Confinement-sensitive volume regulation dynamics via high-speed nuclear morphological measurements.

Diverse tissues in vivo present varying degrees of confinement, constriction, and compression to migrating cells in both homeostasis and disease. The nucleus in particular is subjected to external forces by the physical environment during confined migration. While many systems have been developed to induce nuclear deformation and analyze resultant functional changes, much remains unclear about dynamic volume regulation in confinement due to limitations in time resolution and difficulty imaging in PDMS-based microfluidic chips.

Nucleus size and its effect on nucleosome stability in living cells.

DNA architectural proteins play a major role in organization of chromosomal DNA in living cells by packaging it into chromatin, whose spatial conformation is determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints applied to the DNA by a tight nuclear space. Yet, the exact effects of the nucleus size on DNA-protein interactions and chromatin structure currently remain obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells.

Modulating binding affinity, specificity, and configurations by multivalent interactions.

Biological functions of proteins rely on their specific interactions with binding partners. Many proteins contain multiple domains, which can bind to their targets that often have more than one binding site, resulting in multivalent interactions. While it has been shown that multivalent interactions play a crucial role in modulating binding affinity and specificity, other potential effects of multivalent interactions are less explored.

Application of piconewton forces to individual filopodia reveals mechanosensory role of L-type Ca channels.

Filopodia are ubiquitous membrane projections that play crucial role in guiding cell migration on rigid substrates and through extracellular matrix by utilizing yet unknown mechanosensing molecular pathways. As recent studies show that Ca channels localized to filopodia play an important role in regulation of their formation and since some Ca channels are known to be mechanosensitive, force-dependent activity of filopodial Ca channels might be linked to filopodia's mechanosensing function. We tested this hypothesis by monitoring changes in the intra-filopodial Ca level in response to application of stretching force to individual filopodia of several cell types using optical tweezers.

Effects of size, cooperativity, and competitive binding on protein positioning on DNA.

Accurate positioning of proteins on chromosomal DNA is crucial for its proper organization as well as gene transcription regulation. Recent experiments revealed existence of periodic patterns of nucleoprotein complexes on DNA, which frequently cannot be explained by sequence-dependent binding of proteins. Previous theoretical studies suggest that such patterns typically emerge as a result of the proteins' volume-exclusion effect.

Effects of Mechanical Stimuli on Profilin- and Formin-Mediated Actin Polymerization.

Self-assembling actin filaments not only form the basis of the cytoskeleton network in cells but also are utilized as nanosized building blocks to make novel active matter in which the dynamic polymerization and depolymerization of actin filaments play a key role. Formins belong to a main family of actin nucleation factors that bind to the barbed end of actin filaments and regulate actin polymerization through an interaction with profilin. Due to actomyosin contractility and relative rotation between formin and actin filaments, formin-dependent actin polymerization is subject to force and rotation constraints.

Transfer-matrix calculations of the effects of tension and torque constraints on DNA-protein interactions.

Organization and maintenance of the chromosomal DNA in living cells strongly depends on the DNA interactions with a plethora of DNA-binding proteins. Single-molecule studies show that formation of nucleoprotein complexes on DNA by such proteins is frequently subject to force and torque constraints applied to the DNA. Although the existing experimental techniques allow to exert these type of mechanical constraints on individual DNA biopolymers, their exact effects in regulation of DNA-protein interactions are still not completely understood due to the lack of systematic theoretical methods able to efficiently interpret complex experimental observations.

mDia1 senses both force and torque during F-actin filament polymerization.

Formins, an important family of force-bearing actin-polymerizing factors, function as homodimers that bind with the barbed end of actin filaments through a ring-like structure assembled from dimerized FH2 domains. It has been hypothesized that force applied to formin may facilitate transition of the FH2 ring from an inhibitory closed conformation to a permissive open conformation, speeding up actin polymerization. We confirm this hypothesis for mDia1 dependent actin polymerization by stretching a single-actin filament in the absence of profilin using magnetic tweezers, and observe that increasing force from 0.

Theoretical Methods for Studying DNA Structural Transitions under Applied Mechanical Constraints.

Recent progress in single-molecule manipulation technologies has made it possible to exert force and torque on individual DNA biopolymers to probe their mechanical stability and interaction with various DNA-binding proteins. It was revealed in these experiments that the DNA structure and formation of nucleoprotein complexes by DNA-architectural proteins can be strongly modulated by an intricate interplay between the entropic elasticity of DNA and its global topology, which is closely related to the mechanical constraints applied to the DNA. Detailed understanding of the physical processes underlying the DNA behavior observed in single-molecule experiments requires the development of a general theoretical framework, which turned out to be a rather challenging task.

Transfer-matrix calculations of DNA polymer micromechanics under tension and torque constraints.

