PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.05.283432v1?rss=1

Authors: Pertici, I., Bianchi, G., Bongini, L., Lombardi, V., Bianco, P.

Abstract:

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine, made by an ensemble of eight HMM myosin fragments from rabbit psoas muscle carried on a piezoelectric nanopositioner and brought to interact with a correctly oriented actin filament attached via gelsolin (a Ca2+-regulated actin binding protein) to a bead trapped by a Dual Laser Optical Tweezers. The application of the original version of the nanomachine to investigation of the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them on at a time, was anyway prevented by the impossibility to preserve Ca2+ as a free parameter. Here the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined either in the absence or in the presence of 0.1 mM Ca2+ (the concentration required for BTA preparation with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.05.284026v1?rss=1

Authors: Wang, Q., Asarnow, D. E., Ding, K., Zhang, Y., Ma, Y., Cheng, Y., Beachy, P. A.

Abstract:

The DISP1 protein, related to the NPC1 and PTCH1 cholesterol transporters and to H+-driven transporters of the RND family, enables tissue patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly-localized sites of embryonic expression. A 2.5 [A] resolution cryo-EM structure of DISP1 revealed three Na+ ions coordinated within a channel that traverses its transmembrane domain. This channel and Na+-binding are disrupted in DISP1-NNN (3.4 [A] resolution), a variant with isosteric substitutions for three intramembrane aspartates that each coordinate and neutralize charge for one of the three Na+ ions. DISP1-NNN and other variants that singly disrupt the Na+ sites retain binding to the lipid-modified Hedgehog protein, but most fail to export Hedgehog. The Sonic hedgehog signal (ShhN) interacts with the DISP1 extracellular domains (2.7 [A] complex structure), including an unusual extended DISP1 arm, at a location well above the membrane. Variability analysis reveals a dynamic series of DISP1 conformations, only a restricted subset of which appear to bind ShhN. The bound and unbound DISP1 conformations display distinct Na+ site occupancies; these differences, in conjunction with the unusual locations of certain lipids bound to DISP1, suggest a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane, thus enabling its tissue patterning activity. The Na+-coordinating residues resolved in our DISP1 structures are wholly or partly conserved in some of the other metazoan RND family members, such as PTCH1 and NPC1, suggesting the utilization of Na+ flux to power their conformationally-driven activities.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.04.282509v1?rss=1

Authors: Cheng, J., Li, B., Si, L., Zhang, X.

Abstract:

Cryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the model dependence and improved the resolution during the subsequent structure refinement procedure. By eliminating model bias, our method allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.04.282921v1?rss=1

Authors: Huertas, J., Schöler, H. R., Cojocaru, V.

Abstract:

Genomic DNA is packaged in chromatin, a dynamic polymer that forms fibers variable in size and compaction. Chromatin's monomeric unit, the nucleosome, is formed by wrapping ~ 146 basepairs of DNA around histone proteins. Dynamic transitions in the chromatin structure, which depend on intra- and inter-nucleosome dynamics and on chemical modifications of histones, are key to initiating gene regulation programs. The histone modifications occur in unstructured terminal regions known as tails and are key to epigenetic regulation of gene expression. Here we elucidate how the interplay between the tails of two histones, H3 and H2A modulate ample breathing motions of genomic nucleosomes. From 18 s of atomistic molecular dynamics simulations of two genomic and one engineered nucleosomes, we observed two rare, large opening events, one in each genomic nucleosome, and only small amplitude breathing in the engineered nucleosome. Clustering of the sampled structures revealed a strong correlation between DNA conformations with different degree of opening and histone tail conformations and positions. The ample nucleosome motions were caused by changes in the interaction profiles between the DNA and clusters of lysine and arginine residues in these tails, many of which are targets for epigenetic regulatory modifications. Given that histone tail modulated nucleosome breathing can change the structure and compaction of chromatin fibers, the mechanism we describe provides the basis for understanding chromatin dynamics at atomic resolution.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.04.283184v1?rss=1

Authors: Banterle, N., Nievergelt, A. P., de Buhr, S., Hatzopoulos, G. N., Brillard, C., Andany, S., Hübscher, T., Sorgenfrei, F., Schwarz, U. S., Gräter, F., Fantner, G. E., Gönczy, P.

