Biology subjects

Walcott, S.

Publications and source records attributed to Walcott, S..

6 recordsLinked to original sources

bioRxiv2025

Kinesin-1 Autoinhibition Tunes Cargo Transport by Motor Ensembles

Intracellular vesicular transport by kinesin-1 motors through numerous 3-dimensional (3D) microtubule (MT) intersections must be regulated to support proper vesicle delivery. Knowing kinesin-1 can be regulated via autoinhibition, does kinesin-1 exhibit autoinhibition on cargo, and could this regulate vesicular transport through 3D MT intersections in vitro? To answer this question, we compared liposome transport by [~]10 nearly full-length kinesin-1 motors with KLC bound (Kin{Delta}C) versus constitutively active control (K543). In 3D MT intersections, Kin{Delta}C-liposomes terminate (48%), go straight (43%), but rarely turn (9%), starkly contrasting K543-liposomes which go straight (57%), turn (31%), but rarely terminate (12%). On single MTs, Kin{Delta}C-liposomes have reduced run lengths and detachment forces versus K543-liposomes, suggesting autoinhibition reduces MT engagement, as supported by 3-fold lower Kin{Delta}C MT landing rates versus K543, and mechanistic in silico modeling. Furthermore, kinesore, a small molecule that overcomes kinesin-1 autoinhibition, restores Kin{Delta}Cs MT engagement. Thus, we propose that partial kinesin-1 autoinhibition while cargo-bound may fine-tune cargo delivery to support physiological demands. Statement of significanceKinesin-1 vesicular transport through the cells complex microtubule (MT) network must be spatially and temporally regulated to meet the cells physiological demands. With kinesin-1 being autoinhibited, does this form of regulation impact the directional outcomes of vesicles encountering 3-dimensional (3D) MT-MT intersections as they do in cells? To address this in vitro, liposomes transported by kinesin-1 motors (team of [~]10), which exhibit autoinhibition, are challenged with 3D MT intersections and interestingly, preferentially terminate or go straight and rarely turn, while liposomes transported by constitutively active motors preferentially go straight or turn but rarely terminate. Therefore, autoinhibition reduces the number of MT-engaged motors within the team and provides the cell a means of fine-tuning cargo transport to its destination.

biophysics↗

bioRxiv2023

Metabolic cost for isometric force scales nonlinearlyand predicts how humans distribute forces across limbs

Muscles consume metabolic energy for force production and movement. A mathematical model of metabolic energy cost will be useful in predicting instantaneous costs during human exercise and in computing effort-minimizing movements via simulations. Previous in vivo data-derived models usually assumed either zero or linearly increasing cost with force, but a nonlinear relation could have significant metabolic or behavioural implications. Here, we show that metabolic cost scales nonlinearly with joint torque with an exponent of about 1.64, using calorimetric measurements of isometric squats. We then demonstrate that this metabolic nonlinearity is reflected in human behaviour: minimizing this nonlinear cost predicts how humans share forces between limbs in additional experiments involving arms and legs. This shows the utility of the nonlinear energy cost in predictive models and its generalizability across limbs. Finally, we show mathematical evidence that the same nonlinear metabolic objective may underlie force sharing at the muscle level.

biophysics↗

bioRxiv2023

Spatial Relationships Matter: Kinesin-1 Molecular Motors Transport Liposome Cargo Through 3D Microtubule Intersections In Vitro

