Comment in Biophys J. 2023 Sep 5;122(17):3544-3548.

E-cadherin plays a central role in cell-cell adhesion. The ectodomains of wild-type cadherins form a crystalline-like two-dimensional lattice in cell-cell interfaces mediated by both trans (apposed cell) and cis (same cell) interactions. In addition to these extracellular forces, adhesive strength is further regulated by cytosolic phenomena involving α and β catenin-mediated interactions between cadherin and the actin cytoskeleton. Cell-cell adhesion can be further strengthened under tension through mechanisms that have not been definitively characterized in molecular detail. Here we quantitatively determine the role of the cadherin ectodomain in mechanosensing. To this end, we devise an E-cadherin-coated emulsion system, in which droplet surface tension is balanced by protein binding strength to give rise to stable areas of adhesion. To reach the honeycomb/cohesive limit, an initial emulsion compression by centrifugation facilitates E-cadherin trans binding, whereas a high protein surface concentration enables the cis-enhanced stabilization of the interface. We observe an abrupt concentration dependence on recruitment into adhesions of constant crystalline density, reminiscent of a first-order phase transition. Removing the lateral cis interaction with a "cis mutant" shifts this transition to higher surface densities leading to denser, yet weaker adhesions. In both proteins, the stabilization of progressively larger areas of deformation is consistent with single-molecule experiments that show a force-dependent lifetime enhancement in the cadherin ectodomain, which may be attributed to the "X-dimer" bond.

In the presence of polyvalent cations, long double-stranded DNA (dsDNA) in dilute solution undergoes a single-molecule, first-order, phase transition ("condensation"), a phenomenon that has been documented and analyzed by many years of experimental and theoretical studies. There has been no systematic effort, however, to determine whether long single-stranded RNA (ssRNA) shows an analogous behavior. In this study, using dynamic light scattering, analytical ultracentrifugation, and gel electrophoresis, we examine the effects of increasing polyvalent cation concentrations on the effective size of long ssRNAs ranging from 3000 to 12,000 nucleotides. Our results indicate that ssRNA does not undergo a discontinuous condensation as does dsDNA but rather a "continuous" decrease in size with increasing polyvalent cation concentration. And, instead of the 10-fold decrease in size shown by long dsDNA, we document a 50% decrease, as demonstrated for a range of lengths and sequences of ssRNA.

The heat shock protein 90 (Hsp90) is a molecular chaperone, which plays a key role in eukaryotic protein homeostasis. Co-chaperones assist Hsp90 in client maturation and in regulating essential cellular processes such as cell survival, signal transduction, gene regulation, hormone signaling, and neurodegeneration. Aha1 (activator of Hsp90 ATPase) is a unique co-chaperone known to stimulate the ATP hydrolysis of Hsp90, but the mechanism of their interaction is still unclear. In this report, we show that one or two Aha1 molecules can bind to one Hsp90 dimer and that the binding stoichiometry affects Hsp90's conformation, kinetics, ATPase activity, and stability. In particular, a coordination of two Aha1 molecules can be seen in stimulating the ATPase activity of Hsp90 and the unfolding of the middle domain, whereas the conformational equilibrium and kinetics are hardly affected by the stoichiometry of bound Aha1. Altogether, we show a regulation mechanism through the stoichiometry of Aha1 going far beyond a regulation of Hsp90's conformation.

Chromosomal dynamics plays a central role in a number of critical biological processes, such as transcriptional regulation, genetic recombination, and DNA replication. However, visualization of chromatin is generally limited to live imaging of a few fluorescently labeled chromosomal loci or high-resolution reconstruction of multiple loci from a single time frame. To aid in mapping the underlying chromosomal structure based on parsimonious experimental measurements, we present an exact analytical expression for the evolution of the polymer configuration based on a flexible-polymer model, and we propose an algorithm that tracks the polymer configuration from live images of chromatin marked with several fluorescent marks. Our theory identifies the resolution of microscopy needed to achieve high-accuracy tracking for a given spacing of markers, establishing the statistical confidence in the assignment of genome identity to the visualized marks. We then leverage experimental data of locus-tracking measurements to demonstrate the validity of our modeling approach and to establish a basis for the design of experiments with a desired resolution. Altogether, this work provides a computational approach founded on polymer physics that vastly improves the interpretation of in vivo measurements of biopolymer dynamics.

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we report the development and proof-of-concept results of a single-molecule approach to monitor the catalytic activity of human telomerase in real time and with single-nucleotide resolution. Using zero-mode waveguides and multicolor FRET, we recorded the processive addition of multiple telomeric repeats to individual DNA primers. Unlike existing biophysical and biochemical tools, the novel approach enables the quantification of nucleotide-binding kinetics before nucleotide incorporation. Moreover, it provides a means to dissect the unique translocation dynamics that telomerase must undergo after synthesis of each hexameric DNA repeat. We observed an unexpectedly prolonged binding dwell time of dGTP in the enzyme active site at the start of each repeat synthesis cycle, suggesting that telomerase translocation is composed of multiple rate-contributing sub-steps that evade classical biochemical analysis.

