Session G06: Molecular Machines I

Molecular Cutting Machine: Katanin Microtubule Severing Enzyme

Microtubules are required to perform multidues of essential cellular processes. For these funcations, they must form and fall apart in a regulated manner. The microtubule severing enzymes are a family of molecular machines from the ATPases associated with various cellular Activities (AAA+) family of enzymes that use ATP to cut microtubules anywhere along their length. Our recent work on katanin has revealed surprising results including that katanin is inhibited by the presence of free tubulin, katanin can depolymerize microtubules lacking the carboxy-terminal tail, and katanin only mildly disrupts cell division, even when tethered directly to the kinetochore.   

Exploring secondary structural transitions in microtubule severing motors with machine learning

Katanin is a microtubule severing nanomachine that becomes fully functional in a hexameric assembly driven by binding to co-factors; however, it has been observed to populate lower order oligomeric states. Literature findings suggest that the hexameric state of katanin is prone to disassembly into lower order oligomers and furthermore that this disassembly may be accompanied by a secondary structure change in the protomers. To gain insight into the molecular factors driving these two processes, we carried out long all-atom molecular dynamics simulations on various order oligomeric states of katanin in the presence or absence of binding partners. To characterize changes to the secondary structure of the protomers, we developed a centroid-based clustering method applied directly to the Ramachandran plot to analyze structural changes occurring in simulations. We evaluated the performance of our method through the application of established clustering methods to our simulations and of our methodology to existing simulations of intrinsically disordered proteins, which are known to populate varied secondary structures. We will discuss the results of our analysis, which highlight significant conformational transitions and their link to underlying allosteric networks in oligomers.   

Motor guidance by long-range communication through the microtubule highway

Coupling of motor proteins within arrays drives muscle contraction, flagellar beating, chromosome segregation, and other biological processes. Current models of motor coupling invoke either direct mechanical linkage or protein crowding, which rely on short-range motor-motor interactions. In contrast, coupling mechanisms that act at longer length scales remain largely unexplored. Here we report that microtubules can physically couple motor movement in the absence of short-range interactions. The human kinesin-4 Kif4A changes the run-length and velocity of other motors on the same microtubule in the dilute binding limit, where 10-nm-sized motors are separated by microns. This effect does not depend on specific motor-motor interactions because similar changes in Kif4A motility are induced by kinesin-1 motors. In a computational model of motor coupling, a long-range (micron-scale) interaction between motors can recreate the experimental results, while models with short- and intermediate-range interactions cannot. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling affects not only binding kinetics but also motor mechanochemistry. Therefore, long-range coupling allows motors to sense and respond to motors bound several microns away on a microtubule. These results suggest a paradigm in which the microtubule lattice, rather than being merely a passive track, is a dynamic medium responsive to binding proteins to enable new forms of collective motor behavior.   

Microtubule Studies on Lattice Dynamics and Severing Machine Guided Regulation

Microtubules (MTs) are dynamically unstable biofilaments composed of tubulin dimers that undergo stochastic phases of polymerization and depolymerization, induced by single-event transitions known as catastrophe and rescue. Due to low kinetic resolution, studies on MTs have yet to agree on what drives catastrophe and rescue events. Here, we used very long (20 seconds) coarse-grained simulations of MT assembly and disassembly to collect parameters for the characterization of catastrophe, rescue, growth, and shortening of MTs. Utilizing Machine Learning techniques, we found that features related to the energetics of the lattice are crucial to distinguish between the four kinetic states. Catastrophe and rescue can also arise due to MT interactions with severing enzymes, such as spastin. Spastin is a complex machine made of an ATPase motor, a flexible linker, and a MIT domain that collectively form a hexamer. It is known to have a specific binding and alignment to tubulin monomer terminal tails and a non-specific orientation onto the MT lattice, and that it applies mechanical force to remove dimers from the MT. Employing different coarse-grained model simulations, we found strong correlations between the strength of the interactions between the MIT domains and an MT lattice, and the orientation of spastin on the MT surface, the tubulin extraction pathways, and the fate of the machine upon the start of severing. In turn, these results can shed light on this machine's role in catastrophe and rescue events.   

A Magnetic Tweezers-Based Study of DNA-Histone Interactions

We report data from single-molecule forcemediated DNAcore histone assembly and disassembly processes using horizontal magnetic tweezers. The tweezers can apply stresses ranging from 0.01 to 100 pN and generate highresolution, low-noise data. DNA tethers, core histones, and NAP1 were used to create DNAcore histone complexes that were subjected to forces ranging from 2 pN to 80 pN. We noticed that the length of the DNA decreased in approximate integer multiples of 50 nm during the assembly process, suggesting histone octamers were bound to the DNA tether. We also found disruption lengths distributed around near integer multiples of 50 nm during mechanically induced disassembly events, again suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Further, we present preliminary data from studies comparing the mechanical stability of native and post-translationally modified histones bound to DNA, which will help to quantify the relative affinities of histoneDNA interactions in nucleosomes.   

