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bioRxiv2020

An urn model approach to stochastic modeling of regulated gene expression yields physically intuitive solutions for probability distributions

Fitting the probability mass functions from analytical solutions of stochastic models of gene expression to the count distributions of mRNA and protein molecules in single cells can yield valuable insights into mechanisms of gene regulation. Solutions of chemical master equations are available for various kinetic schemes but, even for the models of regulation with a basic ON-OFF switch, they take complex forms with generating functions given as hypergeometric functions. Gene expression studies that have used these to fit the data have interpreted the parameters as burst size and frequency. However, this is consistent with the hypergeometric functions only if a gene stays active for short time intervals separated by relatively long intervals of inactivity. Physical insights into the probability mass functions are essential to ensure proper interpretations but are lacking for models of gene regulation. We fill this gap by developing urn models for regulated gene expression, which are of immense value to interpret probability distributions. Our model consists of a master urn, which represents the cytosol. We sample RNA polymerases and ribosomes from it and assign them to recipient urns of two or more colors, which represent time intervals with a homogeneous propensity for gene expression. Colors of the recipient urns represent sub-systems of the promoter states, and the assignments to urns of a specific color represent gene expression. We use elementary principles of discrete probability theory to derive the solutions for a range of kinetic models, including the Peccoud-Ycart model, the Shahrezaei-Swain model, and models with an arbitrary number of promoter states. For activated genes, we show that transcriptional lapses, which are events of gene inactivation for short time intervals separated by long active intervals, quantify the transcriptional dynamics better than bursts. Our approach reveals the physics underlying the solutions, which has important implications for single-cell data analysis.

biophysics↗

bioRxiv2020

Differences in the free energies between the excited states of Aβ40 and Aβ42 monomers encode their distinct aggregation propensities

The early events in the aggregation of the intrinsically disordered peptide, A{beta}, involve transitions from the disordered lowest free energy ground state to assembly-competent states. Are the finger-prints of order found in the amyloid fibrils encoded in the conformations that the monomers access at equilibrium? If so, could the enhanced aggregation rate of A{beta}42 compared to A{beta}40 be rationalized from the sparsely populated high free energy states of the monomers? Here, we answer these questions in the affirmative using coarse-grained simulations of the SOP-IDP model of A{beta}40 and A{beta}42. Although both the peptides have practically identical ensemble-averaged properties, characteristic of random coils (RCs), the conformational ensembles of the two monomers exhibit sequence-specific heterogeneity. Hierarchical clustering of conformations reveals that both the peptides populate high free energy aggregation-prone (N*) states, which resemble the monomers in the fibril structure. The free energy gap between the ground (RC) and the N* states of A{beta}42 peptide is smaller than for A{beta}40. By relating the populations of excited states of the two peptides to the fibril formation time scales using an empirical formula, we explain nearly quantitatively the faster aggregation rate of A{beta}42 relative to A{beta}40. The N* concept accounts for fibril polymorphs, leading to the prediction that the less stable N* state of A{beta}42, encoding for the U-bend fibril, should form earlier than the structure with the S-bend topology, which is in accord with the Ostwalds rule rationalizing crystal polymorph formation. Significance StatementAlzheimers disease (AD), a rampant neurodegenerative disorder, is caused by the accumulation of pathological aggregates, primarily composed of the two isoforms A{beta}40 and A{beta}42. Experiments have shown that A{beta}42 is more aggregation-prone compared to A{beta}40. However, the molecular origin of this apparent anomaly remains elusive. Here, we provide a microscopic basis for the different aggregation rates in terms of the distinct populations of high free energy excited fibril-like states (N*) that are encoded in the monomer spectra. The N* theory explains the emergence of fibril polymorphs, and predicts the relative kinetic stabilities of A{beta}42 fibrils using Ostwalds rule of stages. Our work shows that sequence-specific conformational heterogeneity of the monomer ensembles provides important cues for understanding protein aggregation.