Optical trapping in biophysics typically uses micron-scale beads made of materials like polystyrene or glass to probe the target of interest. Using smaller beads made of higher-index materials could increase the time resolution of these measurements. We characterized the trapping of nanoscale beads made of diamond and titanium dioxide (TiO2) in a single-beam gradient trap. Calculating theoretical expectations for the trapping stiffness of these beads, we found good agreement with measured values. Trap stiffness was significantly higher for TiO2 beads, owing to notable enhancement from nonlinear optical effects, not previously observed for continuous-wave trapping. Trap stiffness was over 6-fold higher for TiO2 beads than polystyrene beads of similar size at 70 mW laser power. These results suggest that diamond and TiO2 nanobeads can be used to improve time resolution in optical tweezers measurements.

Traction patterns of adherent cells provide important information on their interaction with the environment, cell migration, or tissue patterns and morphogenesis. Traction force microscopy is a method aimed at revealing these traction patterns for adherent cells on engineered substrates with known constitutive elastic properties from deformation information obtained from substrate images. Conventionally, the substrate deformation information is processed by numerical algorithms of varying complexity to give the corresponding traction field via solution of an ill-posed inverse elastic problem. We explore the capabilities of a deep convolutional neural network as a computationally more efficient and robust approach to solve this inversion problem. We develop a general purpose training process based on collections of circular force patches as synthetic training data, which can be subjected to different noise levels for additional robustness. The performance and the robustness of our approach against noise is systematically characterized for synthetic data, artificial cell models, and real cell images, which are subjected to different noise levels. A comparison with state-of-the-art Bayesian Fourier transform traction cytometry reveals the precision, robustness, and speed improvements achieved by our approach, leading to an acceleration of traction force microscopy methods in practical applications.

Ryanodine receptors (RyRs) are Ca2+ release channels, gated by Ca2+ in the cytosol and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac and muscle diseases. Although a lot of data is available on the luminal Ca2+ regulation of RyR, its interpretation is complicated by the possibility that the divalent ions used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing the channel pore. In this study, we used Eu3+, an impermeable agonist of Ca2+ binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca2+ sensor. Single-channel currents were measured from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique. We show that RyR2 is activated by the luminal addition of Ca2+, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu3+. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type. RyR1 inhibition by Eu3+ was extremely voltage dependent, whereas RyR2 activation did not depend on the membrane potential. These results suggest that the RyR1 inhibition site is in the membrane's electric field (channel pore), whereas the RyR2 activation site is outside. Using in silico analysis and previous results, we predicted putative Ca2+ binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca2+ binding site near the channel gate.

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

Mechanical stresses generated at the cell-cell level and cell-substrate level have been suggested to be important in a host of physiological and pathological processes. However, the influence various chemical compounds have on the mechanical stresses mentioned above is poorly understood, hindering the discovery of novel therapeutics, and representing a barrier in the field. To overcome this barrier, we implemented two approaches: 1) monolayer boundary predictor and 2) discretized window predictor utilizing either stepwise linear regression or quadratic support vector machine machine learning model to predict the dose-dependent response of tractions and intercellular stresses to chemical perturbation. We used experimental traction and intercellular stress data gathered from samples subject to 0.2 or 2 μg/mL drug concentrations along with cell morphological properties extracted from the bright-field images as predictors to train our model. To demonstrate the predictive capability of our machine learning models, we predicted tractions and intercellular stresses in response to 0 and 1 μg/mL drug concentrations which were not utilized in the training sets. Results revealed the discretized window predictor trained just with four samples (292 images) to best predict both intercellular stresses and tractions using the quadratic support vector machine and stepwise linear regression models, respectively, for the unseen sample images.

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration, and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryoelectron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished this filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament cross-linker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

Cell migration is a complex phenomenon. Not only do different cells migrate in different default modes, but the same cell can also change its migration mode to adapt to different terrains. This complexity has riddled cell biologists and biophysicists for decades in that, despite the development of many powerful tools over the past 30 years, how cells move is still being actively investigated. This is because we have yet to fully understand the mystery of cell migration plasticity, particularly the reciprocal relation between force generation and migration mode transition. Herein we explore the future directions, in terms of measurement platforms and imaging-based techniques, to facilitate the undertaking of elucidating the relation between force generation machinery and migration mode transition. By briefly reviewing the evolution of the platforms and techniques developed in the past, we propose the desirable features to be added to achieve high measurement accuracy and improved temporal and spatial resolution, permitting us to unveil the mystery of cell migration plasticity.

Fluorescence recovery after photobleaching (FRAP) has emerged as one of the most widely utilized techniques to quantify binding and diffusion kinetics of biomolecules in biophysics. Since its inception in the mid-1970s, FRAP has been used to address an enormous array of questions including the characteristic features of lipid rafts, how cells regulate the viscosity of their cytoplasm, and the dynamics of biomolecules inside condensates formed by liquid-liquid phase separation. In this perspective, I briefly summarize the history of the field and discuss why FRAP has proven to be so incredibly versatile and popular. Next, I provide an overview of the extensive body of knowledge that has emerged on best practices for quantitative FRAP data analysis, followed by some recent examples of biological lessons learned using this powerful approach. Finally, I touch on new directions and opportunities for biophysicists to contribute to the continued development of this still-relevant research tool.

