Session E11: Physics of Cytoskeleton Across Scales II

Live

Cellular functions such as autophagy, signaling, and endocytosis involve physical interaction or capture of transported organelles, proteins, and signaling molecules at specific locations within the cell. We model transport-and-capture processes in tubular cells to explore how the spatial arrangement of capture regions affects biological function. In neurons, autophagosomes carrying cellular material for degradation transport over long distances to interact and fuse with acidified lysosomes. Using a simple analytical model, we show that the flux of degraded material can be optimized by regulating the location of organelle interactions. Our model identifies key features and parameters that govern the interplay between autophagy and long-range transport. In contrast to motile lysosomes, stationary microtubule tips can also behave as capture regions for loading motor-driven cargo. Endocytosed vesicles and signaling molecules diffuse from their entry point until captured at a microtubule tip. We model this diffusive capture process and show that a dispersed microtubule tip arrangement can minimize capture time. Our results highlight important physical features of autophagy and microtubule-based transport, providing guiding principles for optimal spatial arrangements within the cell.   

Live

Neurodegenerative diseases like the Alzheimer’s and Parkinson's disease involve disruption of intracellular transport due to the precence of different kinds of roadblocks on microtubule lattice, which include Microtubule Associated Proteins (MAPs) such as the tau protein. More generally, a variety of decorating proteins, stalled motors or cargo or other structures in the crowded cytoplasm can act as roadblocks. To explore the mechanisms available for cargoes to get past such roadblocks, we developed a Brownian dynamics simulation of membrane-bound cargo transport by teams of kinesin motors along microtubules that are populated with different kinds of roadblocks. Our previous work has shown that motor diffusivity on the membrane-bound cargo surface reduces the inter-motor interference and enhances run length in a co-operative and ATP dependent manner. Here we show that membrane-bound cargoes also have a higher probability of crossing roadblocks than membrane-free cargoes under specific conditions. Our results could have implications for the development of efficient treatments for neurodegenerative diseases.   

Live

Actomyosin networks are systems of actin filaments that are actively reorganized by motor proteins. Actomyosin networks are essential for providing structural scaffolds to cells and are involved in cell movement, growth, and division. The dynamics of these networks are regulated by many actin-binding proteins. In this work, we simulate the reorganizations of motorized actin networks with and without the actin-related protein 2/3 (Arp2/3) complex. We include motor proteins (non-muscle myosin IIA heavy chain (NMIIA)) and cross-linking proteins (α-actinin) in both actin systems. We observe that the relaxation times of the branched actomyosin networks are significantly longer than those of their unbranched counterparts, by as much as a factor of 4. We also observe that the branched networks exhibit rare convulsive movements, which we call avalanches. These avalanches release tension in the network. Recent experimental evidence of “cytoquakes” are consistent with the results of our simulations.   

Live

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos.
Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional singlebead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which hasimplications for a range of cellular activities, including cell division and organelle transport.   

Live

Identifying the key factors that affect the transport of a cargo by multiple molecular motors help understand the underlying mechanism of several neurodegenerative diseases arising from defects in motor transport. We hypothesize that the cargo-motor dissociation rate significantly impacts cargo transport by ensembles of molecular motor. To investigate the same, we develop a Markov-chain based simulation strategy, which assumes the number of motors on the cargo to be a stochastic quantity. Motors attach and detach from the cargo with certain rates, in addition to stepping, attaching, and detaching on/or from cytoskeletal tracks. These rates, along with the maximal number of motors possible on a cargo, are varied, and their effect is observed on transport quantities like average run lengths, average velocities, and average number of motors engaged in carrying the cargo. The results are compared with and contrasted against studies which assume a fixed number of motors on the cargo and are used to obtain better insights about experimental findings. Preliminary results show that probability of dissociation has a significant impact on run lengths, with a twofold decrease in average run lengths for a 5-fold increase in probability of dissociation of motor from the cargo.   

