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bioRxiv2020

Computational 4D-OCM for label-free imaging of collective cell invasion and force-mediated deformations in collagen

Traction force microscopy (TFM) is an important family of techniques used to measure and study the role of cellular traction forces (CTFs) associated with many biological processes. However, current standard TFM methods rely on imaging techniques that do not provide the experimental capabilities necessary to study CTFs within 3D collective and dynamic systems embedded within optically scattering media. Traction force optical coherence microscopy (TF-OCM) was developed to address these needs, but has only been demonstrated for the study of isolated cells embedded within optically clear media. Here, we present computational 4D-OCM methods that enable the study of dynamic invasion behavior of large tumor spheroids embedded in collagen. Our multi-day, time-lapse imaging data provided detailed visualizations of evolving spheroid morphology, collagen degradation, and collagen deformation, all using label-free scattering contrast. These capabilities, which provided insights into how stromal cells affect cancer progression, significantly expand access to critical data about biophysical interactions of cells with their environment, and lay the foundation for future efforts toward volumetric, time-lapse reconstructions of collective CTFs with TF-OCM.

biophysics↗

bioRxiv2020

Circadian rhythms of early afterdepolarizations and ventricular arrhythmias in a cardiomyocyte model

AO_SCPLOWBSTRACTC_SCPLOWSudden cardiac arrest is a malfunction of the hearts electrical system, typically caused by ventricular arrhythmias, that can lead to sudden cardiac death (SCD) within minutes. Epidemiological studies have shown that SCD and ventricular arrhythmias are more likely to occur in the morning than in the evening, and laboratory studies indicate that these daily rhythms in adverse cardiovascular events are at least partially under the control of the endogenous circadian timekeeping system. However, the biophysical mechanisms linking molecular circadian clocks to cardiac arrhythmogenesis are not fully understood. Recent experiments have shown that L-type calcium channels exhibit circadian rhythms in both expression and function in guinea pig ventricular cardiomyocytes. We developed an electrophysiological model of these cells to simulate the effect of circadian variation in L-type calcium conductance. We found that there is a circadian pattern in the occurrence of early afterdepolarizations (EADs), which are abnormal depolarizations during the repolarization phase of a cardiac action potential that can trigger fatal ventricular arrhythmias. Specifically, the model produces EADs in the morning but not at other times of day. We show that the model exhibits a codimension-2 Takens-Bogdanov bifurcation that serves as an organizing center for different types of EAD dynamics. We also simulated a 2-D spatial version of this model across a circadian cycle. We found that there is a circadian pattern in the breakup of spiral waves, which represents ventricular fibrillation in cardiac tissue. Specifically, the model produces spiral wave breakup in the morning but not in the evening. Our study is the first to propose a link between circadian rhythms and EAD formation and suggests that the efficacy of drugs targeting EAD-mediated arrhythmias may depend on the time of day that they are administered. Significance StatementWhy are life-threatening cardiac arrhythmias more likely to occur in the morning than in the evening? The electrical properties of the heart exhibit daily rhythms due to molecular circadian clocks within cardiomyocytes. Our computational model of ventricular myocytes shows that clock-controlled expression of a voltage-gated calcium ion channel leads to early afterdepolarizations (EADs) at certain times of the day. EADs, in which the membrane potential of a cardiomyocyte depolarizes a second time before fully repolarizing, can trigger arrhythmias. To our knowledge, this is the first study linking the circadian clock to EAD formation. Our results suggest that the efficacy of anti-arrhythmic medications targeting this calcium ion channel may depend on the time of day the drug is taken.

biophysics↗

bioRxiv2020

A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

biophysics↗

bioRxiv2020

Understanding the photophysics and structural organization of photosynthetic proteins using model lipid membranes assembled from natural plant thylakoids

The light-harvesting (LH) biomembranes from photosynthetic organisms perform solar energy absorption and transfer with high efficiency. There is great interest in the nanoscale biophysics of photosynthesis, however, natural membranes are complex and highly curved so can be challenging to study. Here we present model photosynthetic "hybrid membranes" assembled from a combination of natural LH membranes and synthetic lipids deposited into a patterned polymerized lipid template on glass. This arrangement offers many advantages over previous model systems including: a sufficiently complex mixture of natural proteins to mimic the biological processes, a modular self-assembly mechanism, and a stabilizing template promoting the formation of supported lipid bilayers from complex natural membranes with high protein content (that would not otherwise form). These hybrid membranes can be used as a platform to delineate the complex relationship between LH energy pathways and membrane organization. Atomic force microscopy and fluorescence lifetime microscopy revealed that hybrid membranes have an elongated fluorescence lifetime ([~]4 ns) compared to native membranes ([~]0.5 ns), a direct consequence of reduced protein density and an uncoupling of protein-protein interactions. We observed the real time self-assembly and migration of LH proteins from natural membrane extracts into the hybrid membranes and monitored the photophysical state of the membranes at each stage. Finally, experiments utilizing our hybrid membranes suggest that assays currently used in the photosynthesis community to test the electron transfer activity of Photosystem II may have non-specific interactions with other proteins, implying that new methods are needed for reliable quantification of electron transfers in photosynthesis.

