Biophysical journal最新文献

Cell and extracellular matrix interactions are essential for maintaining tissue function and homeostasis. Changes in the biochemical or mechanical properties of the extracellular matrix can lead to diseases such as fibrosis or cancer. In a 3D microenvironment, cell-matrix interaction is vital to how cells sense and respond to biochemical and biophysical cues. This study examines the reciprocal interactions between fibroblasts and collagen in 3D hydrogels. We quantitatively measured changes in collagen branch number and junctions in 3D hydrogels using confocal reflectance microscopy and existing analysis protocols. This reveals the impact small changes in collagen concertation (1.0 vs. 1.5 mg/mL) over time (15 min-4 h) have on 3D gels. Embedded in 3D hydrogels, wild-type mouse fibroblasts differentially affect collagen organization in their immediate proximity with changing concentration and time. This regulation is interestingly lost in caveolin-1-null fibroblasts with altered stiffness, mechanosensing, and cytoskeletal regulation. Inhibition of the Rho-ROCK pathway (altered in caveolin-1-null fibroblasts) through myosin light chain kinase drives cellular protrusions and concentration-dependent 3D collagen organization in wild-type fibroblasts, but surprisingly not in caveolin-1-null fibroblasts. This depends on dynamin-dependent endocytosis, which, when inhibited, disrupts ROCK-dependent protrusions and alters collagen organization in 3D collagen. Together, these observations quantitatively demonstrate how cells respond at the cell-matrix interphase to subtle changes in collagen concentration and organization in 3D hydrogels, regulated by the presence of caveolin-1.

A living cell is a nonequilibrium thermodynamic system where, nevertheless, a notion of local equilibrium exists. This notion applies to all micro- and nanoscale aqueous volumes, each containing a large number of molecules. This allows one to define sets of local conditions, including thermodynamic ones; for instance, a defined temperature requires thermodynamic equilibrium by definition. Once such a condition is fulfilled, one can control local variables and their gradients to theoretically describe the thermodynamic state of living systems at the micro- and nanoscale. Performing ultralocal experimental manipulations has become possible thanks to the patch-clamp technique, which controls the cell membrane potential, and fluorescence imaging, which monitors molecular concentrations and their intracellular gradients. However, precise temperature gradient control at the micro- and nanoscales has yet to be reliably realized in a living cell. Here, we present a new methodology-microscale control of a temperature gradient profile in aqueous media by a fully optical diamond heater-thermometer in a plug-and-play fiber configuration combined with the patch-clamp technique. In particular, we demonstrate applications of the combined diamond heater-thermometer-patch-clamp approach for fast, reproducible thermal modulation of ionic current from voltage-gated Na_v1.5 sodium channels expressed in HEK293 cells and in freshly isolated ventricular mouse cardiomyocytes. Such an approach of manipulating the ultralocal temperature has the potential to uncover previously inaccessible phenomena in various physiological intracellular processes related to the endogenous nanoscale heat sources, such as open ion channels capable of producing Joule heat.

Mechanical overstretching of individual RNA-DNA hybrids is used as a novel in vitro assay to prepare a co-transcriptional RNA structure and study its interaction with proteins. Dual optical traps hold two microscopic beads linked by an RNA-DNA molecular construct that is designed such that the RNA strand progressively peels off and folds when trap-to-trap distance increases. Subsequent distance reduction leads to duplex reannealing, RNA structure and its interaction with proteins are probed by continuously measuring force during this peeling/reannealing cycle. Focusing on the early stage of E.coli large ribosomal subunit assembly (domains I-II of 23S rRNA and early-binding r-proteins uL4, uL13, bL20, uL22, and uL24), we find that these five r-proteins stabilize the 23S rRNA structure: this property is notably characterized in our experiments by the observation that full reannealing is less frequent when the r-proteins are present than when they are absent. Sites of reannealing blockage were derived from the single-molecule data and compared with known RNA structural elements and r-protein binding sites of the bacterial ribosome. Our results also show that the five early-binding r-proteins bind the 23S rRNA co-transcriptionally, corroborating the classical "assembly gradient" hypothesis.

