The eukaryotic cell's microtubule cytoskeleton is a complex 3D filament network. Microtubules cross at a wide variety of separation distances and angles. Prior studies in vivo and in vitro suggest that cargo transport is affected by intersection geometry. However, geometric complexity is not yet widely appreciated as a regulatory factor in its own right, and mechanisms that underlie this mode of regulation are not well understood. We have used our recently reported 3D microtubule manipulation system to build filament crossings de novo in a purified in vitro environment and used them to assay kinesin-1-driven model cargo navigation. We found that 3D microtubule network geometry indeed significantly influences cargo routing, and in particular that it is possible to bias a cargo to pass or switch just by changing either filament spacing or angle. Furthermore, we captured our experimental results in a model which accounts for full 3D geometry, stochastic motion of the cargo and associated motors, as well as motor force production and force-dependent behavior. We used a combination of experimental and theoretical analysis to establish the detailed mechanisms underlying cargo navigation at microtubule crossings.

Received; accepted 1 Visiting Astronomer, MMT Observatory. Observations reported here were obtained at the MMT Observatory, a joint facility of the University of Arizona and the Smithsonian Institution 2 Visiting Astronomer, Steward Observatory 2.3 m Telescope. ABSTRACTWe present spectra of Eris from the MMT 6.5 meter telescope and Red Channel Spectrograph (5700−9800Å; 5Å pix −1 ) on Mt. Hopkins, AZ, and of Pluto from the Steward Observatory 2.3 meter telescope and Boller and Chivens spectrograph (7100−9400Å; 2Å pix −1 ) on Kitt Peak, AZ. In addition, we present laboratory transmission spectra of methane-nitrogen and methane-argon ice mixtures. By anchoring our analysis in methane and nitrogen solubilities in one another as expressed in the phase diagram of Prokhvatilov & Yantsevich (1983), and comparing methane bands in our Eris and Pluto spectra and methane bands in our laboratory spectra of methane and nitrogen ice mixtures, we find Eris' bulk methane and nitrogen abundances are ∼ 10% and ∼ 90% and Pluto's bulk methane and nitrogen abundances are ∼ 3% and ∼ 97%. Such abundances for Pluto are consistent with values reported in the literature. It appears that the bulk volatile composition of Eris is similar to the bulk volatile composition ofPluto. Both objects appear to be dominated by nitrogen ice. Our analysis also suggests, unlike previous work reported in the literature, that the methane and nitrogen stoichiometry is constant with depth into the surface of Eris. Finally, we point out that our Eris spectrum is also consistent with a laboratory ice mixture consisting of 40% methane and 60% argon. Although we cannot rule out an argon rich surface, it seems more likely that nitrogen is the dominant species on Eris because the nitrogen ice 2.15 µm band is seen in spectra of Pluto and Triton.

Cellular cargos, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies focused on cargos with rigidly attached motors, in contrast to many biological cargos that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical 3D computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Finally, cargos with freely-diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Single-molecule cytoplasmic dynein function is well understood, but there are major gaps in mechanistic understanding of cellular dynein regulation. We reported a mode of dynein regulation, force adaptation, where lipid droplets adapt to opposition to motion by increasing the duration and magnitude of force production, and found LIS1 and NudEL to be essential. Adaptation reflects increasing NudEL-LIS1 utilization; here, we hypothesize that such increasing utilization reflects CDK5-mediated NudEL phosphorylation, which increases the dynein-NudEL interaction, and makes force adaptation possible. We report that CDK5, 14-3-3ε, and CDK5 cofactor KIAA0528 together promote NudEL phosphorylation and are essential for force adaptation. By studying the process in COS-1 cells lacking Tau, we avoid confounding neuronal effects of CDK5 on microtubules. Finally, we extend this in vivo regulatory pathway to lysosomes and mitochondria. Ultimately, we show that dynein force adaptation can control the severity of lysosomal tug-of-wars among other intracellular transport functions involving high force.