Kinesin-1 ensembles maneuver vesicular cargoes through intersections in the 3-dimensional (3D) intracellular microtubule (MT) network. To characterize directional outcomes (straight, turn, terminate) at MT intersections, we challenge 350 nm fluid-like liposomes transported by [~]10 constitutively active, truncated kinesin-1 KIF5B (K543) with perpendicular 2-dimensional (2D) and 3D intersections in vitro. Liposomes frequently pause at 2D and 3D intersections ([~]2s), suggesting that motor teams can simultaneously engage each MT and undergo a tug-of-war. Once resolved, the directional outcomes at 2D MT intersections have a straight to turn ratio of 1.1; whereas at 3D MT intersections, liposomes more frequently go straight (straight to turn ratio of 1.8), highlighting that spatial relationships at intersections bias directional outcomes. Using 3D super-resolution microscopy (STORM), we define the gap between intersecting MTs and the liposome azimuthal approach angle heading into the intersection. We develop an in silico model in which kinesin-1 motors diffuse on the liposome surface, simultaneously engage the intersecting MTs, generate forces and detach from MTs governed by the motors mechanochemical cycle, and undergo a tug-of-war with the winning team determining the directional outcome in 3D. The model predicts that 1-3 motors typically engage the MT, consistent with optical trapping measurements. Modeled liposomes also predominantly go straight through 3D intersections over a range of intersection gaps and liposome approach angles, even when obstructed by the crossing MT. Our observations and modeling offer mechanistic insights into how cells might tune the MT cytoskeleton, cargo, and motors to modulate cargo transport. Significance StatementKinesin-1 molecular motors transport vesicles containing essential cellular resources along the dense 3D microtubule (MT) cytoskeleton, with dysfunctions linked to neurodegenerative diseases such as Alzheimers. Despite its importance, the mechanism by which kinesin-1s maneuver intracellular cargoes through MT-MT intersections towards their destination remains unclear. Therefore, we developed a 3D in vitro model transport system, which challenges kinesin-1 motor teams to maneuver vesicle-like liposomes through MT-MT intersections. Surprisingly, liposomes are biased to pass straight through 3D MT intersections rather than turn, even when the MT intersection presents as a physical barrier. A mechanistic model informs this observation, suggesting that spatial relationships between the cargo and MT intersection influence how molecular motors maneuver intracellular cargoes towards their destination to satisfy cellular demands.

biophysics↗

bioRxiv2023

Modeling Thick Filament Activation Suggests a Molecular Basis for Force Depression

Multiscale models aiming to connect muscles molecular and cellular function have been difficult to develop, in part, due to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single skinned rabbit psoas muscle fibers to ramp shortenings and step stretches performed on the plateau region of the force-length relationship. We isolated myosin from the same muscles and, under similar conditions, performed single molecule and ensemble measurements of myosins ATP-dependent interaction with actin using laser trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of an inhibited state. The model also includes a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force following stretch (residual force enhancement). By optimizing the model fit to a subset of our fiber measurements, we specified seven unknown parameters. The model then successfully predicted the remainder of our fiber measurements and also our molecular measurements from the laser trap and in vitro motility. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale models. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combines with thick filament activation to decrease the number of cycling cross-bridges. SIGNIFICANCEConnecting the molecular and cellular scales of muscle contraction would assist in, e.g., the treatment of genetic muscle diseases, the development of heart drugs, and the design of prostheses. The history dependence of muscle contraction, having no clear molecular basis, has remained an obstacle in making this connection for the seventy years since its discovery. We measured the force- and motion-generating capacity of rabbit psoas muscle from the scale of single molecules to single cells. We developed a mathematical model that, when fit to some of the cellular measurements, predicted the remaining cellular measurements and also the molecular measurements. The models ability to capture muscles history dependence suggests a unified description of muscle contraction from the molecular to cellular scale.

biophysics↗

bioRxiv2021

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350nm vs. 1,750nm) transport by myoVa motors. 5,000 simulated liposomes transported within each network adopted one of three states: transport, tug of war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug of wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, Nc. Nc, is a geometric property of the actin network that depends only on the position and polarity of the actin filaments and the liposome diameter, as evidenced by a five-fold increase in liposome diameter resulting in a five-fold decrease in Nc. Thus, in cells, actin network density and cargo size may be regulated to match cargo delivery to the cells physiological demands.

molecular biology↗

bioRxiv2019

MYOSIN VA TRANSPORT OF LIPOSOMES IN THREE-DIMENSIONAL ACTIN NETWORKS IS MODULATED BY ACTIN FILAMENT DENSITY, POSITION, AND POLARITY

The cell's dense three-dimensional (3D) actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position, using STORM microscopy, and its polarity by observing the movement of single fluorescent, reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350 nm fluid-like, liposomes transported by MyoVa teams (~10 motors) moving within each of the two networks. Compared to the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine-tune MyoVa-based intracellular cargo transport.

biophysics↗