biophysics↗

bioRxiv2020

Measuring aptamer folding energy using a molecular clamp

Folding energy offers a useful metric for characterizing the stability and function of aptamers. However, experimentally measuring the folding energy is challenging and there is currently no general technique to measure this parameter directly. In this work, we present a simple approach for measuring aptamer folding energy. First, the aptamer is stretched under equilibrium conditions with a double-stranded DNA "molecular clamp" that is coupled to the aptamer ends. We then measure the total internal energy of stressed DNA molecules using time-lapse gel electrophoresis and compare the folding and unfolding behavior of molecular clamp-stressed molecules that incorporate either the aptamer or unstructured random single-stranded DNA in order to derive the aptamer folding energy. Using this approach, we measured a folding energy of 10.40 kJ/mol for the HD22 thrombin aptamer, which is consistent with other predictions and estimates. We also analyzed a simple hairpin structure, generating a folding energy result of 9.05 kJ/mol, consistent with the value predicted by computational models (9.24 kJ/mol). We believe our strategy offers an accessible and generalizable approach for obtaining such measurements with virtually any aptamer.

biophysics↗

bioRxiv2020

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

Nuclei are constantly under external stress - be it during migration through tight constrictions or compressive pressure by the actin cap - and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope (AFM-LS) to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

biophysics↗

bioRxiv2020

Mobility of lower MA-helices for ion-conduction through lateral portals in 5-HT3A receptors

The intracellular domain of the serotonin type 3A receptor, a pentameric ligand-gated ion channel, is crucial for regulating conductance. However, the specific ion conduction pathway through this domain is less clear. The intracellular domain starts with a short loop after the third transmembrane segment, followed by a short -helical segment, a large unstructured loop, and finally the membrane-associated MA-helix that continues into the last transmembrane segment. The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a "closed vestibule", with their lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. The question remains whether the lateral portals or cytosolic constriction conduct ions upon channel opening. In the present study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of segments of the MA-helices most distal to the membrane bilayer. Our results indicate that the proximity and orientation for cysteine pairs at I409C/R410C, in close proximity to the lateral windows, and L402C/L403C, at the cytosolic ends of the MA-helices, are conducive for disulfide bond formation. While conformational changes associated with gating promote crosslinking for I409C/R410C, which in turn decreases channel currents, crosslinking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely. SignificanceThe intracellular domain (ICD) of pentameric ligand-gated ion channels (pLGICs) is the most diverse domain within receptors of the Cys-loop superfamily. Despite being the least understood domain of pLGICs, its impact on ion-channel function and contribution to the cytosolic exit pathway of the channel have been investigated. X-ray and cryo-EM structures have captured the structured segments of the ICD of 5-HT3A receptors in different conformational states with lower resolution of the ICD as compared to the other domains. Here, we provide experimentally derived evidence for the importance of the differential mobility of the cytosolic segment of the MA-helices, which supports the existence of lateral portals as opposed to a vertical pathway for 5-HT3A receptors.

biophysics↗

bioRxiv2020

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

Super-resolution imaging allows for visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA Point Accumulation In Nanoscale Topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), that allows for faster binding. Ago pre-orders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude while maintaining the high spatial resolution. We envision this tool to be useful not only for super-resolution imaging, but also for other techniques that rely on nucleic-acid interactions.

biophysics↗

bioRxiv2020

Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM based SCFS

Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. Single molecule force spectroscopy experiments in in-vitro experiments can reveal the cadherin-cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin homophilic and heterophilic binding in their native conformational and functional state in living cells has rarely been done. Here, we used Atomic Force Microscopy (AFM) based Single cell force Spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N-, OB- and E-cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e. between same type of cells and between cells of different type. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E cadherin homophilic bindings are stronger than N/OB, E/N and E/OB heterophilic bindings. Disassembly of intracellular actin filaments reduces the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements. Whereas, myosin inactivation particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins homophilic bonds.

biophysics↗

bioRxiv2020

Investigating the conformational ensembles of intrinsically-disordered proteins with a simple physics-based model

