Biology subjects

Hancock, W. O.

Publications and source records attributed to Hancock, W. O..

28 records · Page 2Linked to original sources

bioRxiv2021

Nanoscale dynamics of cellulase TrCel7A digesting cellulose

Understanding how cellulases catalyze the digestion of lignocellulose is a major goal of bioenergy research. Cel7A from Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. We used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A binding to and moving processively along immobilized Gluconoacetobacter cellulose. Enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzymes bound into a static state and dissociated without detectable movement. Processive enzymes moved an average distance of 39 nm at an average speed of 3.2 nm/s. Static binding episodes preceding and following processive runs were of similar duration to static binding events that lacked any processive movement. Transient jumps of >20 nm were observed, but no diffusive behavior indicative of a diffusive search of the enzyme for a free cellulose strand end was observed. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either a static or a processive state, and can reversibly switch between static and processive states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state either by dissociation from the cellulose surface or initiation of a processive run.

plant biology↗

bioRxiv2020

The fast and superprocessive KIF1A predominately resides in a vulnerable one-head-bound state during its chemomechanical cycle

Kinesin-3 are the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to wild-type KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and-2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate limiting transition in the KIF1A stepping cycle. The fast speed, superprocessivity and load sensitivity of KIF1A can be explained by a fast rear head detachment rate, a rate-limiting step of tethered head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly-bound post-hydrolysis state.

cell biology↗

bioRxiv2019

Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport

The construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors line up in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each others way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

biophysics↗

bioRxiv2019

Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein

Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used Interferometric Scattering (iSCAT) microscopy to track dynein-dynactin-BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB - kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity, in quantitative agreement with the increase in diffusivity seen in isolated DDB. The data suggest a DDB activation model in which engagement of dynactin p150 with the microtubule promotes dynein processivity, serves as an allosteric activator of dynein, and enhances processive minus-end motility during intracellular bidirectional transport.\n\nTOC HighlightDynein-dynactin-BicD2 (DDB) is highly processive, but also shows transient pausing and diffusion, which we analyzed using iSCAT microscopy. Blocking dynactin p150 results in more diffusion of isolated DDB and a plus-end shift of kinesin-1 - DDB complexes. Thus, we conclude that p150 is an allosteric activator of dynein in the DDB complex.

cell biology↗

bioRxiv2019

Slow Microtubule Binding Kinetics of Membrane-bound Kinesin Predicts High Motor Copy Numbers on Intracellular Cargo

Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rate constants (koff) can be measured via single-molecule experiments, but motor reattachment rate constants (kon) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand motor attachment dynamics during vesicle transport, we quantified the microtubule accumulation rate of fluorescently-labeled kinesin-1 motors in a 2D system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a koff that matched single-molecule measurements, and measured a two-dimensional kon for membrane-bound kinesin-1 motors binding to the microtubule. This kon is consistent with kinesin-1 being able to reach roughly 20 tubulin subunits when attaching to a microtubule. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this kon is not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this two-dimensional kon predicts that long-range transport of 100 nm diameter vesicles requires 35 kinesin-1 motors, suggesting that teamwork between different motor classes and motor clustering may play significant roles in long-range vesicle transport.\n\nSignificance StatementLong-distance transport of membrane-coated vesicles involves coordination of multiple motors such that at least one motor is bound to the microtubule at all times. Microtubule attachment of a membrane-bound motor comprises two steps - diffusing through the lipid bilayer to a binding zone near the microtubule, followed by binding. Using a 2D supported lipid bilayer system, we show that membrane diffusion is not the limiting factor for motor attachment. This result suggests that in cells kinesin-1 binding kinetics are not altered by the membrane composition of vesicle cargos. The intrinsically slow binding properties of kinesin-1 suggest that divergent motor binding kinetics and motor clustering regulate long-range vesicle transport.

biophysics↗

bioRxiv2019

Kinesin-5 promotes microtubule nucleation and assembly by stabilizing a lattice-competent conformation of tubulin

