PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.03.281576v1?rss=1

Authors: Budaitis, B. G., Jariwala, S., Rao, L., Sept, D., Verhey, K., Gennerich, A.

Abstract:

The kinesin-3 motor KIF1A functions in neurons where its fast and superprocessive motility is thought to be critical for long-distance transport. However, little is known about the force-generating properties of kinesin-3 motors. Using optical tweezers, we demonstrate that KIF1A and its C. elegans homolog UNC-104 undergo force-dependent detachments at ~3 pN and then rapidly reattach to the microtubule to resume motion, resulting in a sawtooth pattern of clustered force generation events that is unique among the kinesin superfamily. Whereas UNC-104 motors stall before detaching, KIF1A motors do not. To examine the mechanism of KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders, V8M and Y89D, based on their location in structural elements required for force generation in kinesin-1. Molecular dynamics simulations predict that the V8M and Y89D mutations impair docking of the N-terminal ({beta}9) or C-terminal ({beta}10) portions of the neck linker, respectively, to the KIF1A motor domain. Indeed, both mutations dramatically impair force generation of KIF1A but not the motor's ability to rapidly reattach to the microtubule track. Homodimeric and heterodimeric mutant motors also display decreased velocities, run lengths, and landing rates and homodimeric Y89D motors exhibit a higher frequency of non-productive, diffusive events along the microtubule. In cells, cargo transport by the mutant motors is delayed. Our work demonstrates the importance of the neck linker in the force generation of kinesin-3 motors and advances our understanding of how mutations in the kinesin motor domain can manifest in disease.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.02.279398v1?rss=1

Authors: Kamal, M. A., Janes, J. A., Li, L., Thibaudau, F., Smith, A.-S., Sengupta, K.

Abstract:

The interactions between different intra-cellular organelles, including the endoplasmic reticulum, have recently been in focus thanks to the tremendous progress in imaging them using cryogenic transmission electron microscopy. However, they are still difficult to study in cellulo, and reconstituting these systems has been a standing challenge. Here we achieve this task using a giant unilamellar vesicle (GUV) and supported lipid bilayer (SLB) system. The tethers, which may reside in the cytosol when unbound, are mimicked by single (or double) stranded DNA sequences of two different lengths with ends that are self-sticky, and with terminal cholesterol moieties which insert into GUV or SLB membranes. The DNA-tethers, bound by their sticky-end, can exist in two possible states - either with both cholesterols in the same membrane or each cholesterol in a different membrane, the latter conformation leading to adhesion. Exchange of tether-molecules between the membranes occurs through the aqueous phase. By developing theoretical arguments that are supported in our experiments, we show that this possibility of exchange and the relative difference in the projected area between the two states drives the adhesion due to collective entropic considerations, rather than the usually considered enthalpy of binding. The establishment of this fundamentally different interaction between two membranes suggests that in physiological conditions, the regulation of contact formation inside cells may be very different from the case of the much studied ligand-receptor pairing on the external cell membrane.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.02.279794v1?rss=1

Authors: Alibert, C., Pereira, D., Lardier, N., Etienne-Manneville, S., Goud, B., Asnacios, A., Manneville, J.-B.

Abstract:

Cancer cell softening increases with the progression of the disease, suggesting that mechanical phenotyping could be used as a diagnostic or prognostic method. Here we investigate the cell mechanics of gliomas, brain tumors that originate from glial cells or glial progenitors. We use two microrheology techniques, a single cell parallel plates rheometer to probe whole cell mechanics and optical-tweezers to probe intracellular rheology. We show that cell mechanics discriminates human glioma cells of different grades. When probed globally, grade IV glioblastoma cells are softer than grade III astrocytoma cells, while they are surprisingly stiffer at the intracellular level. We explain this difference between global and local intracellular behaviours by changes in the composition and spatial organization of the cytoskeleton and by changes in nuclear mechanics. Our study highlights the need to combine rheology techniques for potential diagnostic or prognostic methods based on cancer cell mechanophenotyping.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.02.868505v1?rss=1

Authors: Francois, J., Kandasamy, A., Yeh, Y.-T., Ayala, C., Meili, R., Chien, S., Lasheras, J. C., del Alamo, J. C.

