PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.310144v1?rss=1

Authors: Aramesh, M., Mergenthal, S., Issler, M., Weber, F., Plochberger, B., Qin, X.-H., Liska, R., Duda, G., Huppa, J., Ries, J., Schuetz, G., Klotzsch, E.

Abstract:

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination process. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Common approaches such as traction force microscopy (TFM) employ a global readout of the force fields, e.g. by measuring the displacements of hydrogel-embedded marker beads. Recent data, however, indicated that T-cells exert tensile forces locally via TCR-enriched microvilli while scanning the surface of antigen-presenting cells. Here, we developed a traction force microscopy platform, which allows for quantifying the pulls exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. For this, we immobilized specific T-cell activating antibodies directly on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas, CD45 is excluded from bead-microvilli contacts.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.309898v1?rss=1

Authors: Hub, J. S.

Abstract:

Topological transitions of membranes, such as pore formation or membrane fusion, play key roles in biology, biotechnology, and in medical applications. Calculating the related free energy landscapes has been complicated by the fact that such processes involve a sequence of transitions along highly distinct directions in conformational space, making it difficult to define good reaction coordinates (RCs) for the overall process. In this study, we present a new RC capable of driving both pore nucleation and pore expansion in lipid membranes. The potential of mean force (PMF) along the RC computed with molecular dynamics (MD) simulations provides a comprehensive view on the free-energy landscape of pore formation, including a barrier for pore nucleation, the size, free energy, and metastability of the open pore, and the energetic cost for further pore expansion against the line tension of the pore rim. We illustrate the RC by quantifying the effects (i) of simulation system size and (ii) of the addition of dimethyl sulfoxide (DMSO) on the free energy landscape of pore formation. PMF calculations along the RC provide mechanistic and energetic understanding of pore formation, hence they will be useful to rationalize the effects of membrane-active peptides, electric fields, and membrane composition on transmembrane pores.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.310391v1?rss=1

Authors: Jensen, S. B., Thodberg, S., Parween, S., Moses, M. E., Hansen, C. C., Thomsen, J., Sletfjerding, M. B., Knudsen, C., Giudice, R. D., Lund, P. M., Castano, P. R., Bustamante, Y. G., Jorgensen, F. S., Pandey, A. V., Laursen, T., Moller, B. L., Hatzakis, N. S.

Abstract:

Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identified ligands that dock on POR and bias its specificity towards CYP redox partners. Single molecule FRET studies revealed ligand docking to alter POR conformational sampling, which resulted in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand docking on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease related, metabolic pathways.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.309609v1?rss=1

Authors: Beton, J. G., Helfmann, L., Gray, R., Hoogenboom, B., Joseph, A. P., Topf, M., Pyne, A. L. B.

Abstract:

We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program includes identification and tracing of individual molecules in both circular and linear conformations without user input. The program is freely available via GitHub (https://github.com/afmstats/TopoStats), and is intended to be modified and adapted for use if required. TopoStats can identify individual molecules and molecular assemblies within a wide field of view, without the need for prior processing. We demonstrate its power by identifying and tracing individual biomolecules, including DNA origami, pore-forming proteins, and DNA molecules in both closed circular and linear form.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.309492v1?rss=1

Authors: Thallmair, V., Schultz, L., Marrink, S.-J., Oliver, D., Thallmair, S.

Abstract:

Phosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic. Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico. Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.22.309005v1?rss=1

Authors: Bromberg, R., Guo, Y., Plymire, D. A., Emde, T., Puzio, M., Borek, D., Otwinowski, Z.

Abstract:

The His-tag is a widely used affinity tag that facilitates purification of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the water-air interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. The analysis of particle orientations suggests that His-tag presence can mask the hydrophobic patches on a protein's surface that mediate the interactions with the water-air interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with water-air interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the water-air interface.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.309575v1?rss=1

Authors: Liao, X., Purohit, P.