Intrinsically disordered proteins (IDPs) play an important role in an array of biological processes but present a number of fundamental challenges for computational modeling. Recently, simple polymer models have re-gained popularity for interpreting the experimental characterization of IDPs. Homopolymer theory provides a strong foundation for understanding generic features of phenomena ranging from single-chain conformational dynamics to the properties of entangled polymer melts, but is difficult to extend to the copolymer context. This challenge is magnified for proteins due to the variety of competing interactions and large deviations in side-chain properties. In this work, we apply a simple physics-based coarse-grained model for describing largely disordered conformational ensembles of peptides, based on the premise that sampling sterically-forbidden conformations can compromise the faithful description of both static and dynamical properties. The Hamiltonian of the employed model can be easily adjusted to investigate the impact of distinct interactions and sequence specificity on the randomness of the resulting conformational ensemble. In particular, starting with a bead-spring-like model and then adding more detailed interactions one by one, we construct a hierarchical set of models and perform a detailed comparison of their properties. Our analysis clarifies the role of generic attractions, electrostatics and side-chain sterics, while providing a foundation for developing efficient models for IDPs that retain an accurate description of the hierarchy of conformational dynamics, which is nontrivially influenced by interactions with surrounding proteins and solvent molecules.

biophysics↗

bioRxiv2020

Cryo-EM structures and functional properties of CALHM channels of the human placenta

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we found that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences.

biophysics↗

bioRxiv2020

Use of Bioelectric Impedance Analysis (BIA) as a new method to detect prostate cancer

BackgroundTo determine the accuracy of a novel BIA test endorectal probe. MethodsOne hundred-forty consecutive patient candidates to prostate biopsy and 40 healthy volunteers were selected (NCT03428087). Total PSA and PSA density (PSAD) determinations, digital rectal examination (DRE), and the BIA test were analysed in patients and controls. A 16 cores trans rectal prostate biopsy was performed on all patients with clinical suspicion of PCa after a multiparametric MRI (mMRI) test. The study endpoints were to determine accuracy of BIA test in comparison to PSA, PSAD levels, and mMRI and obtain PCa prediction in candidates to prostate biopsy by BIA test. The Mann-Withney U test, the Wilkoxon rank test, and Holm-Bonferronis method were adopted for statistical analyses, and a computational approach was also applied to differentiate patients with PCa from those with benign disease (BPH). ResultsCombined DRE, TRUS, PSA, and PSAD alone failed to satisfactorily discern patients with PCa from those with BPH (62.86% of discrimination accuracy) and mMRI PIRADS [≥]3 showed a sensitivity of 83% and a specificity of 59%. The accuracy in discerning PCa and BPH increased up to 75% by BIA test (sensitivity 63.33% and specificity 83.75%). ConclusionsThe BIA test is a simple, promising, cheap, and reliable test for PCa non-invasive diagnosis. The novel finger probe may improve PCa detection also in patients with low-risk PCa, thus reducing the need of useless biopsies.

biophysics↗

bioRxiv2020

Emergence of ribozyme and tRNA-like structures from mineral-rich muddy pools on prebiotic earth

AO_SCPLOWBSTRACTC_SCPLOWThe RNA world hypothesis, although a viable one regarding the origin of life on earth, has so far failed to provide a compelling explanation for the synthesis of RNA molecules with catalytic functions, from free nucleotides via abiotic processes. To tackle this long-standing problem, we develop a realistic model for the onset of the RNA world, using experimentally determined rates for polymerization reactions. We start with minimal assumptions about the initial state that only requires the presence of short oligomers or just free nucleotides and consider the effects of environmental cycling by dividing a day into a dry, semi-wet and wet phases that are distinguished by the nature of reactions they support. Long polymers, with maximum lengths sometimes exceeding 100 nucleotides, spontaneously emerge due to a combination of non-enzymatic, non-templated polymer extension and template-directed primer extension processes. The former helps in increasing the lengths of RNA strands, whereas the later helps in producing complementary copies of the strands. Strands also undergo hydrolysis in a structure-dependent manner that favour breaking of bonds connecting unpaired nucleotides. We identify the most favourable conditions needed for the emergence of ribozyme and tRNA-like structures and double stranded RNA molecules, classify all RNA strands on the basis of their secondary structures and determine their abundance in the population. Our results indicate that under suitable environmental conditions, non-enzymatic processes would have been sufficient to lead to the emergence of a variety of ribozyme-like molecules with complex secondary structures and potential catalytic functions.

