Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin?biotin bond. Implications for the molecular mechanism of unbinding of HA?hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA?hyaladherin complexes and HA-rich extracellular matrices.     

Dynamic Model for Characterizing Contractile Behaviors and Mechanical Properties of a Cardiomyocyte

Studies on the contractile dynamics of heart cells have attracted broad attention for the development of both heart disease therapies and cardiomyocyte-actuated micro-robotics. In this study, a linear dynamic model of a single cardiomyocyte cell was proposed at the subcellular scale to characterize the contractile behaviors of heart cells, with system parameters representing the mechanical properties of the subcellular components of living cardiomyocytes. The system parameters of the dynamic model were identified with the cellular beating pattern measured by a scanning ion conductance microscope. The experiments were implemented with cardiomyocytes in one control group and two experimental groups with the drugs cytochalasin-D or nocodazole, to identify the system parameters of the model based on scanning ion conductance microscope measurements, measurement of the cellular Young’s modulus with atomic force microscopy indentation, measurement of cellular contraction forces using the micro-pillar technique, and immunofluorescence staining and imaging of the cytoskeleton. The proposed mathematical model was both indirectly and qualitatively verified by the variation in cytoskeleton, beating amplitude, and contractility of cardiomyocytes among the control and the experimental groups, as well as directly and quantitatively validated by the simulation and the significant consistency of 90.5% in the comparison between the ratios of the Young’s modulus and the equivalent comprehensive cellular elasticities of cells in the experimental groups to those in the control group. Apart from mechanical properties (mass, elasticity, and viscosity) of subcellular structures, other properties of cardiomyocytes have also been studied, such as the properties of the relative action potential pattern and cellular beating frequency. This work has potential implications for research on cytobiology, drug screening, mechanisms of the heart, and cardiomyocyte-based bio-syncretic robotics.     

3D Protein Dynamics in the Cell Nucleus

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.     

Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.     

Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

The efficiency of intracellular cargo transport from specific sources to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance results in a spatially inhomogeneous intermittent search strategy. To analyze the efficiency of such intracellular search strategies, we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: 1) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane; 2) the reaction problem, which considers the binding time of two particles within the cell; and 3) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.     

Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization

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Shear-Induced Migration of a Transmembrane Protein within a Vesicle

Biomembranes feature phospholipid bilayers and serve as the interface between cells or organelles and the extracellular and/or cellular environment. Lipids can move freely throughout the membrane; the lipid bilayer behaves like a fluid. Such fluidity is important in terms of the actions of membrane transport proteins, which often mediate biological functions; membrane protein motion has attracted a great deal of attention. Because the proteins are small, diffusion phenomena are often in play, but flow-induced transport has rarely been addressed. Here, we used a dissipative particle dynamics approach to investigate flow-induced membrane protein transport. We analyzed the drift of a membrane protein located within a vesicle. Under the influence of shear flow, the protein gradually migrated toward the vorticity axis via a random walk, and the probability of retention around the axis was high. To understand the mechanism of protein migration, we varied both shear strength and protein size. Protein migration was induced by the balance between the drag and thermodynamic diffusion forces and could be represented by the Péclet number. These results improve our understanding of flow-induced membrane protein transport.