biophysics↗

bioRxiv2020

A torsion-based rheometer for measuring viscoelastic material properties

Rheology and the study of viscoelastic materials is an integral part of engineering and the study of biophysical systems however the cost of a rheometer is only feasible for colleges, universities and research laboratories. Even if a rheometer can be purchased it is bulky and delicately calibrated limiting its usefulness to the laboratory itself. The design presented here is less than a tenth of the cost of a professional rheometer and portable making it the ideal solution for high school students as a way to introduce viscoelasticity at a younger age as well as for use in the field for obtaining preliminary rheological data.

biophysics↗

bioRxiv2020

The flexibility of ACE2 in the context of SARS-CoV-2 infection

The COVID-19 pandemic has swept over the world in the past months, causing significant loss of life and consequences to human health. Although numerous drug and vaccine developments efforts are underway, many questions remain outstanding on the mechanism of SARS-CoV-2 viral association to angiotensin-converting enzyme 2 (ACE2), its main host receptor, and entry in the cell. Structural and biophysical studies indicate some degree of flexibility in the viral extracellular Spike glycoprotein and at the receptor binding domain-receptor interface, suggesting a role in infection. Here, we perform all-atom molecular dynamics simulations of the glycosylated, full-length membrane-bound ACE2 receptor, in both an apo and spike receptor binding domain (RBD) bound state, in order to probe the intrinsic dynamics of the ACE2 receptor in the context of the cell surface. A large degree of fluctuation in the full length structure is observed, indicating hinge bending motions at the linker region connecting the head to the transmembrane helix, while still not disrupting the ACE2 homodimer or ACE2-RBD interfaces. This flexibility translates into an ensemble of ACE2 homodimer conformations that could sterically accommodate binding of the spike trimer to more than one ACE2 homodimer, and suggests a mechanical contribution of the host receptor towards the large spike conformational changes required for cell fusion. This work presents further structural and functional insights into the role of ACE2 in viral infection that can be exploited for the rational design of effective SARS-CoV-2 therapeutics. Statement of SignificanceAs the host receptor of SARS-CoV-2, ACE2 has been the subject of extensive structural and antibody design efforts in aims to curtail COVID-19 spread. Here, we perform molecular dynamics simulations of the homodimer ACE2 full-length structure to study the dynamics of this protein in the context of the cellular membrane. The simulations evidence exceptional plasticity in the protein structure due to flexible hinge motions in the head-transmembrane domain linker region and helix mobility in the membrane, resulting in a varied ensemble of conformations distinct from the experimental structures. Our findings suggest a dynamical contribution of ACE2 to the spike glycoprotein shedding required for infection, and contribute to the question of stoichiometry of the Spike-ACE2 complex.

biophysics↗

bioRxiv2020

The Dynamic Influence of Linker Histone Saturation within the Poly-Nucleosome Array

Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction. SignificanceLinker histones dynamically bind to DNA in chromatin fibers and serve as epigentic regulators. However, the extent to which they influence the gamut of chromatin architecture is still not well understood. Using molecular dynamics simulations, we studied compact octa-nucleosome arrays with and without the H1 linker histone to better understand the mechanisms dictating the structure of the chromatin fiber. Inclusion of H1 results in stabilization of the compact chromatin structure, while its removal results in a major conformational change towards an untwisted ladder-like state. The increased rigidity and correlations within the H1-bound array suggests that H1-saturated chromatin fibers are better suited to transferring long-range epigentic information.

biophysics↗

bioRxiv2020

Coexisting lipid phases alter lipid dynamics and sort on nanoscale membrane curvature

