Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e1 bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.
Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.
We demonstrate optical manipulation of nanoliter aqueous droplets containing surfactant or lipid molecules and immersed in an organic liquid using near infrared light. The resulting emulsion droplets are manipulated using both the thermocapillary effect and convective fluid motion. Droplet pair-interactions induced in the emulsion upon optical initiation and control provide direct observations of the coalescence steps in intricate detail. Droplet-droplet adhesion (bilayer formation) is observed under several conditions. Selective bilayer rupture is also realized using the same infrared laser. The technique provides a novel approach to study thin film drainage and interface stability in emulsion dynamics. The formation of stable lipid bilayers at the adhesion interface between interacting water droplets can provide an optical platform to build droplet-based lipid bilayer assays. The technique also has relevance for understanding and improving microfluidics applications by devising Petri dish based droplet assays requiring no substrate fabrication.
Understanding the relationship between intracellular motion and macromolecular structure remains a challenge in biology. Macromolecular structures are assembled from numerous molecules, some of which cannot be labeled. Most techniques to study motion require potentially cytotoxic dyes or transfection, which can alter cellular behavior and are susceptible to photobleaching. Here we present a multimodal label-free imaging platform for measuring intracellular structure and macromolecular dynamics in living cells with a sensitivity to macromolecular structure as small as 20 nm and millisecond temporal resolution. We develop and validate a theory for temporal measurements of light interference. In vitro, we study how higher-order chromatin structure and dynamics change during cell differentiation and ultraviolet (UV) light irradiation. Finally, we discover cellular paroxysms, a near-instantaneous burst of macromolecular motion that occurs during UV induced cell death. With nanoscale sensitive, millisecond resolved capabilities, this platform could address critical questions about macromolecular behavior in live cells.
Chromatin is the macromolecular assembly containing the cell’s genetic information, and its architectural conformation facilitates accessibility to activation sites and thus gene expression. We have developed an analytical framework to quantify chromatin structure with spectral microscopy. Chromatin structure can be described as a mass fractal, with packing scaling D up to specific genomic length scales. Considering various system geometries, we established a model to measure D with the interferometric technique partial wave spectroscopy (PWS) and validated the analysis using finite difference time domain to simulate the PWS system. Calculations of D were consistent with ground truth electron microscopy measurements, enabling a high-throughput, label-free approach to quantifying chromatin structure in the nanometer length scale regime.
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