PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.31.275321v1?rss=1

Authors: Goldfarb, D., Bahrenberg, T., Yardeni, E. H., Feintuch, A., Bibi, E.

Abstract:

MdfA, a member of the major facilitator superfamily (MFS), is a multidrug/proton antiporter from E. coli that has been considered a model for secondary multidrug (Mdr) transporters. Its transport mechanism, driven by a proton gradient, is associated with conformational changes, which accompany the recruitment of drugs and their release. In this work, we applied double-electron electron resonance (DEER) spectroscopy to locate the binding site of one of its substrates, tetraphenylphosphonium (TPP) within available crystal structures. We carried out Gd(III)-nitroxide distance measurements between MdfA labeled with a Gd(III) tag and the TPP analog mito-TEMPO (bearing the nitroxide moiety). Data were obtained both for MdfA solubilized in detergent micelles (n-dodecyl-{beta}-D-maltopyranoside )DDM)), and reconstituted into lipid nanodiscs (ND). For both DDM and ND, the average position of the substrate at a neutral pH was found to be close to the ligand position in the If (inward facing) crystal structure, with the DDM environment exhibiting a somewhat better agreement than the ND environment. We therefore conclude that the If structure provides a good description for substrate-bound MdfA in DDM solution, while in ND the structure is slightly modified. A second binding site was found for the ND sample situated at the cytoplasmic side, towards the end of transmembrane helix 7 (TM7). In addition, we used DEER distance measurements on Gd(III) doubly labeled MdfA to track conformational changes within the periplasmic and cytoplasmic sides associated with substrate binding. We detected significant differences in the periplasmic side of MdfA, with the ND featuring a more closed conformation than in DDM, in agreement with earlier reports. The addition of TPP led to a noticeable conformational change in the periplasmic face in ND, attributed to a movement of TM10. This change was not observed in DDM.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.29.273185v1?rss=1

Authors: Honda, G., Saito, N., Fujimori, T., Hashimura, H., Nakamura, M. J., Nakajima, A., Sawai, S.

Abstract:

In fast moving cells such as amoeba and immune cells, spatio-temporal regulation of dendritic actin filaments shapes large-scale plasma membrane protrusions. Despite the importance in migration as well as in particle and liquid ingestion, how these processes are affected by the micrometer-scale surface features is poorly understood. Here, through quantitative imaging analysis of Dictyostelium on micro-fabricated surfaces, we show that there is a distinct mode of topographically guided cell migration (phagotaxis) directed by the macropinocytic Ras/PI3K signaling patches. The topography guidance was PI3K-dependent and involved nucleation of a patch at the convex curved surface and confinement at the concave surface. Due to the topography-dependence, constitutive cup formation for liquid uptake in the axenic strain is also destined to trace large surface features. Given the fact that PI3K-dependency of phagocytosis are restricted to large particles in both Dictyostelium and immune cells, topography-dependency and the dual-use of membrane cups may be wide-spread.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.30.266148v1?rss=1

Authors: Hou, W., Ma, D., He, X., Han, W., Ma, J., Xu, C., Xie, R., Fan, Q.-h., Ye, F., Hu, S., Lu, Y., Li, M.

Abstract:

It is a big challenge to measure position changes of biomolecules in the direction normal to the plasma membranes of living cells. We developed a one donor-multiple quenchers Forster resonance energy transfer method by using non-fluorescent quenchers in the extracellular environment. It senses subnanometer position changes of a fluorophore-labeled biomolecule in the plasma membrane. The method was validated by monitoring flip-flops of individual lipid molecules incorporated in plasma membranes. We studies membrane perforation by a host defense peptide from the extracellular side and found that the pore-forming peptide is dynamic, switching among different insertion depths. The method is especially useful in studying interactions of membrane proteins with the inner surfaces of plasma membranes. Our method will find wide applications in systematic analysis of fundamental cellular processes at plasma membranes.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.30.273748v1?rss=1

Authors: Kaur, T., Raju, M., Alshareedah, I., Davis, R. B., Potoyan, D. A., Banerjee, P. R.

Abstract:

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Bio-condensates typically contain a dense network of multiple proteins and RNAs, yet the role of overlapping molecular interactions in regulating the condensate composition and structure is not well understood. Employing a ternary system comprising of a prion-like polypeptide (PLD), arginine-rich polypeptide (RLD), and RNA, here we show that competition between the PLD and RNA for a single shared partner, the RLD, leads to PLD-RLD demixing and spontaneous formation of biphasic condensates. Combining experiments with simulations, we show that the topology of coexisting condensates is regulated via mixture composition and the nature of protein-protein and protein-RNA interactions, giving rise to a diverse set of multiphasic patterns including completely separated, partially and completely engulfed droplet morphologies, and Janus droplets. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic bio-condensates.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.28.273052v1?rss=1

Authors: McCreery, K., Xu, X., Scott, A., Fajrial, A. K., Calve, S., Ding, X., Neu, C.

Abstract:

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. The extracellular environment is physically linked to the nuclear envelope and provides cues to maintain nuclear structure and cellular homeostasis regulated in part by mechanotransduction mechanisms. However, little is known about how structure and properties of the extracellular matrix in living tissues impacts nuclear mechanics, and current experimental challenges limit the ability to detect and directly measure nuclear mechanics while cells are within the native tissue environment. Here, we hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. We aimed to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, we expanded an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue. We validated this technique by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, we found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, we demonstrated the capability for cell and nucleus elastography using the AFM needle-tip technique. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.30.274373v1?rss=1

Authors: Tompkins, K. J., Berscheid, E. T., Venkatesh, N., Andrew, A. J., Beckman, A. P., Esler, M. A., Evans, A. C., Everett, B. A., Houtti, M., Koo, H., Litzau, L. A., Nelson, A. T., Peterson, T. M., Reid, T. A., Evans, R. L., Gordon, W. R.

