Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 62,102 bioRxiv papers from 275,834 authors.
Most downloaded bioRxiv papers, since beginning of last month
in category biophysics
1,816 results found. For more information, click each entry to expand.
124 downloads biophysics
Immune cells carry out dynamic search processes for antigen and pathogens. T cells in vivo migrate primarily via undirected random walks, but it remains unresolved how these random walks generate a characteristic search behavior. Here we use light sheet microscopy to observe the native population of T cells in the larval zebrafish tail and fin fold over long times. We find that cell-to-cell variability is amplified by a correlation between speed and directional persistence, generating a characteristic cell behavioral manifold that is preserved under a perturbation to cell speeds. Our results suggest that speed-persistence coupling may be an intrinsic feature of T cell migration that provides an alternative to Levy flight for accessing a broad range of length scales in vivo .
124 downloads biophysics
Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in a z-range of over 10 μm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required.
123 downloads biophysics
Carcinoma, the most common type of cancer, develops in the sheets of cells forming the epithelium and lining our organs and cavities. It usually begins with the transformation of a single cell via the activation of oncogenes such as Ras. The capacity of epithelia to eliminate newly transformed cells via apical extrusion is believed to be a critical defense mechanism to eradicate initial stages of carcinoma. Our organs and tissues are in constant motion, exposing epithelial cells to mechanical stretch. How these external forces impact the onset and progression of tumor growth is thus of primary interest, but little is known currently. Here we show that mechanical strains jeopardize the epithelial defense mechanisms against RasV12-transformed MDCK cells by impeding their apical extrusion. Concurrently, they prevent the formation of strong circumferential belts of actin in RasV12 cells, previously established as a primary step of apical extrusion under static conditions. Cyclic stretching also changes the metastatic phenotype of newly transformed cells by greatly promoting the formation of RasV12 protrusions. We show that RasV12 and wild type MDCK cells possess distinct sensitivity to strain. External forces remodel their actin cytoskeletons and adhesion complexes differently, resulting in a more invasive system dynamic. Our work also shows that the Rho-ROCK mechanotransduction pathway is involved in regulating the mechanically-induced switch to a more aggressive phenotype. Such insight may lead to the targeting of mechanotransduction pathways in innovative future therapies.
123 downloads biophysics
Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.
122 downloads biophysics
MicroRNAs (miRNAs) are a class of short non-coding RNAs that function in RNA silencing and post-transcriptional gene regulation. Besides their participation in regulating normal physiological activities, specific miRNA types could act as oncogenes, tumor suppressors or metastasis regulators, which are critical biomarkers for cancer. However, direct characterization of miRNA is challenging due to its unique properties such as its low abundance, sequence similarities and short length. Nanopore Induced Phase Shift Sequencing (NIPSS), which is a variant form of nanopore sequencing, could directly sequence any short analytes including miRNA. In practice, NIPSS clearly discriminates between different identities, isoforms and epigenetic variants of model miRNA sequences. This work demonstrates the first report of direct miRNA sequencing, which serves as a complement to existing miRNA sensing routines by the introduction of single molecule resolution. Future engineering of this technique may assist miRNA based early stage diagnosis or inspire novel cancer therapeutics.
120 downloads biophysics
Cancer's cellular behavior is driven by alterations in the processes that cells use to sense and respond to diverse stimuli. Underlying these processes are a series of chemical processes (enzyme-substrate, protein-protein, etc.). Here we introduce a set of mathematical techniques for describing and characterizing these processes.
120 downloads biophysics
Condensin, a key member of the Structure Maintenance of Chromosome (SMC) protein complexes, has recently been shown to be a motor that extrudes loops of DNA. It remains unclear, however, how condensin complexes work together to collectively package DNA into the chromosomal architecture. Here, we use time-lapse single-molecule visualization to study mutual interactions between two DNA-loop-extruding yeast condensins. We find that these one-side-pulling motor proteins are able to dynamically change each other's DNA loop sizes, even when located large distances apart. When coming into close proximity upon forming a loop within a loop, condensin complexes are, surprisingly, able to traverse each other and form a new type of loop structure, which we term Z loop, three double-stranded DNA helices aligned in parallel with one condensin at each edge. These Z-loops can fill gaps left by single loops and can form symmetric dimer motors that reel in DNA from both sides. These new findings indicate that condensin may achieve chromosomal compaction using a variety of looping structures.