Live

Cargos in biological cells are often carried along microtubules by small ensembles of molecular motors. Predictive understanding of motility in cells therefore requires quantitative insights into how molecular motors function in the small-ensemble limit. Toward this goal, here we employed Monte Carlo simulations to examine the run length of cargos carried by 1-2 motors. Focusing on the key microtubule-based motor kinesin-1, our simulations utilized experimentally determined single-kinesin characteristics, and included alterations in single-motor on- and off-rates caused by cellular factors and physical load. We found that, compared to the single-motor case, a fractional increase in kinesin number enhances cargo run length and amplifies sensitivity to changes in single-kinesin on-rate and off-rate. These tuning effects are largely independent of solution viscosity over the range reported for cells. Together, our data indicate that kinesin number in the small-ensemble limit is a sensitive tuning parameter for cargo run length. We anticipate   

Live

Myosin molecular motors actively generate contractile forces in the actin cytoskeleton of animal cells that drive biological processes. These forces can be transmitted at long range through force chains that form in the elastic, disordered, cytoskeletal network. We model the disordered cytoskeletal network around an actin bundle as a mechanical fiber network comprising linear elastic fiber elements that can be bent or stretched, and that buckle above a critical compression threshold, which captures its anisotropic and nonlinear elastic properties.
We show by numerically minimizing the total elastic energy of the network that it mediates mechanical interactions between distant actomyosin units leading to favorable spatial configurations of motors, including possibly the recently observed registry of myosin in cells. We
then extend the model to a line of equally spaced force dipoles representing a stress fiber, and explore its elastic interaction energy with a distant force dipole as a function of its position and orientation, to show that mechanical forces promote stress fiber assembly by promoting
actomyosin alignment and registry. In future, we will extend our model to describe the force-dependent remodeling dynamics of the actomyosin structures of the cytoskeleton.   

Not Participating

Amphiphiles (e.g. membrane scaffold protein, styrene-co-maleic acid, etc.) stabilized lipid nanodiscs have been developed as water-soluble membrane mimetic platforms to stabilize membrane proteins for further structural characterizations. In contrast to detergent micelles, the structures of membrane proteins in nanodiscs are better retained with enhanced long-term stabilities.
In this work, hydrophobically modified polypeptoids (HMPs) have been designed and investigated as facially amphiphiles to fragment liposome to form stable HMP-lipid complexes or nanodiscs. The effects of polymer concentration, chain length, and relative hydrophobicity on liposome fragmentation have been elucidated by a combination of DLS, SLS and EM analysis. It was found that HMPs with relatively higher polymer hydrophobicity (De mol % = 27 %) and longer chain length (DP_n =100) can induce more effective liposome fragmentation relative to short polymers (DP_n = 34-60) and hydrophilic polymers with similar chain length (DP_n = 96 with De mol % = 16 %). It is hypothesized that a high local hydrophobic contact between polymers and lipid membranes is critical to accelerate and enhance the extent of liposome fragmentation.   

Live

Here we combine advances in membrane biophysics with established single-molecule optical-trapping assays to characterize the transport of membrane-enclosed cargos in vitro. Our study employed the major microtubule-based kinesin-1 motor and tau, an important microtubule-associated protein (MAP) that inhibits kinesin-based transport of membrane-free cargos. Remarkably, we found that coupling kinesins via a biomimetic membrane reduces the inhibitory effect of tau, significantly enhancing the transport of membrane-enclosed cargos on tau-decorated microtubules. Further, adding cholesterol to the cargo membrane amplifies the inhibitory effect of tau on kinesin. Combing simulation and modeling approaches, we found that cholesterol hinders the ability of individual motors to search for available binding sites on the microtubule, thereby amplifying tau’s steric inhibition of transport. Our study establishes a direct link between cargo-membrane composition and MAP-based regulation of kinesin-1. The combination of experimental and theoretical approaches we developed is generally applicable for interrogating the regulation of motor proteins in a context directly relevant to in vivo scenarios.   

Live

Recently there has been an explosion of new studies that target transport on networks of filaments. This has been driven by advances in experimental techniques that have provided clearer and more controllable in vivo and in vitro studies. However, one aspect that has received much less attention is the growth/shrinkage and dynamic turnover of the network filaments themselves, which can occur on the same time scale as the transport of cargo on the network. Consequently, the complex intracellular dynamics of the inhomogeneous cytoskeletal structure can have profound impacts on transport. Here, we study transport of cargo carried by myosin motors on a dynamic actin network. We use a stochastic simulation model that accounts for both active transport along filaments as well as passive diffusion and incorporates the dynamics of the explicitly represented actin network. We study how filament growth/shrinkage velocity, for actin treadmilling, effect cargo transport along with motor attachment/detachment rates. We will vary the density and the location and distribution of F-actin to ascertain how these variations change the optimal transport of cargo. This analysis will illuminate how actin dynamics can be used to modulate optimality in the molecular transport of cellular cargo.   

On Demand

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 relies 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, when 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. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling not only affects binding kinetics but also motor mechanochemistry. Therefore motors can 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.