Nascent adhesions differentially regulate lamellipodium velocity and persistence

Cell migration is essential to physiological and pathological biology. Migration is driven by the motion of a leading edge, in which actin polymerization pushes against the edge and adhesions transmit traction to the substrate while membrane tension increases. How the actin and adhesions synergistically control edge protrusion remains elusive. We addressed this question by developing a computational model in which the Brownian ratchet mechanism governs actin filament polymerization against the membrane and the molecular clutch mechanism governs adhesion to the substrate (BR-MC model). Our model predicted that actin polymerization is the most significant driver of protrusion, as actin had a greater effect on protrusion than adhesion assembly. Increasing the lifetime of nascent adhesions also enhanced velocity, but decreased the protrusion's motional persistence, because filaments maintained against the cell edge ceased polymerizing as membrane tension increased. We confirmed the model predictions with measurement of adhesion lifetime and edge motion in migrating cells. Adhesions with longer lifetime were associated with faster protrusion velocity and shorter persistence. Experimentally increasing adhesion lifetime increased velocity but decreased persistence. We propose a mechanism for actin polymerization-driven, adhesion-dependent protrusion in which balanced nascent adhesion assembly and lifetime generates protrusions with the power and persistence to drive migration.

Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

Carboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. McdA, a ParA-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to carboxysome-localized McdB. As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. How the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos. [Media: see text] [Media: see text] [Media: see text]

Bending-torsional elasticity and energetics of the plus-end microtubule tip

Microtubules (MTs), mesoscopic cellular filaments, grow primarily by the addition of GTP-bound tubulin dimers at their dynamic flaring plus-end tips. They operate as chemomechanical energy transducers with stochastic transitions to an astounding shortening motion upon hydrolyzing GTP to GDP. Time-resolved dynamics of the MT tip - a key determinant of this behavior - as a function of nucleotide state, internal lattice strain, and stabilizing lateral interactions have not been fully understood. Here, we use atomistic simulations to study the spontaneous relaxation of complete GTP-MT and GDP-MT tip models from unfavorable straight to relaxed splayed conformations and to comprehensively characterize the elasticity of MT tips. Our simulations reveal the dominance of viscoelastic dynamics of MT protofilaments during the relaxation process, driven by the stored bending-torsional strain and counterbalanced by the inter-protofilament interactions. We show that the post-hydrolysis MT tip is exposed to higher activation energy barriers for straight lattice formation, which translates into its inability to elongate. Our study provides an 'information ratchet' mechanism for the elastic energy conversion and release by MT tips and offers new insights into the mechanoenzymatics of MTs.

Quantification and demonstration of the collective constriction-by-ratchet mechanism in the dynamin molecular motor

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.

Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

ABSTRACTCarboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. McdA, a ParA-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to carboxysome-localized McdB. As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. However, how the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos.

Surfactants and rotelles in active chiral fluids

Surfactant molecules migrate to interfaces, reduce interfacial tension, and form micelles. All of these behaviors occur at or near equilibrium. Here, we describe active analogs of surfactants that operate far from equilibrium in active chiral fluids. Unlike molecular surfactants, the amphiphilic character of surfactants in active chiral fluids is a consequence of their activity. Our fluid of choice is a mixture of spinners that demixes into left-handed and right-handed chiral fluid domains. We realize spinners in experiment with three-dimensionally printed vibrots. Vibrot surfactants are chains of vibrots containing both types of handedness. Experiments demonstrate the affinity of double-stranded chains to interfaces, where they glide along and act as mixing agents. Simulations access larger systems in which single-stranded chains form spinning vesicles, termed rotelles. Rotelles are the chiral analogs of micelles. Rotelle formation is a ratchet mechanism catalyzed by the vorticity of the chiral fluid and only exist far from equilibrium.

Mechanisms of transforming DNA uptake to the periplasm of Bacillus subtilis

ABSTRACTWe demonstrate here that the acquisition of DNAase resistance by transforming DNA, often assumed to indicate transport to the cytoplasm, actually reflects uptake to the periplasm, requiring a re-evaluation of conclusions about the roles of several proteins in transformation. The new evidence suggests that the transformation pilus is needed for DNA binding to the cell surface near the cell poles and for the initiation of uptake. The cellular distribution of the membrane-anchored ComEA of B. subtilis does not noticeably change during DNA uptake as does the unanchored ComEA of Vibrio and Neisseria. Instead, our evidence suggests that ComEA stabilizes the attachment of transforming DNA at localized regions in the periplasm and then mediates uptake, probably by a Brownian ratchet mechanism. Following that, the DNA is transferred to periplasmic portions of the channel protein ComEC, which plays a previously unsuspected role in uptake to the periplasm. We show that the transformation endonuclease NucA also facilitates uptake to the periplasm and that the previously demonstrated role of ComFA in the acquisition of DNAase resistance actually derives from the instability of ComGA when ComFA is deleted. These results prompt a new understanding of the early stages of DNA uptake for transformation.IMPORTANCETransformation is a widely distributed mechanism of bacterial horizontal gene transfer that plays a role in the spread of antibiotic resistance and virulence genes and more generally in evolution. Although transformation was discovered nearly a century ago and most, if not all of the proteins required have been identified in several bacterial species, much remains poorly understood about the molecular mechanism of DNA uptake. This study uses epifluorescence microscopy to investigate the passage of labeled DNA into the compartment between the cell wall and the cell membrane of Bacillus subtilis, a necessary early step in transformation. The roles of individual proteins in this process are identified, and their modes of action are clarified.

STRESS ANALYSIS AND TOPOLOGY OPTIMIZATION OF A CHAIN BUCKET ELEVATOR USING ANSYS

A failure of conveyor chain links in a production process can cause unscheduled shutdowns, which increase the throughput time coupled with damaged buckets and chain links, which increase maintenance and repair costs. Since failures of conveyor chains are inevitable, this research aims to modify the design of the chain bucket elevator by incorporating a ratchet mechanism, which will prevent the chain bucket assembly from dropping to the bottom of the chain bucket elevator whenever there is a chain-link failure and also avoid the jamming of the bucket chain assembly against one another when dropping to the bottom of the elevator during failure. The number of damaged buckets and chains will be minimal, thereby reducing the maintenance and repair costs. Also, the time required for replacing the failed chain link will be reduced, which in turn, will reduce the down-time, thereby increasing the production rate. The ratchet mechanism, which can withstand a maximum load of 38.10 kN, comprises a toothed wheel, a pawl, and a spring. An analytical method was employed for the initial analysis and the results were verified using the FEM. Topology Optimization was carried out on the beam and lever with results showing a 20% and 26% weight reduction from the original, respectively. The stresses induced in the beam and lever increased significantly by 36% and 47 %, respectively, because of the optimization, however, they remained within the acceptable limits.

Skyrmion ratchet propagation: utilizing the skyrmion Hall effect in AC racetrack storage devices

Magnetic skyrmions are whirl-like nano-objects with topological protection. When driven by direct currents, skyrmions move but experience a transverse deflection. This so-called skyrmion Hall effect is often regarded a drawback for memory applications. Herein, we show that this unique effect can also be favorable for spintronic applications: We show that in a racetrack with a broken inversion symmetry, the skyrmion Hall effect allows to translate an alternating current into a directed motion along the track, like in a ratchet. We analyze several modes of the ratchet mechanism and show that it is unique for topological magnetic whirls. We elaborate on the fundamental differences compared to the motion of topologically trivial magnetic objects, as well as classical particles driven by periodic forces. Depending on the exact racetrack geometry, the ratchet mechanism can be soft or strict. In the latter case, the skyrmion propagates close to the efficiency maximum.