The precise spatiotemporal control of nanoscale membrane shape and composition is the result of complex interplay of individual and collective molecular behaviors. Here, we employed single-molecule localization microscopy and computational simulations to observe single-lipid diffusion and sorting in model membranes with varying compositions, phase, temperature, and curvature. Supported lipid bilayers were created over 50-nm radius nanoparticles to mimic the size of naturally occurring membrane buds, such as endocytic pits and the formation of viral envelopes. The curved membranes recruited liquid-disordered lipid phases while altering the diffusion and sorting of tracer lipids. Disorder-preferring fluorescent lipids sorted to and experienced faster diffusion on the nanoscale curvature only when embedded in a membrane capable of sustaining lipid phase separation at low temperatures. The curvature-induced sorting and faster diffusion even occurred when the sample temperature was above the miscibility temperature of the planar membrane, implying that the nanoscale curvature could induce phase separation in otherwise homogeneous membranes. Further confirmation and understanding of these results are provided by continuum and coarse-grained molecular dynamics simulations with explicit and spontaneous curvature-phase coupling, respectively. The curvature-induced membrane compositional heterogeneity and altered dynamics were achieved only with a coupling of the curvature with a lipid phase separation. These cross-validating results demonstrate the complex interplay of lipid phases, molecular diffusion, and nanoscale membrane curvature that are critical for membrane functionality. SignificanceNanoscopic membrane organization and dynamics are critical for cellular function but challenging to experimentally measure. This work brings together super-resolution optical methods with multiscale computational approaches to reveal the interplay between curvature, composition, phase, and diffusion in model membranes. We report that curvature can induce phase separation in otherwise homogeneous membranes and that the phase-curvature coupling has a direct implication on lipid mobility. This discovery advances our understanding of the fundamental membrane biophysics that regulate membrane activities such as endocytosis and viral budding.

biophysics↗

bioRxiv2020

Single-molecule light-sheet microscopy with local nanopipette delivery

Detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2{micro}m thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-{beta}, which causes proteotoxicity relevant to Alzheimers disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells. Statement of SignificanceThis paper describes and validates a new method to study biological processes based on the controlled local delivery of molecules onto or into the cell, combined with single molecule imaging using light sheet microscopy. we not only demonstrate the instruments capability of delivering controlled numbers of molecules to a defined position, down to the level of single molecules, but also its potential in study of the triggering of the innate immune response by protein aggregates, a key process in the development of neurodegenerative diseases such as Alzheimers disease. The same approach could be applied to a wide range of other important biological processes allowing them to be followed in live cells in real-time, hence it will be of great interest to the biophysical community.

biophysics↗

bioRxiv2020

Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

biophysics↗

bioRxiv2020

Interconversion of unexpected thiol states affects stability, structure and dynamics in engineered antibody for site-specific conjugation

Antibody drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterise an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts and changes the stability and structural dynamics of the antibody. Insight is gained into the local and global destabilisation associated with the engineering and subsequent disulfide bonded variant that will inform future engineering strategies.

biophysics↗

bioRxiv2020

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

biophysics↗

bioRxiv2020

The genotype-phenotype landscape of an allosteric protein

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. Here we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively measure the dose-response curves for nearly 105 variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to discover a new band-stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.

biophysics↗

bioRxiv2020

Concentration Sensing in Crowded Environments

Signal transduction within crowded cellular compartments is essential for the physiological function of cells and organisms. While the accuracy with which receptors can probe the concentration of ligands has been thoroughly investigated in dilute systems, the effect of macromolecular crowding on the inference of concentration remains unknown. In this work we develop a novel algorithm to simulate reversible reactions between reacting Brownian particles. This facilitates the calculation of reaction rates and correlation times for ligand-receptor systems in the presence of macromolecular crowding. Using this method, we show that it is possible for crowding to increase the accuracy of estimated ligand concentration based on receptor occupancy. In particular, we find that crowding can enhance the effective association rates between small ligands and receptors to a large enough degree to overcome the increased chance of rebinding due to caging by crowding molecules. For larger ligands, crowding decreases the accuracy of the receptors estimate primarily by decreasing the microscopic association and dissociation rates. SIGNIFICANCEDeveloping an understanding of how cells effectively transmit signals within or between compartments under physical constraints is an important challenge for biophysics. This work investigates the effect that macromolecular crowding can have on the accuracy of a simple ligand-receptor signaling system. We show that the accuracy of an inferred ligand concentration based on the occupancy of the receptor can be enhanced by crowding under certain circumstances. Additionally, we develop a simulation algorithm that speeds the calculation of reaction rates in crowded environments and can be readily applied to other, more complex systems.

biophysics↗

bioRxiv2020

The guidance and adhesion protein FLRT2 dimerizes in cis via dual Small-X3-Small transmembrane motifs

