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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↗

bioRxiv2017

High resolution in situ structural determination of heterogeneous specimen

Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single particle cryoEM. Such complexes may be studied in situ using cryo-electron tomography combined with sub-tomogram alignment and classification, which in exceptional cases reaches sub-nanometer resolution, yielding insight into structure-function relationships. All maps currently deposited in the EMDB with resolution < 9 [A] are from macromolecules that form ordered structural arrays, like viral capsids, which greatly simplifies structural determination. Extending this approach to more common specimens that exhibit conformational or compositional heterogeneity, and may be available in limited numbers, remains challenging. We developed emClarity, a GPU-accelerated image processing package, specifically to address fundamental hurdles to this aim, and demonstrate significant improvements in the resolution of maps compared to those generated using current state-of-the-art software. Furthermore, we devise a novel approach to sub-tomogram classification that reveals functional states not previously observed with the same data.\n\nThe software is freely available from https://www.github.com/bHimes/emClarity\n\nTutorial documentation and videos at https://www.github.com/bHimes/emClarity/wiki

biophysics↗

bioRxiv2017

Biphasic mechanosensitivity of TCR mediated adhesion of T lymphocytes

Force sensitivity of the T cell receptor (TCR) is now believed to be essential for immune recognition, but cellular mechanosensitivity of T cells is still poorly understood. Here we show that T cells adhering via the TCR-complex respond to environmental stiffness in an unusual biphasic fashion. As the stiffness increases, adhesion and spreading first increase, then decrease, attaining their maximal values on an optimally stiff surface, with stiffness comparable to certain antigen presenting cells. Remarkably, in presence of additional ligands for the integrin LFA-1, spreading increases monotonously with stiffness up to a saturation value. Using a mesoscopic semi-analytical model linking spreading to molecular characteristics of bonds, we identify force sensitivity of the off-rate and the effective bond stiffness as the crucial parameters that determine monotonic or biphasic mechanosensitive behavior.

biophysics↗

bioRxiv2017

Non-native Interactions Explain the Folding Rate Differences in α-Spectrin Domains and the Origin of Internal Friction Effects

Recent experimental and computational studies have shown the influence of internal friction in protein folding dynamics. However, uncertainty remains over its molecular origin. -spectrin experimental results indicate that R15 domain folds three orders of magnitude faster than its homologous R16 and R17. Such anomalous observations are usually attributed to the influence of internal friction on protein folding rates. To study this phenomenon, we carried out molecular dynamics simulations with structure-based C models, in which the folding process of -spectrin domains was investigated by adding non-native interactions. The simulations take into account the hydrophobic and the electrostatic contributions separately. The folding time results have shown a qualitative agreement with experimental data. We have also investigated mutations in R16 and R17, and the simulation folding time results correlate with the observed experimental ones. We suggest that the origin of the internal friction emerges from a cooperativity effect of these non-native interactions.

biophysics↗

bioRxiv2017

Collective cell migration without proliferation: density determines cell velocity and wave velocity

Collective cell migration contributes to morphogenesis, wound healing or tumor metastasis. Culturing epithelial monolayers on a substrate enables to quantify such tissue migration. By using narrow strips, we stabilise the front shape; by inhibiting cell division, we limit density increase and favor steady migration; by using long strips, we observe a confined cell monolayer migrating over days. A coherent collective movement propagates over millimeters; cells spread and density decreases from the monolayer bulk toward the front. Cell velocity ([~]micrometer per minute) increases linearly with cell radius, and does not depend explicitly on the distance to the front. Over ten periods of backwards propagating velocity waves, with wavelength [~]millimeter, are detected with a signal-to-noise ratio enabling for quantitative spatio-temporal analysis. Their velocity ([~]ten micrometers per minute) is ten times the cell velocity; it increases linearly with the cell radius. Their period ([~]two hours) is spatially homogeneous, and increases with the front density. When we inhibit the formation of lamellipodia, cell velocity drops while waves either disappear, or have a smaller amplitude and slower period. Our phenomenological model assumes that both cell and wave velocities are related with the activity of lamellipodia, and that the local stretching in the monolayer bulk modulates traction stresses. We find that parameter values close to the instability limit where waves appear yield qualitative and quantitative predictions compatible with experiments, including the facts that: waves propagate backwards; wave velocity increases with cell radius; lamellipodia inhibition attenuates, slows down or even suppresses the waves. Together, our experiments and modelling evidence the importance of lamellipodia in collective cell migration and waves.

