protein recruitment, IFT-protein-motor-protein assembly, axonemal engagement of IFT-protein complexes, and transition zone (TZ) gating [2-9]. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is exceedingly challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy (sptPALM) [10, 11], balancing the imaging requirements of tracking speed, tracking duration, and localization precision for IFT88-mEOS4b in live human retinal pigment epithelial (hTERT-RPE-1) cells. Intriguingly, we found that mobile IFT proteins \"switched gears\" multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal TZ, slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures, and reached the space enveloped by the ciliary pocket in the proximal TZ. Together, our live-cell superresolution imaging revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliogenesis homeostasis.

biophysics↗

bioRxiv2017

Cytoskeletal mechanisms of axonal contractility

Mechanotransduction is likely to be an important mechanism of signalling in thin, elongated cells like neurons. Maintenance of pre-stress or rest tension may facilitate mechanotransduction in these cells. In recent years, functional roles for mechanical tension in neuronal development and physiology are beginning to emerge but the cellular mechanisms regulating neurite tension remain poorly understood. Active contraction of neurites is a potential mechanism of tension regulation. In this study, we have explored cytoskeletal mechanisms mediating active contractility of neuronal axons. We have developed a simple assay where we evaluate contraction of curved axons upon trypsin-mediated detachment. We show that curved axons undergo contraction and straighten upon de-adhesion. Axonal straightening was found to be actively driven by actomyosin contractility, while microtubules may subserve a secondary role. We find that while axons show a monotonous decrease in length upon contraction, subcellularly, the cytoskeleton shows a heterogeneous contractile response. Further, using an assay for spontaneous development of tension without trypsin-induced de-adhesion, we show that axons are intrinsically contractile. These experiments, using novel experimental approaches, implicate the axonal cytoskeleton in tension homeostasis. Our data suggest that while globally the axon behaves as a mechanical continuum, locally the cytoskeleton is remodelled heterogeneously.

biophysics↗

bioRxiv2017

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single particle tracking and super-resolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in E. coli, where RNase E co-localizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Moreover, RNase E cluster positions correlate with the subcellular location of chromosomal loci of two highly transcribed ribosomal RNA genes, suggesting that RNase Es function in ribosomal RNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

biophysics↗

bioRxiv2017

Dynamical model of the CLC-2 ion channel exhibits a two-step gating mechanism

This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low ([~]1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population ([~]1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations. Author summaryIn the brain, the roles and mechanisms of sodium-, potassium-, and calcium-selective ion channels are well established. In contrast, chloride-selective channels have been studied much less and are not sufficiently understood, despite known associations of chloride-channel defects with brain disorders. The most broadly expressed voltage-activated chloride channel in the brain is CLC-2 (one of 9 human CLC homologs). In this work, we use simulations to model the conformational dynamics of the CLC-2 chloride ion channel selectivity filter (SF), which is the part of the protein that controls whether the channel is in an ion-conducting or non-conducting state. Our analysis identifies four primary conformational states and a specific progression through these states. Our results are consistent with structural and functional data in the literature and provide a high-resolution model for guiding further studies of CLC-2. These results will inform our understanding of how CLC-2 governs electrical activity and ion homeostasis in the brain.

biophysics↗

bioRxiv2017

On-line Optimization of Hamiltonian Replica Exchange Simulations

Replica exchange is a widely used sampling strategy in molecular simulation. While a variety of methods exist for optimizing temperature replica exchange, less is known about how to optimize more general Hamiltonian replica exchange simulations. We present an algorithm for the on-line optimization of both temperature and Hamiltonian replica exchange simulations that draws on techniques from the optimization of deep neural networks in machine learning. We optimize a heuristic-based objective function capturing the efficiency of replica exchange. Our approach is general, and has several desirable properties, including: (1) it makes few assumptions about the system of interest; (2) optimization occurs on-line wihout the requirement of pre-simulation; and (3) it readily generalizes to systems where there are multiple control parameters per replica. We explore some general properties of the algorithm on a simple harmonic oscillator system, and demonstrate its effectiveness on a more complex data-guided protein folding simulation.

biophysics↗

bioRxiv2017

Static length changes of cochlear outer hair cells can tune low-frequency hearing