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules. FieldsStructural Biology and Biophysics / Computational Biology

biophysics↗

bioRxiv2020

Transfer function for YAP/TAZ nuclear translocation revealed through spatial systems modeling

YAP/TAZ is a master regulator of mechanotransduction whose functions rely on translocation from the cytoplasm to the nucleus in response to diverse physical cues. Substrate stiffness, substrate dimensionality, and cell shape are all input signals for YAP/TAZ, and through this pathway, regulate critical cellular functions and tissue homeostasis. Yet, the relative contributions of each biophysical signal and the mechanisms by which they synergistically regulate YAP/TAZ in realistic tissue microenvironments that provide multiplexed input signals remains unclear. For example, in simple 2D culture, YAP/TAZ nuclear localization correlates strongly with substrate stiffness, while in 3D environments, YAP/TAZ translocation can increase with stiffness, decrease with stiffness, or remain unchanged. Here, we develop a spatial model of YAP/TAZ translocation to enable quantitative analysis of the relationships between substrate stiffness, substrate dimensionality, and cell shape. Our model couples cytosolic stiffness to nuclear mechanics to replicate existing experimental trends, and extends beyond current data to predict that increasing substrate activation area through changes in culture dimensionality, while conserving cell volume, forces distinct shape changes that result in nonlinear effect on YAP/TAZ nuclear localization. Moreover, differences in substrate activation area versus total membrane area can account for counterintuitive trends in YAP/TAZ nuclear localization in 3D culture. Based on this multiscale investigation of the different system features of YAP/TAZ nuclear translocation, we predict that how a cell reads its environment is a complex information transfer function of multiple mechanical and biochemical factors. These predictions reveal design principles of cellular and tissue engineering for YAP/TAZ mechanotransduction. STATEMENT OF SIGNIFICANCEIn chemical engineering, a transfer function is a mathematical function that models the output of a reactor for all possible inputs, and enables the reliable design and operation of complex reaction systems. Here, we apply this principle to cells to derive the transfer function by which substrate stiffness is converted into YAP/TAZ nuclear localization. This function is defined by a spatial model of the YAP/TAZ mechano-chemical sensing network, wherein key spatial and physical inputs to the system, namely cell and nuclear shape, surface area to volume ratios of cytoplasmic and nuclear compartments, substrate dimensionality, substrate activation area, and substrate stiffness, are all integrated. The resulting model accounts for seemingly contradictory experimental trends and lends new insight into controlling YAP/TAZ signalling.

biophysics↗

bioRxiv2020

Computational and experimental analyses of mitotic chromosome formation pathways in fission yeast

Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the long S. pombe chromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin density enrichment. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

biophysics↗

bioRxiv2020

Real-Time Observation of Structure and Dynamics during the Liquid-to-Solid Transition of FUS LC

Many of the proteins found in pathological protein fibrils also exhibit tendencies for liquid-liquid phase separation (LLPS) both in vitro and in cells. The mechanisms underlying the connection between these phase transitions have been challenging to study due to the heterogeneous and dynamic nature of the states formed during the maturation of LLPS protein droplets into gels and solid aggregates. Here, we interrogate the liquid-to-solid transition of the low complexity domain of the RNA binding protein FUS (FUS LC), which has been shown to adopt LLPS, gel-like, and amyloid states. We employ magic-angle spinning (MAS) NMR spectroscopy which has allowed us to follow these transitions in real time and with residue specific resolution. We observe the development of {beta}-sheet structure through the maturation process and show that the final state of FUS LC fibrils produced through LLPS is distinct from that grown from fibrillar seeds. We also apply our methodology to FUS LC G156E, a clinically relevant FUS mutant that exhibits accelerated fibrillization rates. We observe significant changes in dynamics during the transformation of the FUS LC G156E construct and begin to unravel the sequence specific contributions to this phenomenon with computational studies of the phase separated state of FUS LC and FUS LC G156E. SignificanceThe presence of protein aggregates and plaques in the brain is a common pathological sign of neurodegenerative disease. Recent work has revealed that many of the proteins found in these aggregates can also form liquid-liquid droplets and gels. While the interconversion from one state to another can have vast implications for cell function and disease, the molecular mechanisms that underlie these processes are not well understood. Here, we combine MAS NMR spectroscopy with other biophysical and computational tools to follow the transitions of the stress response protein FUS. This approach has allowed us to observe real-time changes in structure and dynamics as the protein undergoes these transitions, and to reveal the intricate effects of disease-relevant mutations on the transformation process.

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