biophysics↗

bioRxiv2020

Stem cell lineage survival as a noisy competition for niche access

Understanding to what extent stem cell potential is a cell-intrinsic property, or an emergent behavior coming from global tissue dynamics and geometry, is a key outstanding question of stem cell biology. Here, we propose a theory of stem cell dynamics as a stochastic competition for access to a spatially-localized niche, giving rise to a "stochastic conveyor-belt" model. Cell divisions produce a steady cellular stream which advects cells away from the niche, while random rearrangements enable cells away from the niche to be favourably repositioned. Importantly, even when assuming that all cells in a tissue molecularly equivalent, the model predicts a common ("universal") functional dependence of the long-term clonal survival probability on the position within the niche, as well as the emergence of a well-defined number of "functional" stem cells, dependent only on the rate of random movements vs. mitosis-driven advection. We test the predictions of this theory on datasets on pubertal mammary gland tips, embryonic kidney tips as well homeostatic intestinal crypt, and find good quantitative agreement for the number of functional stem cells in each organ, as well as the predicted functional dependence of the competition.

biophysics↗

bioRxiv2020

AFM characterization of the interaction of PriA helicase with stalled DNA replication forks

In bacteria, the restart of stalled DNA replication forks requires the PriA DNA helicase. PriA recognizes and remodels abandoned DNA replication forks performing the DNA unwinding in 3 to 5-direction and facilitates loading of the DnaB helicase onto the DNA to restart replication. The single stranded DNA binding protein (SSB) is typically present at the abandoned forks, but there is gap in the knowledge on the interaction between SSB and PriA protein. Here, we used atomic force microscopy (AFM) to visualize the interaction of PriA with DNA substrates in the absence or presence of SSB. Results show that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Preferential binding occurs only on forked DNA structures as 5- and 3-tailed duplexes were bound equally well. Furthermore, in the absence of SSB, PriA bound exclusively to the fork regions of substrates. In contrast, fork bound SSB loads PriA onto the duplex DNA arms of forks. When the fork has a gap in the leading strand, PriA localizes to both the parental and lagging strand arms. When the gap is present in the lagging strand, PriA is loaded preferentially onto the leading strand arm of the fork. Remodeling of PriA requires a functional C-terminal domain of SSB.

biophysics↗

bioRxiv2020

Nanoscale Molecular Characterisation of Hair Cuticles using Integrated AFM-IR

The nanometre-scale topography and chemical structure of hair cuticles has been investigated by vibrational spectroscopy and imaging in two spectral regions. The combination of Atomic Force Microscopy with a tuneable infrared laser (AFM-IR) circumvents the diffraction limit that has impaired traditional infrared spectroscopy, facilitating surface spectroscopy at ultra-spatial resolution. The variation in protein and lipid content of the cuticle cell surface approaching its edge, as well as the exposed layered structure of the cell at the edge itself, was investigated. Furthermore, the contribution of cystine-related products to the cuticle layers was determined. The variation of protein, lipid and cystine composition in the observed layers, as well as the measured dimensions of each, correspond closely to that of the epicuticle, A-layer, exocuticle and endocuticle layers of the cuticle cell sub-structure. Statement of SignificanceUsing AFM-IR to analyse the nanoscale cuticle features is both significant and novel in the field. Thus far, the great majority of work on the chemical investigation of the structure of hair has been limited to bulk measurements, or subject to the diffraction limit associated with traditional IR spectroscopies and microscopies. AFM-IR circumvents this diffraction limit and allows nanometre-scale, localised chemical investigation with high surface selectivity. While non-chemical investigations, e.g. those using Transmission Election Microscopy, have previously shown cuticles to have a layered substructure, AFM-IR sheds light on significant chemical variations of protein and lipid compositions within such layers, enabling their quantification.