Recent development of single-molecule manipulation technologies has made it possible to exert constant force and torque on individual DNA biopolymers to probe their elastic characteristics and structural stability. It has been previously shown that depending on the nature of applied mechanical constraints, DNA can exist in several forms including B-, L-, and P-DNA. However, there is still a lack of understanding of how structural heterogeneity of DNA, which may naturally arise due to sequence-dependent DNA properties, protein binding, or DNA damage, influences local stability of the above DNA states.

Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies.

Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions-diameter (D): 40-50 μm, height (H): 100 μm; H:D∼2:1).

Transcriptional Repressor TrmBL2 from Thermococcus kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt- and DNA Supercoiling-dependent Manner.

Architectural DNA proteins play important roles in the chromosomal DNA organization and global gene regulation in living cells. However, physiological functions of some DNA-binding proteins from archaea remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon Thermococcus kodakarensis.

From bistate molecular switches to self-directed track-walking nanomotors.

Track-walking nanomotors and larger systems integrating these motors are important for wide real-world applications of nanotechnology. However, inventing these nanomotors remains difficult, a sharp contrast to the widespread success of simpler switch-like nanodevices, even though the latter already encompasses basic elements of the former such as engine-like bistate contraction/extension or leg-like controllable binding. This conspicuous gap reflects an impeding bottleneck for the nanomotor development, namely, lack of a modularized construction by which spatially and functionally separable "engines" and "legs" are flexibly assembled into a self-directed motor.

Force-dependent conformational switch of α-catenin controls vinculin binding.

Force sensing at cadherin-mediated adhesions is critical for their proper function. α-Catenin, which links cadherins to actomyosin, has a crucial role in this mechanosensing process. It has been hypothesized that force promotes vinculin binding, although this has never been demonstrated.

Dynamics and stability of polymorphic human telomeric G-quadruplex under tension.

As critical DNA structures capping the human chromosome ends, the stability and structural polymorphism of human telomeric G-quadruplex (G4) have drawn increasing attention in recent years. This work characterizes the equilibrium transitions of single-molecule telomeric G4 at physiological K(+) concentration. We report three folded states of telomeric G4 with markedly different lifetime and mechanical stability.

Analysis of Cooperative Behavior in Multiple Kinesins Motor Protein Transport by Varying Structural and Chemical Properties.

Intracellular transport is a fundamental biological process during which cellular materials are driven by enzymatic molecules called motor proteins. Recent optical trapping experiments and theoretical analysis have uncovered many features of cargo transport by multiple kinesin motor protein molecules under applied loads. These studies suggest that kinesins cooperate negatively under typical transport conditions, although some productive cooperation could be achieved under higher applied loads.

Delineating cooperative responses of processive motors in living cells.

Characterizing the collective functions of cytoskeletal motors is critical to understanding mechanisms that regulate the internal organization of eukaryotic cells as well as the roles various transport defects play in human diseases. Though in vitro assays using synthetic motor complexes have generated important insights, dissecting collective motor functions within living cells still remains challenging. Here, we show that the protein heterodimerization switches FKBP-rapalog-FRB can be harnessed in engineered COS-7 cells to compare the collective responses of kinesin-1 and myosinVa motors to changes in motor number and cargo size.

Directional fidelity of nanoscale motors and particles is limited by the 2nd law of thermodynamics--via a universal equality.

Directional motion of nanoscale motors and driven particles in an isothermal environment costs a finite amount of energy despite zero work as decreed by the 2nd law, but quantifying this general limit remains difficult. Here we derive a universal equality linking directional fidelity of an arbitrary nanoscale object to the least possible energy driving it. The fidelity-energy equality depends on the environmental temperature alone; any lower energy would violate the 2nd law in a thought experiment.

Probing the cytoadherence of malaria infected red blood cells under flow.

Malaria is one of the most widespread and deadly human parasitic diseases caused by the Plasmodium (P.) species with the P. falciparum being the most deadly.

Bipedal nanowalker by pure physical mechanisms.

Artificial nanowalkers are inspired by biomolecular counterparts from living cells, but remain far from comparable to the latter in design principles. The walkers reported to date mostly rely on chemical mechanisms to gain a direction; they all produce chemical wastes. Here we report a light-powered DNA bipedal walker based on a design principle derived from cellular walkers.

How the interplay between mechanical and nonmechanical interactions affects multiple kinesin dynamics.

Intracellular transport is supported by enzymes called motor proteins that are often coupled to the same cargo and function collectively. Recent experiments and theoretical advances have been able to explain certain behaviors of multiple motor systems by elucidating how unequal load sharing between coupled motors changes how they bind, step, and detach. However, nonmechanical interactions are typically overlooked despite several studies suggesting that microtubule-bound kinesins interact locally via short-range nonmechanical potentials.