The cochlea not only transduces sound-induced vibration into neural spikes, it also amplifies weak sound to boost its detection. Actuators of this active process are sensory outer hair cells in the organ of Corti, whereas the inner hair cells transduce the resulting motion into electric signals that propagate via the auditory nerve to the brain. However, how the outer hair cells modulate the stimulus to the inner hair cells remains unclear. Here, we combine theoretical modeling and experimental measurements near the cochlear apex to study the way in which length changes of the outer hair cells deform the organ of Corti. We develop a geometry-based kinematic model of the apical organ of Corti that reproduces salient, yet counter-intuitive features of the organs motion. Our analysis further uncovers a mechanism by which a static length change of the outer hair cells can sensitively tune the signal transmitted to the sensory inner hair cells. When the outer hair cells are in an elongated state, stimulation of inner hair cells is largely inhibited, whereas outer hair cell contraction leads to a substantial enhancement of sound-evoked motion near the hair bundles. This novel mechanism for regulating the sensitivity of the hearing organ applies to the low frequencies that are most important for the perception of speech and music. We suggest that the proposed mechanism might underlie frequency discrimination at low auditory frequencies, as well as our ability to selectively attend auditory signals in noisy surroundings.\n\nAuthor summaryOuter hair cells are highly specialized force producers inside the inner ear: they can change length when stimulated electrically. However, how exactly this electromotile effect contributes to the astonishing sensitivity and frequency selectivity of the inner ear has remained unclear. Here we show for the first time that static length changes of outer hair cells can sensitively regulate how much of a sound signal is passed on to the inner hair cells that forward the signal to the brain. Our analysis holds for the apical region of the inner ear that is responsible for detecting the low frequencies that matter most in speech and music. This shows a mechanisms for how frequency-selectivity can be achieved at low frequencies. It also opens a path for the efferent neural system to regulate hearing sensitivity.

biophysics↗

bioRxiv2017

Collective cell behaviour in mechanosensing of substrate thickness

Extracellular matrix stiffness has a profound effect on the behaviour of many cell types. Adherent cells apply contractile forces to the material on which they adhere, and sense the resistance of the material to deformation - its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 {micro}m) thicknesses. Here, we hypothesised that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic ECM-functionalised polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker labelled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 m. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater halfmaximal response at 54 m. Depth-sensing was dependent on ROCK-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.

biophysics↗

bioRxiv2017

Towards simple kinetic models of functional dynamics for a kinase subfamily

Kinases are ubiquitous enzymes involved in the regulation of critical cellular pathways and have been implicated in several cancers. Consequently, the kinetics and thermodynamics of prototypical kinases are of interest and have been the subject of numerous experimental studies. In-silico modeling of the conformational ensembles of these enzymes, on the other hand, is lacking due to inherent computational limitations. Recent algorithmic advances combined with homology modeling and parallel simulations allow us to address this computational sampling bottleneck. Here, we present the results of molecular dynamics (MD) studies for seven Src family kinase (SFK) members Fyn, Lyn, Lck, Hck, Fgr, Yes, and Blk. We present a sequence invariant extension to Markov state models (MSMs), which allows us to quantitatively compare the structural ensembles of the seven kinases. Our findings indicate that in the absence of their regulatory partners, SFK members have similar in-silico dynamics with active state populations ranging from 4-40% and activation timescales in the hundreds of microseconds. Furthermore, we observe several potentially druggable intermediate states, including a pocket next to the ATP binding site that could be potentially targeted via a small molecule inhibitors. These results establish the utility of MSMs for studying protein families.

biophysics↗

bioRxiv2017

Specific cholesterol binding drives drastic structural alterations in apolipoprotein A1

Protein adopts multitude of flexible and rapidly interconverting conformers, many which are governed by specific protein-interaction domains. ApoA1, a key player involved in high-density lipoprotein (HDL) regulation exists in structurally diverse forms with varying degree of cholesterol association, and each state is associated with different functional properties. While disc-shaped HDL and its oligomeric ApoA1 protein components have been the focus of several investigations, structural properties of monomeric ApoA1 are poorly understood. Here, we undertook large-scale structural analysis of ApoA1 in apo and cholesterol-bound forms using tens of independent simulations with total computing time exceeding 50 s. Examination of multiple lipid-free trajectories of monomeric ApoA1 revealed a common conformation, with distinct spatial proximity between N- and C-terminal domains. With incorporation of physiologically known cholesterol concentration ({approx}100 cholesterol molecules) in ApoA1 simulations, the monomeric protein spontaneously formed an open circular topology. Remarkably, these drastic structural perturbations are driven by specific binding site at C-terminal and a novel cholesterol binding site at the N-terminal. We proposed a mechanism of stage-wise opening of ApoA1 and demonstrated that less cholesterol concentration around interaction sites or mutation within N-terminal binding sites does not lead to open bell-shaped topology. The kinetic barriers between open and closed-states also showed an ensemble of loosely packed helix bundle (H1-H7; H4-H7) that posed as a slow-intermediate step. Lastly we performed complementary experiments, including ITC and CD measurements to confirm that structural changes are induced by ligand association and not driven by random hydrophobic effect. Collectively, our study suggests a previously unknown mechanism of cholesterol sequestering by ApoA1 that could directly aid in developing modulators for cholesterol efflux with chronic cardiovascular diseases.