Abstract:

Discovering the physical principles directing organelle assembly is a fundamental pursuit in biology. Centrioles are evolutionarily conserved organelles with a 9-fold rotational symmetry of chiral microtubules imparted onto the cilia they template. Centriole assemble from likewise symmetrical ring polymers of SAS-6 proteins, orthogonal to a toroidal surface surrounding the resident centriole. How surface properties ensure ring assembly with proper symmetry and orthogonal arrangement is not known. Here, we deployed photothermally-actuated off-resonance tapping high-speed atomic force microscopy (PORT-HS-AFM) to decipher physical principles of surface-guided SAS-6 self-assembly. Using machine learning to quantify the polymerization reaction and developing a coagulation-fragmentation model, we discovered that the surface shifts the reaction equilibrium by ~104 compared to the solution situation, explaining orthogonal organelle emergence. Moreover, molecular dynamics and PORT-HS-AFM revealed that the surface converts helical SAS-6 polymers into 9-fold ring polymers with residual asymmetry, which may impart chiral features to centrioles and cilia. Overall, we discovered two fundamental physical principles directing robust centriole organelle assembly.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.02.279729v1?rss=1

Authors: Vemula, V., Huber, T., Usaj, M., Bugyi, B., Mansson, A.

Abstract:

Actin is a major intracellular protein with key functions in cellular motility, signalling and structural rearrangements. Its dynamic behavior with actin filaments (F-actin) polymerising and depolymerising in response to intracellular changes, is controlled by actin-binding proteins (ABPs). Gelsolin is one of the most potent filament severing ABPs. However, myosin motors that interact with actin in the presence of ATP also produce actin filament fragmentation through motor induced shearing forces. To test the idea that gelsolin and myosin cooperate in these processes we used the in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin (5 nM) at very low [Ca2+] (free [Ca2+] ~6.8 nM) appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM [MgATP], an effect that was increased at increased HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. As further support of myosin-gelsolin cooperativity we observed reduced sliding velocity of the HMM propelled filaments in the presence of gelsolin. Overall, the results corroborate ideas for cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity, leading among other effects to enhanced F-actin severing of possible physiological relevance.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.02.259200v1?rss=1

Authors: Zhang, M., Tang, W., Ying, W.

Abstract:

Autofluorescence (AF) of skin holds great promise for non-invasive diagnosis of diseases. Our previous study has indicated that keratin-based AF of skin holds great promise to become a novel biomarker for diagnosis of multiple major diseases, including lung cancer, acute ischemic stroke, Parkinson diseases, stable coronary artery disease and myocardial infarction. It is critical to investigate the mechanisms underlying the increased AF of the skin of the patients. Our previous study has shown that development of lung cancer can induce increased green AF of the skin of mice, while the mechanisms underlying the increased AF have remained unclear. We hypothesized that the inflammation in the mice with lung cancer is causative to the increased AF. Our study found that the anti-inflammation drug indomethacin virtually abolished the lung cancer-induced increase in the green AF of the skin. We further found that the AF was originated from the epidermis of the mice. Our findings have provided critical information for understanding the mechanisms underlying the lung cancer-induced increase in the epidermal green AF, indicating that at least for lung cancer, the disease-induced increase in the AF of the skin mainly results from increased inflammation in the body.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.03.281394v1?rss=1

Authors: Medarametla, P., Laitinen, T., Poso, A.

Abstract:

Quorum sensing is being investigated as an alternative therapeutic strategy in antibacterial drug discovery to combat bacterial resistance. LsrK is an autoinducer-2 kinase, playing a key role in the phosphorylation of autoinducer-2 (AI-2) signalling molecules involved in quorum sensing. Inhibiting LsrK could result in reduced pathogenicity by interfering with the quorum sensing signalling. Previously, we have generated homology models to identify LsrK inhibitors using structure-based virtual screening and successfully found the first class of LsrK inhibitors. While conducting these studies, the crystal structure of LsrK was released providing us an opportunity to inspect the reliability and quality of our models. Structural analysis of crystal structure and homology models revealed the consistencies of constructed models with crystal structure in the structural fold and binding site. Further, binding characteristics and conformational changes are investigated using molecular dynamics. These simulations provided us insights into the protein function and flexibility that need to be considered during the structure-based drug design studies targeting LsrK.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.03.281956v1?rss=1

Authors: Heiligenstein, X., de Beer, M., Heiligenstein, J., Eyraud, F., Manet, L., Schmitt, F., Lamers, E., Lindenau, J., Kea-te Lindert, M., Salamero, J., Raposo, G., sommerdijk, N., Belle, M., Akiva, A.

Abstract:

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To explore further the potential of cryo-fixation, we developed a high pressure freezing (HPF) system that allows vitrification under different environmental parameters and apply it in different CLEM workflows. In this chapter we introduce our novel HPF live instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.03.280222v1?rss=1

Authors: Riley, B. T., Wankowicz, S. A., de Oliveira, S. H. P., van Zundert, G. C. P., Hogan, D., Fraser, J. S., Keedy, D. A., van den Bedem, H.

Abstract:

New X-ray crystallography and cryo-electron microscopy (cryo-EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open-source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0.

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