120 downloads biophysics
Cryo-electron tomography (cryo-ET) provides a promising technique to study high resolution structures of macromolecules in situ, opening a new era of structural biology. One major bottleneck of this technique is to prepare suitable cryo-lamellas of cell/tissue samples. The emergence of cryo-focused ion beam (cryo-FIB) milling technique provides a good solution of this bottleneck. However, there are still large limitations of using cryo-FIB to prepare cryo-lamella of tissue specimen because the thickness of tissue increases the difficulty of specimen freezing and cryo-FIB milling. Here we report a new workflow, VHUT-cryo-FIB (Vibratome - High pressure freezing - Ultramicrotome Trimming - cryo-FIB), aiming for efficient preparation of frozen hydrated tissue lamella for subsequent cryo-ET data collection. This workflow includes tissue slicing using vibratome, high pressure freezing, ultramicrotome cryo-trimming, cryo-FIB milling and the subsequent cryo-electron microscopy (cryo-EM). The modification of equipment in this workflow is highly eliminated. We developed two strategies with a special cryo-holder tip or carrier for loading cryo-lamella into side entry cryo-holder or Autoloader catridge. We tested this workflow using the tissue sample of rat skeleton muscle and spinach leaf and collected high quality cryo-ET tilt series, which enabled us to obtain an in situ structure of spinach ribosome by sub-tomogram averaging.
117 downloads biophysics
Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture, at the expense of image detail. Here we show that retinal optical quality improves 2-fold during terminal development, which, confirmed by a mouse model, happens due to nuclear inversion. We further reveal that improved retinal contrast-transmission, rather than photon-budget or resolution, leads to enhanced contrast sensitivity under low light condition. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast-transmission as a decisive determinant of mammalian visual perception.
116 downloads biophysics
Experiments have suggested that bacterial mechanosensitive channels separate into 2D clusters, the role of which is unclear. By developing a coarse-grained computer model we find that clustering promotes the channel closure, which is highly dependent on the channel concentration and membrane stress. This behaviour yields a tightly regulated gating system, whereby at high tensions channels gate individually, and at lower tensions the channels spontaneously aggregate and inactivate. We implement this positive feedback into the model for cell volume regulation, and find that the channel clustering protects the cell against excessive loss of cytoplasmic content.
115 downloads biophysics
Directed cell motion is essential in physiological and pathological processes such as morphogenesis, wound healing and cancer spreading. Chemotaxis has often been proposed as the driving mechanism, even though evidence of long-range gradients is often lacking in vivo. By patterning adhesive regions in space, we control cell shape and the associated potential to move along one direction in another mode of migration coined ratchetaxis. We report that focal contacts distributions collectively dictate cell directionality, and bias is non-linearly increased by gap distance between adhesive regions. Focal contact dynamics on micro-patterns allow to integrate these phenomena in a consistent model where each focal contact can be translated into a force with known amplitude and direction, leading to quantitative predictions for cell motion in every condition. Altogether, our study shows how local and minutes timescale dynamics of focal adhesions and their distribution lead to long term cellular motion with simple geometric rules.
114 downloads biophysics
Experiments on tumor spheroids have shown that compressive stress from their environment can reversibly decrease tumor expansion rates and final sizes. Stress release experiments show that nonuniform anisotropic elastic stresses can be distributed throughout. The elastic stresses are maintained by structural proteins and adhesive molecules, and can be actively relaxed by a variety of biophysical processes. In this letter, we present a new continuum model to investigate how the instantaneous elastic moduli and active stress relaxation, in conjunction with mechanical feedback machinery within cells, regulate the sizes of and stress distributions within growing tumors in the presence of external physical confinement and gradients of growth-promoting chemical fields. We introduce an adaptive reference map that relates the current position with the reference position but adapts to the current position in the Eulerian frame (lab coordinates) via relaxation. This type of stress relaxation is similar to but simpler than the classical Maxwell model of viscoelasticity. By fitting the model to experimental data from two independent studies of tumor spheroid growth, treating the tumors as incompressible, neo-Hookean elastic materials, we find that the rates of stress relaxation of tumor tissues can be comparable to volumetric growth rates. Our study provides insight on how the biophysical properties of the tumor and host microenvironment, mechanical feedback control and diffusion-limited differential growth act in concert to regulate spatial patterns of stress and growth. When the tumor is stiffer than the host, our model predicts tumors are more able to change their size and mechanical state autonomously, which may help to explain why increased tumor stiffness is an established hallmark of malignant tumors.