Mechanical properties of cells are important features that are tightly regulated and are dictated by various pathologies. Deformability cytometry allows for the characterization of the mechanical properties at a rate of hundreds of cells per second, opening the way to differentiating cells via mechanotyping. A remaining challenge for detecting and classifying rare sub-populations is the creation of a combined experimental and analysis protocol that approaches the maximum potential classification accuracy for single cells. In order to find this maximum accuracy, we designed a microfluidic channel that subjects each cell to repeated deformations and relaxations and provides a comprehensive set of mechanotyping parameters. We track the shape dynamics of individual cells with high time resolution and apply sequence-based deep learning models for feature extraction. In order to create a dataset based solely on differing mechanical properties, a model system was created with treated and untreated HL60 cells. Treated cells were exposed to chemical agents that perturb either the actin or microtubule networks. Multiple recurrent and convolutional neural network architectures were trained using time sequences of cell shapes and were found to achieve high classification accuracy based on cytoskeletal properties alone. The best model classified two of the sub-populations of HL60 cells with an accuracy over 90%, significantly higher than the 75% we achieved with traditional methods. This increase in accuracy corresponds to a fivefold increase in potential enrichment of a sample for a target population. This work establishes the application of sequence-based deep learning models to dynamic deformability cytometry.

Formation of fluid filled lumen by epithelial tissues is a fundamental process for organ development. How epithelial cells regulate the hydraulic and cortical forces to control lumen morphology is not completely understood. Here, we quantified the mechanical role of tight junctions in lumen formation using genetically modified MDCKII cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for hydraulic inflation of lumen. However, depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements and perturbations of hydrostatic lumen pressure and junctional tension with modelling, we were able to predict lumen morphologies from the pressure-tension force balance. We found that in MDCK tissue the tight junction promotes formation of spherical lumen by decreasing cortical tension via inhibition of myosin. In addition, we found that the apical surface area of cells is largely uncoupled from lumen volume changes, suggesting that excess apical area contributes to lumen opening in the low-pressure regime. Overall, our findings provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

Cellular cargos, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. In the cell, it is unclear how motors are coordinated to achieve transport outcomes that are cargo-specific. One possibility is that transport is modulated by differences in organization and mobility of motors on the cargo's surface. We use mechanochemical 3D computational modeling to compare different motor anchoring modes, and find that organizational changes can optimize for different objectives. Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules. Cargos with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Cargos with freely-diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results point to a functional role for observed changes in motor organization on cargos, and suggest motor diffusivity as a control point for transport, either by modulation of adaptor proteins or changes in lipid composition. Author summaryThe molecular motors of the kinesin family are responsible for moving the parts of cells 1 to their subcellular destinations, organizing the cell interior. Computational modeling 2 has historically played an important role in understanding how these motors work. But, 3 strikingly, most models assume the subcellular cargos have rigid surfaces. Simulating 4 fluid surfaces is more challenging but more applicable to biological cargo. In this work, 5 we build a computational model of molecular motor transport of cargo with fluid 6 surfaces. Surface fluidity gives cargo transport new properties, like enhanced transport 7 efficiency, and the ability to push against opposing forces. Because the effects of surface 8 fluidity vary strongly with some of the properties of the cargo, like its size, our results 9 hint at one of the ways that the same transport machinery can distinguish different 10 cargo despite using the same motors. 13 kinesin and dynein superfamilies to transport organelles and other cargo along 14 microtubules. Despite having only a limited set of cargo transport motors (kinesin-1, 15 kinesin-2 and kinesin-3 families [1], along with cytoplasmic dynein), different cargos are 16 transported to different locations, even though they are transported along the same set 17 of microtubule "roads". For example, under normal conditions COS-7 cells direct lipid 18 droplets toward microtubule plus end, localizing them near the plasma membrane, and 19 mitochondria toward the minus end, localizing them near the nucleus. Under glucose 20 starvation, localization of both organelles changes to spread them out around the cell, 21 allowing them to come into contact with each other [2]. How do cells achieve these 22 cargo-specific routing outcomes? In some cases, cells use molecular specificity to achieve 23 cargo specificity, such as using specific linkers or cargo-bound regulators [3,4]. 24 However, recent experiment...

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.