Cytoplasmic proteins must recruit to membranes to function in processes such as endocytosis and cell division. Many of these proteins recognize not only the chemical structure of the membrane lipids, but the curvature of the surface, binding more strongly to more highly curved surfaces, or curvature sorting. Curvature sorting by amphipathic helices is known to vary with membrane bending rigidity, but changes to lipid composition can simultaneously alter membrane thickness, spontaneous curvature, and leaflet symmetry, thus far preventing a systematic characterization of lipid composition on such curvature preferences through either experiment or simulation. Here we develop and apply a bilayer continuum membrane model that can tractably address this gap, quantifying how controlled changes to each material property can favor or disfavor protein curvature sorting. We evaluate both energetic and structural changes to vesicles upon helix insertion, with strong agreement to new in vitro experiments and all-atom MD simulations, respectively. Our membrane model builds on previous work to include both monolayers of the bilayer via representation by continuous triangular meshes. We introduce a coupling energy that captures the incompressibility of the membrane and approximates the established energetics of lipid tilt without using an explicit tilt field. In agreement with experiment, our model predicts stronger curvature sorting in membranes with distinct tail groups (POPC vs DOPC vs DLPC), despite having identical head-group chemistry; the model shows that the primary driving force for weaker curvature sorting in DLPC is that it is thinner, and more wedge shaped. Somewhat surprisingly, asymmetry in lipid shape composition between the two leaflets has a negligible contribution to membrane mechanics following insertion. Our multi-scale approach can be used to quantitatively and efficiently predict how changes to membrane composition in flat to highly curved surfaces alter membrane energetics driven by proteins, a mechanism that helps proteins target membranes at the correct time and place.

Hepatitis C virus (HCV) genome codes for various proteins essential to its replication cycle. Among these, the core protein (HCC) forms the capsid via organized multimerization, thereby guiding viral assembly. This process depends on the dynamic localization of HCC between the endoplasmic reticulum bilayer membrane and the lipid droplet monolayer. Although past studies have examined the role of some HCC structural properties and other viral and host proteins in the assembly process, the contribution of lipid molecules has not been thoroughly investigated yet. Since specific lipids are upregulated during HCV infection, it is reasonable that these molecules might play an important role for virus fitness and infection progression. In this study, we have addressed this topic and, specifically, how HCC-lipid interactions might impact HCC-HCC interactions. Using in vitro models and quantitative fluorescence microscopy techniques, we investigated HCC binding affinity to lipid membranes, multimerization, and lateral diffusivity. Our results reveal the effect of HCC interactions with anionic phospholipids (PLs) on the binding to monolayers and bilayers. Furthermore, our analysis shows that PI(4)P enhances HCC multimerization and, therefore, might drive lateral assembly for capsid formation. These results not only provide insights into the lipid-driven regulation of HCV assembly and identify anionic PLs as potential modulators of the viral replications cycle, but they also highlight a previously unrecognized role of PL composition in HCC-membrane interaction.

Plectin is a giant protein of the plakin family that cross-links the cytoskeleton of mammalian cells. It is expressed in virtually all tissues, and its dysfunction is associated with various diseases such as skin blistering. There is evidence that plectin regulates the mechanical integrity of the cytoskeleton in diverse cell and tissue types. However, it is unknown how plectin modulates the mechanical response of cells depending on the frequency and amplitude of mechanical loading. Here we demonstrate the role of plectin in the viscoelastic properties of fibroblasts at small and large deformations by quantitative single-cell compression measurements. To identify the importance of plectin, we compared the mechanical properties of wild-type (Plec^+/+) fibroblasts and plectin knockout (Plec^-/-) fibroblasts. We show that plectin knockout cells are nearly twofold softer than wild-type cells, but their strain-stiffening behavior is similar. Plectin deficiency also caused faster viscoelastic stress relaxation at long times. Fluorescence recovery after photobleaching experiments indicated that this was due to threefold faster actin turnover. Short-time poroelastic relaxation was also faster in Plec^-/- cells compared with Plec^+/+ cells, suggesting a more sparse cytoskeletal network. Confocal imaging indicated that this was due to a marked change in the architecture of the vimentin network, from a fine meshwork in wild-type cells to a bundled network in the plectin knockout cells. Our findings therefore indicate that plectin is an important regulator of the organization and viscoelastic properties of the cytoskeleton in fibroblasts. Our findings emphasize that mechanical integration of the different cytoskeletal networks present in cells is important for regulating the versatile mechanical properties of cells.