Abstract:

Three-dimensional (3-D) neutrophil migration is essential for immune surveillance and inflammatory responses. During 3-D migration, especially through extravascular spaces, neutrophils rely on frontal protrusions and rear contractions to squeeze and maneuver through extracellular matrices containing narrow pores. However, the role of matrix density and the cells' ability to probe and remodel matrix pores during 3-D chemotaxis are far from being understood. We investigated these processes by tracking the trajectories of over 20,000 neutrophils in a 3-D migration device containing collagen matrices of varying concentrations and analyzing the shape of these trajectories at multiple scales. Additionally, we quantified the transient 3-D matrix deformations caused by the migrating cells. The mean pore size of our reconstituted collagen matrices decreased when the collagen concentration ([col]) was increased. In low-[col] matrices, neutrophils exerted large transient deformations and migrated in relatively straight trajectories. In contrast, they were not able to appreciably deform high-[col] matrices and adapted to this inability by turning more often to circumvent these narrow matrix pores. While this adaptation resulted in slower migration, the cells were able to balance the more frequent turning with the long-range directional bias necessary for chemotaxis. Based on our statistical analysis of cell trajectories, we postulate that neutrophils achieve this balance by using matrix obstacles as pivoting points to steer their motion towards the chemoattractant. Inhibiting myosin-II contractility or Arp2/3-mediated pseudopod protrusions not only compromised the cells' ability to deform the matrix, but also made them switch to increased turning in more restrictive matrices when compared to untreated control cells. Both myosin-II contractility and Arp2/3-mediated branched polymerization of actin played a role in fast migration, but Arp2/3 was also crucial for neutrophils when coordinating the orientations of successive turns to prevent veering away from the chemotactic path. These results may contribute to an improved understanding of the mechanisms employed by migrating neutrophils in confined 3-D environments, as well as the molecular and environmental regulators for maintaining persistent motion.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.01.278812v1?rss=1

Authors: Yang, Y. X., Li, P., Wang, P., Zhu, B. T.

Abstract:

Insulin receptor plays an important role in regulation of energy metabolism. Dysfunction of insulin receptor (IR) can lead to many disease states, such as diabetes mellitus. Deciphering the complex dynamic structure of human IR and its mechanism of activation would greatly aid in understanding IR-mediated signaling pathways and in particular, in designing new drugs (including nonpeptidal insulin analogs) to treat diabetes mellitus. Experimental evidence about IR structure has been gradually obtained by biologists over the past three decades. Based on the available experimental structures of IR in different states, here we employ molecular modeling approach to construct the full-length IR structures in different states and model its structural and conformational changes during insulin-induced IR activation. Several key possible intermediate states are constructed based on structural alignment, rotation and computational modeling. Based on the structures of the full-length IR in different states, it appears that there are two possible conformational transition pathways: one is symmetric, and the other one is asymmetric. Structural changes and motions of different domains of the full-length IR along the pathways are analyzed. The role of insulin binding to IR in facilitating the conformational transition of the receptor is modeled. Information and insights derived from our present structural modeling analyses may aid in understanding the complex dynamic, structural and conformational changes during the process of IR activation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.01.277756v1?rss=1

Authors: Yang, D., Lyashkov, A. E., Morrell, C. H., Zahanich, I., Yaniv, Y., Vinogradova, T. M., Ziman, B. D., Maltsev, V. A., Lakatta, E. G.

Abstract:

