Session U20: Physics of the Cytoskeleton Across Scales III: Transport

Microtubules Regulate Localization and Availability of Insulin Granules in Pancreatic Beta Cells

Two key prerequisites for glucose stimulated insulin secretion (GSIS) in beta cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). To this point, it is unclear what regulates localization of insulin granules and their interactions with the PM within single cells. We demonstrate that microtubule (MT) motor transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that while the MT cytoskeleton resembles a random meshwork in the cells’ interior, MTs near the cell surface are aligned with the PM. Computational modeling suggests two consequences of this. First, this structured MT network withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. These findings suggest the MT cytoskeleton may negatively regulate GSIS by both limiting the amount of insulin proximal to the PM and preventing stable docking of insulin granules to the PM. These results predict that altering MT network structure in beta cells can be used to tune GSIS. Thus, our study points to a potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.   

Anomalous transport across scales in crosslinked actin-microtubule composites

The diffusion of microscopic particles through the cell is largely controlled by the cytoskeletal network, comprised of semiflexible actin filaments, rigid microtubules, and crosslinking proteins. Yet, how the interactions between actin and microtubules and the various types of filament crosslinking affect particle transport remain unresolved. In our experiments, we couple single-particle tracking (SPT) with differential dynamic microscopy (DDM) to characterize the transport of micron-sized particles diffusing through crosslinked composite networks of actin filaments and microtubules. Specifically, we investigate the impact of permanently crosslinking actin to actin, microtubules to microtubules, and actin to microtubules. By combining SPT and DDM, we are able to couple single-particle dynamics to ensemble transport phenomena, and link particle diffusion to the viscoelastic characteristics of the networks. We find that subtle changes to the crosslinking interactions between cytoskeleton filaments play surprisingly important roles in the anomalous subdiffusion that particles exhibit within a composite cytoskeletal system.   

Spreading through the cell via non-canonical modes of transport

Cells rely on a variety of mechanisms for delivering particles ranging from small molecules to micron-sized organelles to their various destinations. We use physical modeling coupled with live-cell imaging data from collaborating groups to explore the interplay of different transport modes in distributing particles through the complex intracellular environment. This talk will focus on two examples of multi-modal transport: combining motor-driven motion, diffusion, and fluid flow for efficient dispersion of particles within the cell.

Organelle hitchhiking is a non-canonical form of transport, which relies on a cargo organelle attaching to a motor-driven ''carrier'' with the aid of linker proteins. We quantify the effect of physical parameters on hitchhiking efficiency, demonstrating an insensititivy to linker density and a substantial benefit from tethering of cargo organelles to microtubule tracks. Our model is parameterized and validated against dynamic imaging data in fungal hyphae.

In addition, we investigate the role of advective transport for proteins and ions within the endoplasmic reticulum (ER). Capitalizing on recent experimental evidence of contraction-driven uncoordinated flow within ER tubules, we show that rapid flow combine with mobile calcium-binding buffer proteins can substantially enhance the distribution of calcium ions within the active network structure of the ER.   

MEMBRANE CHOLESTEROL IS A NOVEL CONTROL FOR KINESIN-BASED TRANSPORT

Motor protein-based transport in cells underlies all eukaryotic cell function and survival; dysfunctions in this transport are implicated in many diseases, including neurodegeneration. While the properties of motor proteins have been extensively studied both in vivo and in vitro, many important questions remain, including how the properties of the cargo itself impact motor function. In cells, cargos are often membrane-bound; the composition of the cargo membrane has long been hypothesized to impact motor protein-based transport. Here we combined advances in membrane biophysics with single-molecule optical-trap experiments to characterize the transport of membrane-enclosed cargos in vitro. We found that coupling motors via a biomimetic membrane significantly enhanced the transport of cargos along tau-decorated microtubules. This effect diminished when we added cholesterol to our model membrane. To our knowledge, our study uncovers the first direct link between cargo-membrane composition and kinesin function. The experimental approach employed here is generally applicable as a controlled experimental platform for interrogating the control of motor proteins in a context directly relevant to in vivo scenarios.   

Effect of membrane fluidity on the multi-motor transport of intracellular cargoes

In eukaryotic cells, membrane-bound cargoes are transported by teams of molecular motors that diffuse on the lipid membrane. The effect of the cargo fluidity on transport properties is unclear. In particular, it's unknown whether the motor diffusivity helps cargoes navigate the crowded cellular environment especially in the presence of heterogeneity in motor properties. We developed a stochastic dynamical simulation of kinesin-based cargo transport along microtubules that explicitly considers the Langevin dynamics of motors on the cargo surface to answer these questions. Our previous work showed that motor diffusivity reduces inter-motor interference and enhances cargo runlength even in the absence of crowding and heterogeneity. Here we study the effect of motor diffusivity on multi-motor transport when the motor population has a distribution of velocities and also explore how cargoes navigate roadblocks (like Microtubule Associated Proteins) that are typically present on microtubules in vivo.   