Abstract:

Self-assembly of proteins on lipid membranes underlies many important processes in cell biology, such as, exo- and endo-cytosis, assembly of viruses, etc. An attractive force that can cause self-assembly is mediated by membrane thickness interactions between proteins. The free energy profile associated with this attractive force is a result of the overlap of thickness deformation fields around the proteins. The thickness deformation field around proteins of various shapes can be calculated from the solution of a boundary value problem and is relatively well understood. Yet, the time scales over which self-assembly occurs has not been explored. In this paper we compute this time scale as a function of the initial distance between two inclusions by viewing their coalescence as a first passage time problem. The first passage time is computed using both Langevin dynamics and a partial differential equation, and both methods are found to be in excellent agreement. Inclusions of three different shapes are studied and it is found that for two inclusions separated by about hundred nanometers the time to coalescence is hundreds of milliseconds irrespective of shape. Our Langevin dynamics simulation of self-assembly required an efficient computation of the interaction energy of inclusions which was accomplished using a finite difference technique. The interaction energy profiles obtained using this numerical technique were in excellent agreement with those from a previously proposed semi-analytical method based on Fourier-Bessel series. The computational strategies described in this paper could potentially lead to efficient methods to explore the kinetics of self-assembly of proteins on lipid membranes.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.310201v1?rss=1

Authors: Hunter, M., Liu, T., Möbius, W., Fusco, D.

Abstract:

Reaction-diffusion waves have long been used to describe the growth and spread of populations undergoing a spatial range expansion. Such waves are generally classed as either pulled, where the dynamics are driven by the very tip of the front and stochastic fluctuations are high, or pushed, where cooperation in growth or dispersal results in a bulk-driven wave in which fluctuations are suppressed. These concepts have been well studied experimentally in populations where the cooperation leads to a density-dependent growth rate. By contrast, relatively little is known about experimental populations that exhibit a density-dependent dispersal rate. Using bacteriophage T7 as a test organism, we present novel experimental measurements that demonstrate that the diffusion of phage T7, in a lawn of host E. coli, is hindered by the physical presence of the host bacteria cells. The coupling between host density, phage dispersal and cell lysis caused by viral infection results in an effective density-dependent diffusion rate akin to cooperative behavior. Using a system of reaction-diffusion equations, we show that this effect can result in a transition from a pulled to pushed expansion. Moreover, we find that a second independent density-dependent effect on phage dispersal spontaneously emerges as a result of the viral incubation period, during which phage is trapped inside the host unable to disperse. Our results indicate both that bacteriophage can be used as a controllable laboratory population to investigate the impact of density-dependent dispersal on evolution, and that the genetic diversity and adaptability of expanding viral populations could be much greater than is currently assumed.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.22.308320v1?rss=1

Authors: Lam, A. J., Rao, L., Anazawa, Y., Okada, K., Chiba, K., Niwa, S., Gennerich, A., Nowakowski, D. W., McKenney, R. J.

Abstract:

KIF1A, a kinesin-3 family member, plays critical roles as a long-distance cargo-transporter within neurons. Over 100 known KIF1A mutations in humans result in KIF1A Associated Neurological Disease (KAND), developmental and degenerative neurological conditions for which there is no cure. A de novo missense mutation, P305L, was recently identified in several children diagnosed with KAND, but the underlying molecular basis for the disease phenotype is unknown. Interestingly, this residue is highly conserved in kinesin-family proteins, and together with adjacent conserved residues also implicated in KAND, forms an unusual 310-helical element immediately C-terminal to loop-12 (L12, also known as the K-loop in KIF1A) in the conserved kinesin motor core. In KIF1A, the disordered K-loop contains a highly charged insertion of lysines that is thought to endow the motor with a high microtubule-association rate. Here, we characterize the molecular defects of the P305L mutation in KIF1A using genetic, biochemical, and single-molecule approaches. We find the mutation negatively impacts the velocity, run-length, and force generation of the motor. However, a much more dramatic effect is observed on the microtubule-association rate of the motor, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that an elusive K-loop conformation, mediated by formation of a highly conserved adjacent 310-helix that is modulated via P305, is critically important for the kinesin-microtubule interaction. Importantly, we find that the function of this proline is conserved in the canonical kinesin, KIF5, revealing a fundamental principle of the kinesin motor mechanism.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.21.306605v1?rss=1

Authors: Perera, T., Gunasekara, H., Hu, Y. S.

Abstract:

Single-molecule imaging has provided new insights on weak transient biomolecular interactions with micromolar to millimolar affinity. However, the limited duration of observation has hindered the study of strong and reversible interactions with sub-nanomolar affinity. We report single-molecule interaction microscopy (SMIM), which combines point accumulation for imaging in nanoscale topography (PAINT) with extended imaging durations that enables the study of antibody binding kinetics in the cellular environment. SMIM revealed heterogeneous binding kinetics and the effect of concentration and antibody valency on the association and dissociation rates on antibody-antigen interactions in their cellular environments. We thereby demonstrate SMIM as a versatile single-molecule technique for studying strong, transient biomolecular interactions.

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