biophysics↗

bioRxiv2017

Recovery of equilibrium free energy from non-equilibrium thermodynamics with mechanosensitive ion channels in E. coli

Bacterial mechanosensitive channels are major players in cells ability to cope with hypo-osmotic stress. Excess turgor pressure due to fast water influx is reduced as the channels, triggered by membrane tension, open and release osmolytes. However, in vitro measurements of the free energy difference between the open and closed states of ion channels are challenging due to hysteresis effects and inactivation. Exploiting recent developments in statistical physics, we present a general formalism to extract the free energy difference between the closed and open states of mechanosensitive ion channels from non-equilibrium work distributions associated with the channels gating recorded in native patches under ramp stimulation protocols. We show that the work distributions obtained from the gating of MscS channels in E. coli membrane satisfy the strong symmetry relations predicted by the fluctuation theorems and recover the equilibrium free energy difference between the closed and open states of the channel within 1 kB T of its best estimate obtained from an independent experiment.

biophysics↗

bioRxiv2017

A physics-explicit model of bacterial conjugation shows the stabilizing role of the conjugative junction

Conjugation is a process in which bacteria exchange DNA through a physical connection (conjugative junction) between mating cells. Despite its significance for processes such as the spread of antibiotic resistance, the role of physical forces in conjugation is poorly understood. Here we use computer models to show that the conjugative junction not only serves as a link to transfer the DNA but it also mechanically stabilises the mating pair which significantly increases the conjugation rate. We discuss the importance of our findings for biological evolution and suggest experiments to validate them.

biophysics↗

bioRxiv2017

Quantitative mass imaging of single molecules in solution

The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo- and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

biophysics↗

bioRxiv2017

Conformational Ensemble of RNA Oligonucleotides from Reweighted Molecular Simulations

We determine the conformational ensemble of four RNA tetranucleotides by using available nuclear magnetic spectroscopy data in conjunction with extensive atomistic molecular dynamics simulations. This combination is achieved by applying a reweighting scheme based on the maximum entropy principle. We provide a quantitative estimate for the population of different conformational states by considering different NMR parameters, including distances derived from nuclear Overhauser effect intensities and scalar coupling constants. We show the usefulness of the method as a general tool for studying the conformational dynamics of flexible biomolecules as well as for detecting inaccuracies in molecular dynamics force fields.

biophysics↗

bioRxiv2017

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

biophysics↗

bioRxiv2017

Formation of correlated chromatin domains at nanoscale dynamic resolution during transcription

Intrinsic dynamics of chromatin contribute to gene regulation. How chromatin mobility responds to genomic processes and whether this response relies on coordinated movement is still unclear. Here, we introduce an approach called Dense Flow reConstruction and Correlation (DFCC) to quantify correlation of chromatin motion with sub-pixel sensitivity at the level of the whole nucleus. DFCC is based on reconstructing dense global flow fields of fluorescent images acquired in real-time. By simulating variations in microscopic and dynamic parameters, we demonstrate that our approach is robust and more accurate than other methods to estimate flow fields and spatial correlations of dense structures such as chromatin. We applied our approach to analyze stochastic movements of DNA and histones based on direction and magnitude at different time lags in human cells. We observe long-range correlations extending over several m between coherently moving regions over the entire nucleus. Spatial correlation of global chromatin dynamics was reduced by inhibiting elongation by RNA polymerase II and abolished in quiescent cells. Furthermore, quantification of spatial smoothness over time intervals up to 30 seconds points to clear-cut boundaries between distinct regions, while smooth transitions in small (<1 m) neighborhoods dominate for short time intervals. Clear transitions between regions of coherent motion indicate directed squeezing or stretching of chromatin boundaries suggestive of changes in local concentrations of actors regulating gene expression. The DFCC approach hence allows characterizing stochastically forming domains of specific nuclear activity.\n\nSignificance StatementControl of gene expression relies on modifications of chromatin structure and activity of the transcription machinery. However, how chromatin responds dynamically to this genomic process and whether this response is coordinated in space is still unclear. We introduce a novel approach called Dense Flow reConstruction and Correlation (DFCC) to characterize spatially correlated dynamics of chromatin in living cells at nanoscale resolution. DFCC allows us to detect chromatin domains in living cells with long range correlations over the entire nucleus. Furthermore, transitions between domains can be quantified by the newly introduced smoothness parameter of local chromatin motion. The DFCC approach permits characterizing stochastically forming domains of other DNA dependent activity in any cell type in real time imaging.