The Dynein Catch Bond: Implications for cooperative transport

Intracellular bidirectional transport of cargo on microtubule filaments is achieved by the collective action of oppositely directed dynein and kinesin motors. Recent experiments have demonstrated that unline kinesin, dynein motor exhibits catch bonding behaviour, in which the unbinding rate of a single dynein decreases with increasing force, for a certain range of force. Motivated by these experiments, we propose a phenomenological model for catch bonding in dynein. We study the implications of the dynein catch bond for unidirectional and bidirectional cargo transport. We show that the functional divergence of the two motor species manifests itself as an internal regulatory mechanism, and can lead to codependent transport behavior in biologically relevant regimes.

Reference: Dynein catch bond as a mediator of codependent bidirectional cellular transport, Puri et. al. Phys. Rev. Research, 1, 023019 (2019)   

Tracking Down the Fast and Superprocessive KIF1A with Gold Scattering Microscopy

The kinesin-3 family member KIF1A is a neuronal kinesin that performs long-distance anterograde vesicle transport in axons and dendrites. Single molecule studies observe KIF1A velocities > 1 mm/s and average run lengths > 5 mm, making KIF1A one of the fastest and most processive members of the kinesin superfamily; however, the mechanistic basis of these high speeds and long run lengths is unknown. One prevailing model for superprocessivity holds that the positively-charged “K-loop” in the KIF1A motor domain diffusively tethers the motor to the negatively-charged microtubule, which prevents complete dissociation of the motor and effectively links together short runs. However, this model does not account for how KIF1A reaches such high speeds, or what role the K-loop plays in the ATP-driven stepping mechanism. To address these questions, we used biochemical assays in conjunction with direct observations of stepping of wild type KIF1A and k-loop mutants. We captured the transient states of the stepping cycle by tracking a 30-nm gold nanoparticle-functionalized motor domain via interferometric scattering microscopy (iSCAT), which enables fast acquisition of gold particles < 40 nm and simultaneous visualization of the microtubule tracks. We find that the chemomechanical cycle of KIF1A is distinct from other neuronal transport kinesins.   

Investigating the Effect of Cargo-Motor Linkage Stiffness on Cellular Functions of Myosin VI

We examine the effect of cargo-motor linkage stiffness on the mechanobiological properties of the molecular motor Myosin VI. We use the programmability of DNA nanostructures to systematically modulate cargo-motor linkage stiffness and combine it with high precision optical trapping measurements to measure the effect of linkage stiffness on motile properties of Myosin VI. Our single molecule experiments reveal that a stiff cargo-motor linkage leads to shorter step sizes and load-induced anchoring of Myosin VI, while a flexible linkage results in longer steps with frequent detachments from the actin filament under load. These findings suggest a novel regulatory mechanism for tuning the dual cellular roles of anchor and transporter ascribed to Myosin VI [1]. We further make an attempt to investigate the physical basis of our findings using simulations and thermodynamics principles.

[1] Shrivastava, R., Rai, A., Salapaka, M., & Sivaramakrishnan, S. (2019). Stiffness of cargo-motor linkage tunes Myosin VI motility and response to load. Biochemistry   

Deviations from Arrhenius behavior of Kinesin-1 at low temperatures

Kinesin-1 is a mechanochemical enzyme that is essential for executing long-distance transport of cargos in eukaryotic cells via processive motility along the microtubule network. KIF5A is a conventional kinesin in the Kinesin-1 family. The temperature dependence of enzymatic activity for several kinesin-1 motors has been reported to follow a simple Arrhenius trend. The range for this observation has been gradually extended to higher temperatures, as it became possible to circumvent and more recently control kinesin degradation. However, both biophysical and biochemical measurements to date have been limited down to ~5 °C. We investigated the enzymatic activity of KIF5A at even lower temperatures and have observed a break in the Arrhenius trend, corresponding to higher activation energy at lower temperature. We will report our investigations of this phenomenon in different biochemical backgrounds and discuss its cause as it relates to the nature of the rate-limiting step of kinesin’s enzymatic cycle.   

Cargo diffusion shortens single-kinesin runs at low viscous drag

Molecular motors such as kinesin-1 drive active, long-range transport of cargos along microtubules in cells. Thermal diffusion of the cargo can impose a randomly directed, fluctuating mechanical load on the motor carrying the cargo. Recent experiments highlighted a strong asymmetry in the sensitivity of single-kinesin run length to load direction, raising the intriguing possibility that cargo diffusion may non-trivially influence kinesin. To test this possibility, here we employed Monte Carlo-based simulations to evaluate the transport of cargo by a single kinesin. Our simulations included physiologically relevant viscous drag on the cargo and interrogated a large parameter space of cytoplasmic viscosities, cargo sizes, and motor velocities that captures their respective ranges in living cells. We found that cargo diffusion significantly shortens single-kinesin runs. This diffusion-based shortening is countered by viscous drag, leading to an unexpected, non-monotonic variation in run length as viscous drag increases. Our study highlights the importance of cargo diffusion and load-detachment kinetics on single-motor function.