DNA Supercoiling Drives a Transition between Collective Modes of Gene Synthesis

Transcription of genes can be affected by both biochemical and mechanical factors. Recent experiments tie the transition from cooperative to antagonistic group dynamics of RNA polymerases (RNAPs) on promoter repression to the mechanical stresses associated with transcription induced DNA supercoiling. To underpin the mechanism behind this drastic transition, we developed a continuum deterministic model for transcription under torsion with two novel features: (1) the number of RNAPs on the gene affects the torsional stress experienced by individual RNAPs and (2) transcription factors, when bound to the DNA, block the diffusion of supercoils at the promoter. We show that this model generates a fluid mode of RNAP group dynamics when the RNAP flux is continuous, and a torsionally-stressed mode when the flux is interrupted. At low flux conditions, a single RNAP transcribes with the same efficiency irrespective of the continuity in RNAP loading. The results of our minimal model are in quantitative agreement with experimental findings and elucidate the interplay of mechanical and biological factors in the collective dynamics of molecular machines involved in gene expression.   

Atomically precise control over rotation of a rare-earth molecular motor complex on Au(111)

Rare-earth metals are vital for many high technological applications from light emission devices to quantum information science. Here, we have developed a molecular motor formed by rare-earth metal and counterion complex that can be rotated with precise control over rotation angle and direction of rotation. In this system, a triflate counterion attached underneath a tri-blade Europium based molecular rotor acts as a pivot that enables fixed-axis rotation on Au(111) surface. The motor rotates either by thermal activation or by an electric field applied from a scanning tunneling microscope tip. Although the preferential rotation angle should be 60 degrees, an additional counterion attached to a side of the motor acting as a counterweight results in three-fold rotations with precise 120-degree steps. Moreover, attachment of side counterion permits 100% control of rotation direction and induces rotational chirality. This work demonstrates that charged counterions can be used to control dynamics of rare-earth molecular systems on materials surfaces.   

Modelling the step size distribution of myosin VI

Myosin VI is a motor protein that carries intracellular cargo along actin filaments.  The processive motion of myosin VI consists of sequential steps that have been measured to have a large variation in size:  the motor has been measured to make long “hand-over-hand” steps, short “inchworm” steps, as well as backwards steps.  We have developed a single model that allows us to study this multifaceted stepping behavior.   We can study the effects of external parameters on the dynamics of the motor and investigate the evolutionary advantages of the large step size variation.   

ROCK dependent volume regulation of cell shape in confluent epithelial tissue

Epithelial volume regulation is a key component to tissue stability and dynamics. The cortical cytoskeleton plays an integral role in providing mechanical resistance to swelling and shrinkage due to changes in osmotic pressure. In this study, we describe the role of ROCK dependent volume regulation of confluent renal epithelial tissue It is well studied that single cells typically recover their volume in hyperosmotic conditions in under an hour. We show that while this remains true for small epithelial colonies, confluent epithelial height and volume are permanently decreased under hyperosmotic stress. This permanent decrease is dependent on sufficient ROCK activity as well as the presence of an intact tight junction.   

Torques within microtubule bundles generate the curved shape of the mitotic spindle

The mitotic spindle is a complex micro-machine made up of microtubules and associated proteins, which are highly ordered in space and time to ensure its proper biological functioning. A functional spindle has a characteristic shape, which includes curved bundles of microtubules that are twisted around the pole-to-pole axis. An in-depth understanding of both how the linear and rotational forces define the overall shape of the mitotic spindle and how the twisted shapes arise as a result of interactions between microtubules and motor proteins is still unclear. To answer this, we introduce a mean-field approach, in which we describe the dominating forces and torques at the poles, in order to model he entire spindle. The model also includes motor proteins which generate forces and torques within the antiparallel overlap region of microtubule bundles. By comparing theoretical results with experimentally observed shapes of bundles in the mitotic spindle, the model predicts that the shape of the entire spindle is predominately determined by rotational forces, and that a difference in bending forces explains the disparity in the shapes of inner and outer bundles.   

Why exercise builds muscles: Titin mechanosensing controls skeletal muscle growth under load

A eukaryotic cell must be able to quickly respond to mechanical changes to its environment, pulling by neighbouring cells, or changes in its own shape during its cell cycle. Long-lasting mechanical signals can be sensed by molecules placed under load, if a globular domain of the molecule opens or unfolds and reveals a binding site for a signalling molecule. The time-integration of load in particular is a key feature of muscle hypertrophy. Here, I will explain how the opening of kinase domain of the muscle molecule titin under load provides a possible explanation for muscle growth following resistance training. To do this, we modelled the response of titin kinase to exercise and its effects on the synthesis of new proteins. We found that the titin kinase domain acts as a switch, opening and signalling most strongly above a certain force, close to 70% of the maximum muscle fibre force threshold found to be necessary for hypertrophy to occur. In addition, our model predicted that muscles adapt to a new resistance training plan over a few months, matching experimental time courses for hypertrophy. These predictions strongly suggest that muscle cells use cellular mechanical signalling directly to measure and respond to large-scale load.