Coarse-grained chromatin dynamics by tracking multiple similarly labeled gene loci.

The "holy grail" of chromatin research would be to follow the chromatin configuration in individual live cells over time. One way to achieve this goal would be to track the positions of multiple loci arranged along the chromatin polymer with fluorescent labels. Using distinguishable labels would define each locus uniquely in a microscopic image but would restrict the number of loci that could be observed simultaneously due to experimental limits to the number of distinguishable labels.

Coarse-grained chromatin dynamics by tracking multiple similarly labeled gene loci.

The "holy grail" of chromatin research would be to follow the chromatin configuration in individual live cells over time. One way to achieve this goal would be to track the positions of multiple loci arranged along the chromatin polymer with fluorescent labels. Use of distinguishable labels would define each locus uniquely in a microscopic image but would restrict the number of loci that could be observed simultaneously, because of experimental limits to the number of distinguishable labels.

Cohesin distribution alone predicts chromatin organization in yeast via conserved-current loop extrusion.

Background: Inhomogeneous patterns of chromatin-chromatin contacts within 10-100-kb-sized regions of the genome are a generic feature of chromatin spatial organization. These features, termed topologically associating domains (TADs), have led to the loop extrusion factor (LEF) model. Currently, our ability to model TADs relies on the observation that in vertebrates TAD boundaries are correlated with DNA sequences that bind CTCF, which therefore is inferred to block loop extrusion.

Identifying topologically associating domains using differential kernels.

Chromatin is a polymer complex of DNA and proteins that regulates gene expression. The three-dimensional (3D) structure and organization of chromatin controls DNA transcription and replication. High-throughput chromatin conformation capture techniques generate Hi-C maps that can provide insight into the 3D structure of chromatin.

The condensation of HP1α/Swi6 imparts nuclear stiffness.

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains.

Effect of loops on the mean-square displacement of Rouse-model chromatin.

Chromatin polymer dynamics are commonly described using the classical Rouse model. The subsequent discovery, however, of intermediate-scale chromatin organization known as topologically associating domains (TADs) in experimental Hi-C contact maps for chromosomes across the tree of life, together with the success of loop extrusion factor (LEF) model in explaining TAD formation, motivates efforts to understand the effect of loops and loop extrusion on chromatin dynamics. This paper seeks to fulfill this need by combining LEF-model simulations with extended Rouse-model polymer simulations to investigate the dynamics of chromatin with loops and dynamic loop extrusion.

Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins.

Uncovering diffusive states of the yeast membrane protein, Pma1, and how labeling method can change diffusive behavior.

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2.

Loops and the activity of loop extrusion factors constrain chromatin dynamics.

The chromosomes-DNA polymers and their binding proteins-are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes.

Extrusion of chromatin loops by a composite loop extrusion factor.

Chromatin loop extrusion by structural maintenance of chromosome (SMC) complexes is thought to underlie intermediate-scale chromatin organization inside cells. Motivated by a number of experiments suggesting that nucleosomes may block loop extrusion by SMCs, such as cohesin and condensin complexes, we introduce and characterize theoretically a composite loop extrusion factor (composite LEF) model. In addition to an SMC complex that creates a chromatin loop by encircling two threads of DNA, this model includes a remodeling complex that relocates or removes nucleosomes as it progresses along the chromatin, and nucleosomes that block SMC translocation along the DNA.

Covariance distributions in single particle tracking.

Several recent experiments, including our own experiments in the fission yeast, Schizosaccharomyces pombe, have characterized the motions of gene loci within living nuclei by measuring the locus position over time, then proceeding to obtain the statistical properties of this motion. To address the question of whether a population of such single-particle tracks, obtained from many different cells, corresponds to a single mode of diffusion, we derive theoretical equations describing the probability distribution of the displacement covariance, assuming the displacement itself is a zero-mean multivariate Gaussian random variable. We also determine the corresponding theoretical means, variances, and third central moments.

PAINT using proteins: A new brush for super-resolution artists.