biophysics↗

bioRxiv2017

Interdependence Theory of Tissue Failure: Bulk and Boundary Effects

The mortality rate of many complex multicellular organisms increase with age, which suggests that net aging damage is accumulative, despite remodeling processes. But how exactly do little mishaps in the cellular level accumulate and spread to become a systemic catastrophe? To address this question we present experiments with synthetic tissues, an analytical model consistent with experiments, and a number of implications that follow the analytical model. Our theoretical framework describes how shape, curvature and density influences the propagation of failure in a tissue subject to oxidative damage. We propose that aging is an emergent property governed by interaction between cells, and that intercellular processes play a role that are at least as important as intracellular ones.

biophysics↗

bioRxiv2017

Physical epistatic landscape of antibody binding affinity

Affinity maturation produces antibodies that bind antigens with high specificity by accumulating mutations in the antibody sequence. Mapping out the antibody-antigen affinity landscape can give us insight into the accessible paths during this rapid evolutionary process. By developing a carefully controlled null model for noninteracting mutations, we characterized epistasis in affinity measurements of a large library of antibody variants obtained by Tite-Seq, a recently introduced Deep Mutational Scan method yielding physical values of the binding constant. We show that representing affinity as the binding free energy minimizes epistasis. Yet, we find that epistatically interacting sites contribute substantially to binding. In addition to negative epistasis, we report a large amount of beneficial epistasis, enlarging the space of high-affinity antibodies as well as their mutational accessibility. These properties suggest that the degeneracy of antibody sequences that can bind a given antigen is enhanced by epistasis -- an important property for vaccine design.

biophysics↗

bioRxiv2017

The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

In animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

biophysics↗

bioRxiv2017

Application of the fluctuation theorem for non-invasive force measurement in living neuronal axons

Although its importance is recently widely accepted, force measurement has been difficult in living biological systems, mainly due to the lack of the versatile non-invasive force measurement methods. The fluctuation theorem, which represents the thermodynamic properties of small fluctuating non-equilibrium systems, has been applied to the analysis of the thermodynamic properties of motor proteins in vitro. Here, we extend it to the axonal transport (displacement) of endosomes. The distribution of the displacement fluctuation had three or four distinct peaks around multiples of a unit value, which the fluctuation theorem can convert into the drag force exerted on the endosomes. The results demonstrated that a single cargo vesicle is conveyed by one to three or four units of force production.

biophysics↗

bioRxiv2017

Tissue architectural cues drive the emergence of non-random trafficking of human tumor cells in the larval zebrafish

Sites of metastasis are non-random, with certain types of cancers showing organ preference during distal colonization. Using multiple brain- and bone marrow-seeking human and murine breast cancer subclones, we determined that tumor cells that home to specific murine organs (brain and bone marrow) ultimately colonized analogous tissues (brain and caudal vein plexus [CVP]) in larval zebrafish. We then exploited the zebrafish model to delineate factors leading to differential cell homing and extravasation. Bone marrow-tropic clones showed higher expression of integrins and focal adhesions associated with mechanosensing machinery than brain-tropic clones and were more sensitive to vessel topography during extravasation. Knockdown of {beta}1 integrin reduced extravasation and redistributed organ targeting from disordered vessels in the CVP to the brain. Our results show that organ selectivity is driven by topography- and cell type-dependent extravasation at the tumor-endothelial interface in the larval zebrafish and provide important insights into the early stages of metastasis.

biophysics↗

bioRxiv2017

pytc: a python package for analysis of Isothermal Titration Calorimetry experiments

Here we describe pytc, an open-source Python-package for global fits of thermodynamic models to multiple Isothermal Titration Calorimetry experiments. Key features include simplicity, the ability to implement new thermodynamic models, a robust maximum likelihood fitter, a fast Bayesian Markov-Chain Monte Carlo sampler, rigorous implementation, extensive documentation, and full cross-platform compatibility. pytc can be used as either a programming API or with a GUI. It is available for download at: https://github.com/harmslab/pytc.

biophysics↗

bioRxiv2017

High accuracy measurements of nanometer-scale distances between fluorophores at the single-molecule level

To uncover the mechanisms of molecular machines it is useful to probe their structural conformations. Single-molecule Forster resonance energy transfer (smFRET) is a powerful tool for measuring intra-molecular shape changes of single-molecules, but is confined to distances of 2-8 nm. Current super-resolution measurements are error prone at <25 nm. Thus, reliable high-throughput distance information between 8-25 nm is currently difficult to achieve. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with [~]1 nm accuracy at any distance >2 nm, using a standard TIRF microscope and open-source software. We applied our two-color localization method to uncover a [~]4 nm conformational change in the \"stalk\" of the motor protein dynein, revealing unexpected flexibility in this antiparallel coiled-coil domain. These new methods enable high-accuracy distance measurements of single-molecules that can be used over a wide range of length scales.