Besides sliding apart antiparallel microtubules during spindle elongation, the mitotic kinesin-5, Eg5 promotes microtubule polymerization, emphasizing its importance in mitotic spindle length control. Here, we characterize the Eg5 microtubule polymerase mechanism by assessing motor-induced changes in the longitudinal and lateral tubulin-tubulin bonds that form the microtubule lattice. Isolated Eg5 motor domains promote microtubule nucleation, growth and stability. Eg5 binds preferentially to microtubules over free tubulin, and colchicine-like inhibitors that stabilize the bent conformation of tubulin allosterically inhibit Eg5 binding, consistent with a model in which Eg5 induces a curved-to-straight transition in tubulin. Domain swap experiments establish that the family-specific Loop11, which resides near the nucleotide-sensing Switch-II domain, is necessary and sufficient for the polymerase activity of Eg5. Thus, we propose a microtubule polymerase mechanism in which Eg5 at the plus-end promotes a curved-to-straight transition in tubulin that enhances lateral bond formation and thereby promotes microtubule growth and stability.

biophysics↗

bioRxiv2018

The Orphan Kinesin PAKRP2 Achieves Processive Motility Via Noncanonical Stepping

PAKRP2 is an orphan kinesin in Arabidopsis thaliana that is thought to transport vesicles along phragmoplast microtubules for cell plate formation. Here, using single-molecule fluorescence microscopy, we show that PAKRP2 exhibits processive plus-end-directed motility on single microtubules as individual homodimers despite having an exceptionally long (32 residues) neck linker. Furthermore, using high-resolution nanoparticle tracking to visualize motor stepping dynamics, we find that PAKRP2 achieves processivity via a noncanonical stepping mechanism that includes small step sizes and frequent lateral steps to adjacent protofilaments. We propose that the small steps sizes are due to a transient intermediate step that involves a prolonged diffusional search of the tethered head due to its long neck linker. Despite this different stepping behavior, ATP is tightly coupled to each 8-nm step. Collectively, this study reveals PAKRP2 as the first orphan kinesin to demonstrate processive motility and broadens our understanding of the diverse kinesin stepping mechanisms.

biophysics↗

bioRxiv2018

Direct observation of individual tubulin dimers binding to growing microtubules

The biochemical basis of microtubule growth has remained elusive for over thirty years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make the first direct, single-molecule measurements of tubulin on- and off-rates. We detect two populations of transient dwell times, and determine via binding-interface mutants that they are separated by the formation of inter-protofilament bonds. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data, and furthermore predict plus-end tapering. Overall, we provide the most direct and complete quantification of how microtubules grow to date.\n\nSIGNIFICANCEMicrotubule polymerization dynamics are fundamental to cell migration and cell division, where they are targets for chemotherapy drugs. Despite significant progress, the precise structural and biochemical events occurring at growing microtubule tips are not well defined, and better understanding is necessary for discriminating mechanisms of microtubule dynamics regulation in cells. Here, we visualize individual tubulin subunits reversibly and irreversibly interacting with dynamic microtubule tips, and thereby directly measure tubulin on- and off-rates. By analyzing plus-tip residence times of wild-type and mutant tubulin, we characterize the relative contributions of longitudinal (along protofilaments) and lateral (between protofilaments) bond energies to microtubule growth. This work provides insights into microtubule tip structure and potential modes of microtubule dynamics regulation.

biophysics↗

bioRxiv2018

Measurement of the Persistence Length of Cytoskeletal Filaments using Curvature Distributions

Cytoskeletal filaments such as microtubules and actin filaments play important roles in the mechanical integrity of cells and the ability of cells to respond to their environment. Measuring the mechanical properties of cytoskeletal structures is crucial for gaining insight into intracellular mechanical stresses and their role in regulating cellular processes. One of the ways to characterize these mechanical properties is by measuring their persistence length, the average length over which filaments stay straight. There are several approaches in the literature for measuring filament deformations, including Fourier analysis of images obtained using fluorescence microscopy. Here, we show how curvature distributions can be used as an alternative tool to quantify bio-filament deformations, and investigate how the apparent stiffness of filaments depends on the resolution and noise of the imaging system. We present analytical calculations of the scaling curvature distributions as a function of filament discretization, and test our predictions by comparing Monte Carlo simulations to results from existing techniques. We also apply our approach to microtubules and actin filaments obtained from in vitro gliding assay experiments with high densities of non-functional motors, and calculate the persistence length of these filaments. The presented curvature analysis is significantly more accurate compared to existing approaches for small data sets, and can be readily applied to both in vitro or in vivo filament data through the use of an ImageJ plugin we provide.

biophysics↗

bioRxiv2017

Motor reattachment kinetics play a dominant role in multimotor-driven cargo transport

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multi-motor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be 4-fold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of gold-nanoparticle-labeled cargo with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than do kinesin-1. Finally, cargo transported by kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multi-motor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads while kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

biophysics↗