PAINT (points accumulation for imaging in nanoscale topography) refers to methods that achieve the sparse temporal labeling required for super-resolution imaging by using transient interactions between a biomolecule of interest and a fluorophore. There have been a variety of different implementations of this method since it was first described in 2006. Recent papers illustrate how transient peptide-protein interactions, rather than small molecule binding or DNA oligonucleotide duplex formation, can be employed to perform PAINT-based single molecule localization microscopy (SMLM).

A versatile image analysis platform for three-dimensional nuclear reconstruction.

Nuclear morphology is indicative of cellular health in many contexts. In order to robustly and quantitatively measure nuclear size and shape, numerous experimental methods leveraging fluorescence microscopy have been developed. While these methods are useful for quantifying two-dimensional morphology, they often fail to accurately represent the three-dimensional structure of the nucleus, thus omitting important spatial and volumetric information.

Systems-level approach to uncovering diffusive states and their transitions from single-particle trajectories.

The stochastic motions of a diffusing particle contain information concerning the particle's interactions with binding partners and with its local environment. However, an accurate determination of the underlying diffusive properties, beyond normal diffusion, has remained challenging when analyzing particle trajectories on an individual basis. Here, we introduce the maximum-likelihood estimator (MLE) for confined diffusion and fractional Brownian motion.

Domain morphology, boundaries, and topological defects in biophotonic gyroid nanostructures of butterfly wing scales.

Many organisms in nature have evolved sophisticated cellular mechanisms to produce photonic nanostructures and, in recent years, diverse crystalline symmetries have been identified and related to macroscopic optical properties. However, because we know little about the distributions of domain sizes, the orientations of photonic crystals, and the nature of defects in these structures, we are unable to make the connection between the nanostructure and its development and functionality. We report on nondestructive studies of the morphology of chitinous photonic crystals in butterfly wing scales.

Extracting Diffusive States of Rho GTPase in Live Cells: Towards In Vivo Biochemistry.

Resolving distinct biochemical interaction states when analyzing the trajectories of diffusing proteins in live cells on an individual basis remains challenging because of the limited statistics provided by the relatively short trajectories available experimentally. Here, we introduce a novel, machine-learning based classification methodology, which we call perturbation expectation-maximization (pEM), that simultaneously analyzes a population of protein trajectories to uncover the system of diffusive behaviors which collectively result from distinct biochemical interactions. We validate the performance of pEM in silico and demonstrate that pEM is capable of uncovering the proper number of underlying diffusive states with an accurate characterization of their diffusion properties.

Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology.

The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes.

The tethering of chromatin to the nuclear envelope supports nuclear mechanics.

The nuclear lamina is thought to be the primary mechanical defence of the nucleus. However, the lamina is integrated within a network of lipids, proteins and chromatin; the interdependence of this network poses a challenge to defining the individual mechanical contributions of these components. Here, we isolate the role of chromatin in nuclear mechanics by using a system lacking lamins.

Structural Diversity of Arthropod Biophotonic Nanostructures Spans Amphiphilic Phase-Space.

Many organisms, especially arthropods, produce vivid interference colors using diverse mesoscopic (100-350 nm) integumentary biophotonic nanostructures that are increasingly being investigated for technological applications. Despite a century of interest, precise structural knowledge of many biophotonic nanostructures and the mechanisms controlling their development remain tentative, when such knowledge can open novel biomimetic routes to facilely self-assemble tunable, multifunctional materials. Here, we use synchrotron small-angle X-ray scattering and electron microscopy to characterize the photonic nanostructure of 140 integumentary scales and setae from ∼127 species of terrestrial arthropods in 85 genera from 5 orders.

Routes to DNA accessibility: alternative pathways for nucleosome unwinding.

The dynamic packaging of DNA into chromatin is a key determinant of eukaryotic gene regulation and epigenetic inheritance. Nucleosomes are the basic unit of chromatin, and therefore the accessible states of the nucleosome must be the starting point for mechanistic models regarding these essential processes. Although the existence of different unwound nucleosome states has been hypothesized, there have been few studies of these states.