biophysics↗

bioRxiv2020

Formation of aggregates, icosahedral structures and percolation clusters of fullerenes in lipids bilayers: The key role of lipid saturation

Carbon nanoparticles (CNPs) are attractive materials for a great number of applications but there are serious concerns regarding their influence on health and environment. Here, our focus is on the behavior of fullerenes in lipid bilayers with varying lipid saturations, chain lengths and fullerene concentrations using coarse-grained molecular dynamics (CG-MD) simulations. Our findings show that the lipid saturation level is a key factor in determining how fullerenes behave and where the fullerenes are located inside a lipid bilayer. In saturated and monounsaturated bilayers fullerenes aggregated and formed clusters with some of them showing icosahedral structures. In polyunsaturated lipid bilayers, no such structures were observed: In polyunsaturated lipid bilayers at high fullerene concentrations, connected percolation-like networks of fullerenes spanning the whole lateral area emerged at the bilayer center. In other systems only separate isolated aggregates were observed. The effects of fullerenes on lipid bilayers depend strongly on fullerene aggregation. When fullerenes aggregate, their interactions with the lipid tails change.

biophysics↗

bioRxiv2020

Microtubule instability driven by longitudinal and lateral strain propagation

Tubulin dimers associate longitudinally and laterally to form metastable microtubules (MTs). MT disassembly is preceded by subtle structural changes in tubulin fueled by GTP hydrolysis. These changes render the MT lattice unstable, but it is unclear exactly how they affect lattice energetics and strain. We performed long-time atomistic simulations to interrogate the impacts of GTP hydrolysis on tubulin lattice conformation, lateral inter-dimer interactions, and (non-)local lateral coordination of dimer motions. The simulations suggest that most of the hydrolysis energy is stored in the lattice in the form of longitudinal strain. While not significantly affecting lateral bond stability, the stored elastic energy results in more strongly confined and correlated dynamics of GDP-tubulins, thereby entropically destabilizing the MT lattice.

biophysics↗

bioRxiv2020

Divergent ion selectivity and sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs of DEG/ENaC/ASIC channels in C. elegans

The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form non-voltage gated and sodium-selective channels sensitive to the anti-hypertensive drug, amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). C. elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels, but also in their drug sensitivity. We selected five C. elegans DEG/ENaC/ASICs (DEGT-1, DEL-1, UNC-8, MEC-10 and MEC-4) that are co-expressed in mechanosensory neurons and expressed gain-of-function d mutant isoforms in Xenopus laevis oocytes. We found that only DEGT-1d, UNC-8d, and MEC-4d formed homomeric channels and that, unlike MEC-4d and UNC-8d, DEGT-1d channels were insensitive to amiloride and its analogs. As reported for rat ASIC1a, NSAIDs inhibit DEGT-1d and UNC-8d channels. Unexpectedly, MEC-4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels. SummaryAnimal physiology depends on degenerin, epithelial sodium, and acid-sensing ion channels (DEG/ENaC/ASICs). By measuring the sensitivity of three C. elegans DEG/ENaC/ASICs to five amiloride analogs and five NSAIDs, we show that individual channels have distinct pharmacological footprints.

biophysics↗

bioRxiv2020

The mechanical inhibition of the isolated Vo from V-ATPase for proton conductance

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 moiety, with proton flow through the Vo membrane moiety, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo of eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and the isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo adopts the auto-inhibited form and how the holo complex prevents the formation of this inhibited Vo form. One Sentence SummaryCryo-EM structures of rotary V-ATPase reveal the ON-OFF switching mechanism of H+ translocation in the Vo membrane domain.

biophysics↗