Abstract:

Advanced biological molecule force probing methods such as atomic force microscopy and optical tweezers used to quantify forces at the single-molecule level are expensive and require extensive training and technical knowledge. However, the technologies underlying a centrifuge force microscope (CFM) are relatively straight forward, allowing for construction by labs with relatively low budgets and minimal training. Design ideas from previously constructed CFMs served as a guide in the development of this CFM. There were two primary goals: first, to develop an inexpensive, functional CFM using off-the-shelf and 3D printed parts; and second, to do so in the context of providing an educational experience for a broad range of students. The team included high school students and undergraduates from local high schools, the University of Minnesota, and other local higher education institutions. This project created an environment for student-focused development of the CFM that fostered active learning, individual ownership, as well as excellence in research. The instrument discussed herein represents a fully functional CFM designed and built by a postdoctoral researcher and a graduate student who together mentored several high school and undergraduate students.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.30.274316v1?rss=1

Authors: Chen, W., Mani, N., Karani, H., Li, H., Mani, S., Tang, J. X.

Abstract:

Powered by flagella, many species of bacteria exhibit collective motion on a solid surface commonly known as swarming. Physical changes like cell elongation and hyper flagellation have been shown to accompany swarming phenotype. Less noticeable, however, are the contrasts of collective motion between the swarming cells and the planktonic cells of comparable cell density. Here, we show that when confined by microwells of specific sizes mounted on a soft agar surface, novel bacteria Enterobacter sp. SM3 under swarming condition exhibited 'single-swirl' motion pattern distinct from 'multi-swirl' motion pattern formed by its concentrated planktonic counterpart. We hypothesize that 'rafting behavior' of the swarming bacteria upon dilution might account for the motion pattern difference. This was further validated through computational simulation where swarming cells are modeled by lower repulsion and stronger alignment among them than planktonic cells. Our new technical approach also enabled us to observe swarming on a non-agar tissue surface.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.28.272435v1?rss=1

Authors: Balakrishnan, S., Raju, S. R., Barua, A., Ananthasuresh, G. K.

Abstract:

Tension in actin cytoskeleton regulates many cellular processes and nuclear morphology. Here, we demonstrate a simple computational method for estimating actin cytoskeletal tension from nucleus shape. We first note that mechanics-based modeling defines a relationship among the volume, surface area, and projected area of the nucleus and hence a specific surface in the three-parameter space of the aforementioned geometric quantities. Data of nuclei from multiple cell types lie on such a surface. Furthermore, nuclei from a given cell population lie on a straight line on the surface. The location and orientation of the line varies with cell type. By using a mechanical model, we present two non-dimensional parameters, namely, the flatness and stretch indicators, which serve as curvilinear coordinates on the surface. Flatness indicator defines the extent of nuclear flattening due to actin cytoskeletal tension and the stretch indicator captures the effect of the elastic modulus of the nuclear envelope. We validate our assertions by modulating the actin cytoskeletal tension using three independent mechanisms: (i) direct downregulation by Cytochalasin D, (ii) indirect upregulation using Nocodazole, and (iii) mechanical stimulation by varying substrate stiffness. We also infer that the flatness indicator is equivalent to the ratio of the height to diameter of the nucleus and is related to the Vogel number. By using this geometric insight, we validate the predictions of our model with data from many previous studies. Finally, we present an analytical formula and a correlation for estimating actin cytoskeletal tension from nuclear projected area and volume.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.28.273110v1?rss=1

Authors: To, P., Whitehead, B., Tarbox, H. E., Fried, S. D.

Abstract:

Decades of research on protein folding have primarily focused on a privileged subset of small proteins that can reversibly refold out of a denaturant. However, these studies are not representative of the complexity of natural proteomes, which consist of many proteins with more sophisticated architectures. Here, we introduce an experimental approach to probe protein refolding for whole proteomes. We accomplish this by first unfolding and refolding E. coli lysates, and then interrogating the resulting protein structures using a permissive protease that preferentially cleaves at flexible regions. Using mass spectrometry, we globally analyze the digestion patterns to assess structural differences between native and "refolded" proteins. These studies show that roughly half of the E. coli proteome cannot reassemble their native states following chemical denaturation. Our results imply that thermodynamics alone cannot specify the native structures of all proteins, signaling a pervasive role for kinetic control in shaping protein biogenesis.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.28.267468v1?rss=1

Authors: Kilic, Z., Sgouralis, I., Heo, W., Ishii, K., Tahara, T., Presse, S.

Abstract:

Our goal is to learn kinetic rates from single molecule FRET (smFRET) data even if these exceed the data acquisition rate. To achieve this, we develop a variant of our recently proposed hidden Markov jump process (HMJP) with which we learn transition kinetics from parallel measurements in donor and acceptor channels. Our HMJP generalizes the hidden Markov model (HMM) paradigm in two critical ways: (1) it deals with physical smFRET systems as they switch between conformational states in continuous time; (2) it estimates the transition rates between conformational states directly without having recourse to transition probabilities or assuming slow dynamics (as is necessary of the HMM). Our continuous time treatment learns transition kinetics and photon emission rates for dynamical regimes inaccessible to the HMM which treats system kinetics in discrete time. We validate the robustness of our framework on simulated data and demonstrate its performance on experimental data from FRET labeled Holliday junctions. Keywords: smFRET | fluorescence | hidden Markov jump process | continuous time process | single molecule data analysis

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