biophysics↗

bioRxiv2017

The cooperation of the haves and the have-nots

Upon starvation, Dictyostelium discoideum (D.d.) exhibit social behavior mediated by the chemical messenger cyclic adenosine monophosphate (cAMP). Large scale cAMP waves synchronize the population of starving cells and enable them to aggregate and form a multi-cellular organism. Here, we explore the effect of cell-to-cell variability in the production of cAMP on aggregation. We create a mixture of extreme cell-to-cell variability by mixing a few cells that produce cAMP (haves) with a majority of mutants that cannot produce cAMP (have-nots). Surprisingly, such mixtures aggregate, although each population on its own cannot aggregate. We show that (1) a lack of divalent ions kills the haves at low densities and (2) the have-nots supply the cAMP degrading enzyme, phosphodiesterase, which, in the presence of divalent ions, enables the mixture to aggregate. Our results suggest that a range of degradation rates induces optimal aggregation. The haves and the have-nots cooperate by sharing complementary resources.

biophysics↗

bioRxiv2017

Modulation of formin processivity by profilin and mechanical tension

Formins are major regulators of actin networks. They enhance actin filament dynamics by remaining processively bound to filament barbed ends. How biochemical and mechanical factors affect formin processivity are open questions. Monitoring individual actin filaments in a microfluidic flow, we report that formin mDia1 dissociates faster under higher ionic strength and when actin concentration is increased. Profilin, known to increase the elongation rate of formin-associated filaments, surprisingly decreases the formin dissociation rate, by bringing formin FH1 domains in transient contact with the barbed end. In contrast, piconewton tensile forces applied to actin filaments accelerate formin dissociation by orders of magnitude, largely overcoming profilin-mediated stabilization. We developed a model of formin conformations and its confrontation to our data indicates the existence of two different dissociation pathways, with force favoring one over the other. How cells limit formin dissociation under tension is now a key question for future studies.

biophysics↗

bioRxiv2017

Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies.

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of cytidine to uridine in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis - for example, A3G deaminates the third cytidine in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy, which we have used to capture an A3G-ssDNA interaction. We characterized an A3G-DNA binding pocket that is important for the enzyme to scan the DNA for its hotspot. The results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.

biophysics↗

bioRxiv2017

Complementary studies of lipid membrane dynamics using iSCAT and STED microscopy

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag - gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 [≤] t [≤] 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag - gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

biophysics↗

bioRxiv2017

Riding the Cell Jamming Boundary: Geometry, Topology, and Phase of Human Corneal Endothelium

It is important to assess the viability of eye-banked corneas prior to transplantation due to inherent senescence and known loss of endothelial cells during surgical manipulation. Corneal endothelial cells have a complex basal and paracellular shape making them challenging to accurately measure, particularly in oedematous ex vivo tissue. This study used calibrated centroidal Voronoi Diagrams to segment cells in images of these human corneas, in order to characterize endothelial geometry, topology, and phase.\n\nHexagonal cells dominated the endothelia, with most comprised of five different pleomorphs exhibiting self-similar topological coarsening through most of the endothelial cell density range. There was a linear relationship between cell size and shape, though cells with greater than six sides were present in larger proportions than cells with less. Hexagonal cell regularity was stable and largely independent of density.\n\nCell and tissue phase was also examined, using the cell shape index relative to the recently discovered cell jamming phase transition boundary. Images showed fluid endothelia with a range of shape indices spanning the boundary, independent of density but dependent on hexagonal regularity. The cells showed a bimodal distribution centred at the boundary, with the largest proportion of cells on the fluid side. A shoulder at the boundary suggested phase switching via shape transformation across the energy barrier, with cells either side having distinctly different size and shape characteristics. Regular hexagonal cells were closest to the boundary.\n\nThis study showed the corneal endothelium acts as a glassy viscous foam characterized by well-established physical laws. Endothelial cell death transiently and locally increases cell fluidity, which is subsequently arrested by jamming of the pleomorphically diverse cell collective, via rearrangement and shape change of a small proportion of cells, which become locked in place by their neighbours and maintain endothelial function with little energy expenditure.

biophysics↗