Biophysical journal最新文献

Conserved NF-κB signaling pathways shape immune responses in animals. In mammals, NF-κB activation patterns and downstream transcription vary with stimulus, cell type, and stochastic differences among identically treated cells. Whether animals without adaptive immunity exhibit similar heterogeneity or rely on distinct immune strategies remains unknown. We engineered live Drosophila melanogaster S2^∗ reporter cells as an immune-responsive model to monitor the dynamics of an NF-κB transcription factor, Relish, and downstream transcription in single cells. After immune stimulation, Relish exhibits diverse nuclear localization dynamics, with both the fraction of responsive cells and the speed of Relish activation increasing with stimulus dose. Prestimulus features, including the amount of nuclear Relish under basal conditions, predict a cell's responsiveness to stimulation. Simultaneous measurement of Relish and downstream transcription revealed that the probability of transcriptional bursts from immune-responsive enhancers correlates with Relish nuclear fraction. Enhancers containing more κB binding sites have a higher likelihood of activating at the population level. Our study uncovers heterogeneity in NF-κB activation and target gene expression within Drosophila, illustrating how dynamic NF-κB behavior and enhancer architecture tune gene regulation.

Clathrin-mediated endocytosis (CME) is an important internalization route for macromolecules, lipids, and membrane receptors in eukaryotic cells. During CME, the plasma membrane invaginates and pinches off to form clathrin-coated vesicles (CCVs). This rapid, nanoscale process involves significant changes to plasma membrane shape. We previously found heterogeneity in CCV formation, some vesicles form with simultaneous membrane bending and clathrin assembly (constant curvature) and while others form with membrane bending following the accumulation of flat clathrin lattices (flat-to-curved). These architectural dynamics could be influenced by osmotic pressure, membrane stiffness, or cytoskeletal arrangement. Whether these biophysical factors regulate the heterogeneity of vesicle formation dynamics is not well understood. To address this, we investigated the interconnected roles of actin and membrane tension in CME using simultaneous two-wavelength axial ratiometry (STAR) microscopy with nanometer-scale axial resolution. First, we treated Cos-7 cells stably expressing CLCa-iRFP713-EGFP with latrunculin A (LatA) to inhibit actin polymerization, and found the frequency of CCVs increased significantly, especially for short-lifetime CCVs. The proportion of vesicles following the flat-to-curved model was reduced, the membrane curved sooner after clathrin recruitment, and forming vesicles were less stable in x-y compared to control. Next, we disrupted actin branching with CK869 and found the frequency of CCVs decreased. There was increased delay between membrane invagination and clathrin recruitment, increased x-y plane stability of forming vesicles, and increased proportion of vesicles following the flat-to-curved model compared to control. To address these opposing results, we considered the role of membrane tension. When membrane tension was decreased with high osmolality media, CCV formation mirrored the LatA treated group, except the x-y stability of forming vesicles was unchanged. This suggests the increased CCV frequency following actin filament disruption may be due to reduced membrane tension. We conclude actin polymerization promotes the bending of flat clathrin while actin branching promotes constant curvature.

Proteome maintenance is underpinned by molecular motors from the AAA+ superfamily. E. coli ClpA is a representative AAA+ motor that associates with the tetradecameric serine protease ClpP forming the ATP-dependent protease, ClpAP. ClpA unfolds substrates targeted for degradation and translocates them into the central channel of ClpP where the substrate is degraded. However, when ClpA is not associated with ClpP, the motor uses its unfolding activity to noncovalently remodel protein substrates. Although a large body of work exists on the mechanisms of ClpAP-catalyzed protein unfolding and degradation, much less is known about the mechanisms of protein remodeling reactions. In fact, there is a dearth of mechanistic information to complement the emerging static structural information on many AAA+ family members that remodel proteins without covalent modification. Here, we report results from single-turnover stopped-flow experiments to interrogate the ClpA-catalyzed mechanisms of protein unfolding, both alone and when associated with ClpP. To this end, we used substrates containing tandem repeats of the Titin I27 domain. We show that both ClpA and ClpAP catalyze cooperative protein unfolding of the Titin I27 domain in a single kinetic step. This cooperative unfolding is followed by repeated rounds of translocation on the newly unfolded polypeptide. At saturating [ATP], ClpA and ClpAP catalyze protein unfolding and translocation at (12.0 ± 0.4) aa s^-1 and (33.2 ± 1.1) aa s^-1, respectively. By examining the complete ATP dependence of the reaction, we have deconvoluted the elementary rate constants for unfolding and translocation from the overall rate. At saturating [ATP], the translocation rate constant is approximately eightfold and 24-fold faster than the unfolding rate constant for ClpA and ClpAP, respectively. Furthermore, the unfolding rate constant for ClpAP is about threefold faster than for ClpA alone. This indicates fundamental differences in the unfolding mechanisms between ClpA alone and ClpA associated with ClpP.

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.