Organogenesis arises from the collective arrangement of cells into progressively 3D-shaped tissue. The acquisition of a correctly shaped organ is then the result of a complex interplay between molecular cues, responsible for differentiation and patterning, and the mechanical properties of the system, which generate the necessary forces that drive correct shape emergence. Nowadays, technological advances in the fields of microscopy, molecular biology, and computer science are making it possible to see and record such complex interactions in incredible, unforeseen detail within the global context of the developing embryo. A quantitative and interdisciplinary perspective of developmental biology becomes then necessary for a comprehensive understanding of morphogenesis. Here, we provide a roadmap to quantify the events that lead to morphogenesis from imaging to image analysis, quantification, and modeling, focusing on the discrete cellular and tissue shape changes, as well as their mechanical properties.

Cell science has made significant progress by focusing on understanding individual cellular processes through reductionist approaches. However, the sheer volume of knowledge collected presents challenges in integrating this information across different scales of space and time to comprehend cellular behaviors, as well as making the data and methods more accessible for the community to tackle complex biological questions. This perspective proposes the creation of next-generation virtual cells, which are dynamic 3D models that integrate information from diverse sources, including simulations, biophysical models, image-based models, and evidence-based knowledge graphs. These virtual cells would provide statistically accurate and holistic views of real cells, bridging the gap between theoretical concepts and experimental data, and facilitating productive new collaborations among researchers across related fields.

Research on the locomotion of single cells on hard, flat surfaces brought insight into the mechanisms of leading-edge protrusion, spatially graded adhesion, front-rear coordination, and how intracellular and traction forces are harnessed to execute various maneuvers. Here, we highlight how, by studying a variety of cell types, shapes, and movements, Ken Jacobson and his collaborators made several discoveries that triggered the mechanistic understanding of cell motility. We then review the recent advancements and current perspectives in this field.

Mitochondria adapt to changing cellular environments, stress stimuli, and metabolic demands through dramatic morphological remodeling of their shape, and thus function. Such mitochondrial dynamics is often dependent on cytoskeletal filament interactions. However, the precise organization of these filamentous assemblies remains speculative. Here, we apply cryogenic electron tomography to directly image the nanoscale architecture of the cytoskeletal-membrane interactions involved in mitochondrial dynamics in response to damage. We induced mitochondrial damage via membrane depolarization, a cellular stress associated with mitochondrial fragmentation and mitophagy. We find that, in response to acute membrane depolarization, mammalian mitochondria predominantly organize into tubular morphology that abundantly displays constrictions. We observe long bundles of both unbranched actin and septin filaments enriched at these constrictions. We also observed septin-microtubule interactions at these sites and elsewhere, suggesting that these two filaments guide each other in the cytosolic space. Together, our results provide empirical parameters for the architecture of mitochondrial constriction factors to validate/refine existing models and inform the development of new ones.

Fetal lung fibroblasts contribute dynamic infrastructure for the developing lung. These cells undergo dynamic mechanical transitions, including cyclic stretch and spreading, which are integral to lung growth in utero. We investigated the role of the nuclear envelope protein emerin in cellular responses to these dynamic mechanical transitions. In contrast to control cells, which briskly realigned their nuclei, actin cytoskeleton, and extracellular matrices in response to cyclic stretch, fibroblasts that were acutely downregulated for emerin showed incomplete reorientation of both nuclei and actin cytoskeleton. Emerin-downregulated fibroblasts were also aberrantly circular in contrast to the spindle-shaped controls and exhibited an altered pattern of filamentous actin organization that was disconnected from the nucleus. Emerin knockdown was also associated with reduced myosin light chain phosphorylation during cell spreading. Interestingly, emerin-downregulated fibroblasts also demonstrated reduced fibronectin fibrillogenesis and production. These findings indicate that nuclear-cytoskeletal coupling serves a role in the dynamic regulation of cytoskeletal structure and function and may also impact the transmission of traction force to the extracellular matrix microenvironment.

Imaging two or more fluorescent biosensors in the same living cell can reveal the spatiotemporal coordination of protein activities. However, using multiple Förster resonance energy transfer (FRET) biosensors together is challenging due to toxicity and the need for orthogonal fluorophores. Here we generate a biosensor component that binds selectively to the activated conformation of three different proteins. This enabled multiplexed FRET with fewer fluorophores, and reduced toxicity. We generated this MultiBinder (MB) reagent for the GTPases RhoA, Rac1, and Cdc42 by combining portions of the downstream effector proteins Pak1 and Rhotekin. Using FRET between mCherry on the MB and YPet or mAmetrine on two target proteins, the activities of any pair of GTPases could be distinguished. The MB was used to image Rac1 and RhoA together with a third, dye-based biosensor for Cdc42. Quantifying effects of biosensor combinations on the frequency, duration, and velocity of cell protrusions and retractions demonstrated reduced toxicity. Multiplexed imaging revealed signaling hierarchies between the three proteins at the cell edge where they regulate motility.