Variability of heart pacemaker cell action potential (AP) firing intervals (APFI) means that pacemaker mechanisms do not achieve equilibrium during AP firing. We tested whether mechanisms that underlie APFI, in rabbit sinoatrial cells are self-similar within and across the physiologic range of APFIs effected by autonomic receptor stimulation. Principal Component Analyses demonstrated that means and variabilities of APFIs and local Calcium releases kinetics, of AP induced Calcium-transient decay times, of diastolic membrane depolarization rates, of AP repolarization times, of simulated ion current amplitudes, are self-similar across the broad range of APFIs (264 to 786 ms). Further, distributions of both mean APFIs and mean Calcium Variability of heart pacemaker cell action potential (AP) firing intervals (APFI) means that pacemaker mechanisms do not achieve equilibrium during AP firing. We tested whether mechanisms that underlie APFI, in rabbit sinoatrial cells are self-similar within and across the physiologic range of APFIs effected by autonomic receptor stimulation. Principal Component Analyses demonstrated that means and variabilities of APFIs and local Calcium releases kinetics, of AP induced Calcium-transient decay times, of diastolic membrane depolarization rates, of AP repolarization times, of simulated ion current amplitudes, are self-similar across the broad range of APFIs (264 to 786 ms). Further, distributions of both mean APFIs and mean Calcium and membrane potential dependent coupled-clock function kinetics manifested similar power law behaviors across the physiologic range of mean APFIs. Thus, self-similar variability of clock functions intrinsic to heart pacemaker cells determines both the mean APFI and its interval variability, and vice versa. and membrane potential dependent coupled-clock function kinetics manifested similar power law behaviors across the physiologic range of mean APFIs. Thus, self-similar variability of clock functions intrinsic to heart pacemaker cells determines both the mean APFI and its interval variability, and vice versa.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.02.280008v1?rss=1

Authors: Miller-Vedam, L. E., Bräuning, B., Popova, K. D., Schirle Oakdale, N. T., Bonnar, J. L., Prabu, J. R., Boydston, E. A., Sevillano, N., Shurtleff, M. J., Craik, C. S., Schulman, B., Frost, A., Weissman, J. S.

Abstract:

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies, and human-specific features associated with pathologies. Structure-based functional studies revealed at least two separable EMC activities, as an insertase regulating tail-anchored protein levels and as a polytopic membrane protein holdase chaperone. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities, which confer client-specific regulation, and a novel, non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.01.278671v1?rss=1

Authors: Tompkins, K. J., Houtti, M., Litzau, L. A., Aird, E. J., Everett, B. A., Nelson, A. T., Pornschloegl, L., Limon-Swanson, L. K., Evans, R. L., Evans, K., Shi, K., Aihara, H., Gordon, W. R.

Abstract:

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay termed HUH-seq to interrogate subtleties in Rep specificity, and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed us to engineer a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.01.277616v1?rss=1

Authors: Castillo, M. A., Wardley, W. P., Lopez-Garcia, M.

Abstract:

Chloroplasts, the organelles responsible for photosynthesis in most plants and algae, exhibit a variety of morphological adaption strategies to changing light environments which can have important yet overlooked light scattering effects. This can be even more significant for iridoplasts, specialized chloroplasts whose tissue is arranged as a photonic multilayer producing a characteristic strong blue reflectance associated to a wavelength selective absorption enhancement relevant for photosynthesis. In this work, we study how the photonic properties of iridoplasts are affected by light induced dynamic changes using realistic data extracted from previous reports. Our results show a reflectance red-shift from blue to green under increasing light intensity. Consequently, the light absorption enhancement induced by the photonic nanostructure is also redshifted. We also show that the photonic properties are resilient to biologically realistic levels of disorder in the structure. We extended this analysis to another photonic nanostructure-containing chloroplast, known as a bisonoplast, and found similar results, pointing towards similar properties in different plant species. We finally found that all types of chloroplasts can tune light absorption depending on light conditions. In general, our study opens the door to understanding how dynamic morphologies in chloroplasts can affect light scattering and absorption.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.01.276824v1?rss=1

Authors: Leyden, F., Uthistran, S., Moorthi, U. K., York, H. M., Patil, A., Gandhi, H., Petrov, E. P., Arumugam, S.

Abstract:

Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 control the membrane protrusions that rise and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold number of molecules for sustained actin polymerization leading to ruffling. Our experiments also suggest that Rac1 activation leads to actin assembly at the cell membrane that are intrinsically linear, giving rise to the lamellar nature of the protrusions. Deactivating PA Rac1 leads to complex contractile processes leading to formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically enriches in the ruffles. Overall, a biphasic mechanism can be envisioned; a polymerization phase driven by activated Rac1 resulting in growth of membrane protrusions and subsequent deactivation of Rac1 leading to contractile collapse and engulfment. The morphological dynamics and presence of Myo1e suggests that the general architecture and molecular compositions may be conserved across lamellar membrane protrusion machinery in living cells.

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