biophysics↗

bioRxiv2017

Assessment of hydrophobicity scales for protein stability and folding using energy and RMSD criteria

De novo prediction of protein folding is an open scientific challenge. Many folding models and force fields have been developed, yet all face difficulties converging to native conformations. Hydrophobicity scales (HSs) play a crucial role in such simulations as they define the energetic interactions between protein residues, thus determining the energetically favorable conformation. While many HSs have been developed over the years using various methods, it is surprising that the scales show very weak consensus in their assignment of hydrophobicity indexes to the various residues. In this work, several HSs are systematically assessed via atomistic Monte Carlo simulation of folding of small proteins, by converting the HSs of interest into residue-residue contact energy matrices. HSs that poorly preserve native structures of proteins were tuned by applying a linear transformation. Subsequently, folding simulations were used to examine the ability of the HSs to correctly fold the proteins from a random initial conformation. Root mean square deviation (RMSD) and energy of the proteins during folding were sampled and used to define an ER-score, as the correlation between the 2-dimensional energy-RMSD (ER) histogram with 50% lowest energy conformations and the ER histogram with 50% lowest RMSD conformations. Thus, we were able to compare the ability of the different HSs to predict de novo protein folding quantitatively.

biophysics↗

bioRxiv2017

The Relation Between α-Helical Conformation and Amyloidogenicity

Amyloid fibrils are stable aggregates of misfolded proteins and polypeptides that are insoluble and resistant to protease activity. Abnormal formation of amyloid fibrils in vivo may lead to neurodegenerative disorders and other systemic amyloidosis such as Alzheimers, Parkinsons, and atherosclerosis. Because of their clinical importance amyloids are found under intense scientific research. Amyloidogenic sequences of short polypeptide segments within proteins are responsible for the transformation of correctly folded proteins into parts of larger amyloid fibrils. The -helical secondary structure is believed to host many amyloidogenic sequences and be a key player in different stages of the amyloidogenesis process. Most of the studies on amyloids focus on the role of amyloidogenic sequences. The focus of this study is the relation between amyloidogenicity and the structure of the amyloidogenic -helical sequence. We have previously shown that the -helical conformation may be expressed by two parameters ({theta} and{rho} ) that form orthogonal coordinates based on the Ramachandran dihedrals ({varphi} and{psi} ) and provide an illuminating interpretation of the -helical conformation. By performing statistical analysis on -helical conformations found in the protein data bank, an apparent relation between -helical conformation, as expressed by{theta} and{rho} , and amyloidogenicity is revealed. Remarkably, random amino acid sequences, whose helical structure was obtained from the most probably dihedral angles as obtained from PDB data, revealed the same dependency of amyloidogenicity, suggesting the importance of -helical structure as opposed to sequence.

biophysics↗

bioRxiv2017

Using Topological Rearrangements to Characterize De-mixed Liquid Phases in Bio-membranes

Lateral heterogeneities in biomembranes play a crucial role in various physiological functions of the cell. Such heterogeneities lead to demixing of lipid constituents and formation of distinct liquid domains in the membrane. We study lateral heterogeneities in terms of topological rearrangements of lipids to identify the liquid-liquid phase coexistence in model membranes. By quantifying the degree of non-affine deformation associated with individual lipid, we are able to characterize the liquid ordered (Lo) and liquid disordered (Ld) phases in model lipid bilayers, without any prior knowledge about the chemical identity of the lipids. We explore the usage of this method on all atom and coarse-grained lipid bilayer trajectories. This method is helpful in defining the instantaneous Lo-Ld domain boundaries in complex multi-component bilayer systems.\n\nThe characterization is also used to highlight the effect of line-active molecules on the phase boundaries and domain mixing. Overall, we propose a framework to explore the molecular origin of spatial and dynamical heterogeneity in biomembrane systems, which can be exploited not only in computer simulations but also in experiments.

biophysics↗