114 downloads biophysics
Many eukaryotic cells distribute their intracellular components through asymmetrically regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that these MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors, and reveal how transient interactions between MAPs and motors may promote these effects. From these data, we propose a general MAP code that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.
114 downloads biophysics
The three-dimensional organization of chromatin, on the length scale of a few genes, is crucial in determining the functional state \---| accessibility and the amount of gene expression \---| of the chromatin. Recent advances in chromosome conformation capture experiments provide partial information on the chromatin organization in a cell population, namely the contact count between any segment pairs. However, given the contact matrix, determining the complete 3D organization of the whole chromatin polymer is an inverse problem. In the present work, an Inverse Brownian Dynamics (IBD) method has been proposed to compute the optimal interaction strengths between different segments of chromatin such that the experimentally measured contact count probability constraints are satisfied. Applying this method to the alpha-globin gene locus in two different cell types, we predict the 3D organizations corresponding to active and repressed states of chromatin at the locus. We show that the average distance between any two segments of the region has a broad distribution and cannot be computed as a simple inverse relation based on the contact probability alone. We also address the normalization problem of the contact count matrix and argue that extra measurements of polymer properties such as radius of gyration may be required to resolve the problem.
114 downloads biophysics
Michele Oneto, Lorenzo Scipioni, Maria J Sarmento, Isotta Cainero, Simone Pelicci, Laura Furia, Piergiuseppe Pelicci, Gaetano Ivan Dellino, Paolo Bianchini, Mario R Faretta, Enrico Gratton, Alberto Diaspro, Luca Lanzanò
Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, superresolution microscopy has gained considerable interest as it can be used to probe the spatial organization of functional sites in intact single cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here we combine dual color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that STED-ICCS provides not only a value of colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the Proliferating cell nuclear antigen (PCNA) protein have a high level of proximity and are correlated at a nanometer distance which is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of nanoscale distribution of functional sites in the nucleus.
112 downloads biophysics
The TATA-binding protein (TBP) and a transcription factor (TF) IIB−like factor compound the fundamental core of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional intiation factor Bdp1, which is unique to the RNA polymerase (RNAP) III sytem, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain, that arises from DNA compaction and transcriptional activity, on the efficiency of initiation complex formation. We made use of a new nanotechnological tool − a DNA origami-based force clamp - to follow the assembly of human initiation complexes in the Pol II and Pol III system at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is necessary and sufficient to stabilise TBP on a strained RNAP II promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of Pol III genes for the first time.
112 downloads biophysics
ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.
111 downloads biophysics
Piezo1 is a critical mechanical sensor in many cells. It is activated by mechanical force thus allowing cells to sense the physical environment and respond to stress. Structural data have suggested that Piezo1 has a curved shape. Here, we use computational approaches to model, for the first time, the 3D structure of the full-length Piezo1 in an asymmetric membrane. A number of novel insights emerge: (i) Piezo1 creates a dome in the membrane with a trilobed topology that extends beyond the radius of the protein, (ii) Piezo1 changes the lipid environment in its vicinity via specific interactions with cholesterol and PIP2 molecules, (iii) changes in cholesterol concentration that change the membrane stiffness result in changes in the depth of the dome created by Piezo1, and iv) modelling of the N-terminal region that is missing from current structures modifies Piezo1 membrane footprint, suggesting the importance of this region in Piezo1 function.
111 downloads biophysics
The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.
110 downloads biophysics
Pioneer transcription factors (pTFs) bind to target sites within compact chromatin initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here we reveal, using single-molecule fluorescence approaches, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber, but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting nucleosome-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to destabilize promoter nucleosomes, paving the way to form long-lived bound states on now exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression.
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