An automated microwell platform for large-scale single cell RNA-seq
Yuan, J., & Sims, P. A. Sci. Rep. 6, 33883 (2016). Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue. |
Terrain following and applications: Caenorhabditis elegans swims along the floor using a bump and undulate strategy
Yuan, J., Ko, H., Raizen, D. M. & Bau, H. H. J. R. Soc. Interface, 13, 20160612 (2016). Nematodes such as Caenorhabditis elegans are heavier than water. When submerged in water, they settle to the bottom surface. Observations reveal that the animals do not lie flat on the bottom surface, but remain substantially suspended above the surface through continuous collisions with the surface, while maintaining their swimming gaits. Consequently, the swimming animals follow the bottom surface topography. When the bottom surface is inclined, the animals swim up or down along the incline. As the magnitude of the gravitational force can be easily estimated, this behaviour provides a convenient means to estimate the animal's propulsive thrust. The animals' tendency to follow the surface topography provides a means to control the swimmers' trajectories and direction of motion, which we demonstrate with a saw tooth-like ratchet that biases the animals to swim in a selected direction. The animals can also serve as surface topography probes since their residence time as a function of position provides information on surface features. Finally, we take advantage of surface following to construct a simple motility-based sorter that can sort animals based on genotype and state of health. |
A hydrodynamic mechanism for attraction of undulatory microswimmers to surfaces (bordertaxis)
Yuan, J., Raizen, D. M. & Bau, H. H. J. R. Soc. Interface, 12, 20150227 (2015). Although small nematodes significantly impact human and animal health, agriculture, and ecology, little is known about the role of hydrodynamics in their life cycles. Using the nematode Caenorhabditis elegans as a model undulatory microswimmer, we have observed that animals are attracted to and swim along surfaces. The attraction to surfaces does not require mechanosensory neuron function. In dilute swarms, swimmers aggregate near surfaces. Using resistive force-based theory, symmetry arguments, and direct hydrodynamic simulations, we demonstrate that forces resulting from the interaction between the swimmer-induced flow field and a nearby surface cause a short-range hydrodynamic torque that stirs the swimmers towards the surface. When combined with steric forces, this causes locomotion along the surface. This surface attraction may affect nematode mate and food finding behaviour and, in the case of parasitic nematodes, may facilitate host penetration. Surface attraction must be accounted for when studying animals' responses to various stimuli, and suggests means of controlling undulatory microswimmers. |
High-throughput, motility-based sorter for microswimmers such as C. elegans
Yuan, J., Zhou, J., Raizen, D. M. & Bau, H. H. Lab Chip, 15, 2790-2798 (2015). Animal motility varies with genotype, disease, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method is implemented in a simple microfluidic device capable of sorting thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriches for known C. elegans motility mutants. Furthermore, using this device, we isolate low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates C. elegans sleep. By performing genetic complementation tests, we demonstrate that our motility-based sorting device efficiently isolates mutants for the same gene identified by tedious visual inspection of behavior on an agar surface. Therefore, our motility-based sorter is capable of performing high throughput gene discovery approaches to investigate fundamental biological processes. |
Propensity of undulatory swimmers, such as worms, to go against the flow
Yuan, J., Raizen, D. M. & Bau, H. H. Proc. Natl Acad. Sci. USA 112, 3606-3611 (2015). Highlighted by Nature Physics 11, 297 (2015). The ability to orient oneself in response to environmental cues is crucial to the survival and function of diverse organisms. One such orientation behavior is the alignment of aquatic organisms with (negative rheotaxis) or against (positive rheotaxis) fluid current. The questions of whether low-Reynolds-number, undulatory swimmers, such as worms, rheotax and whether rheotaxis is a deliberate or an involuntary response to mechanical forces have been the subject of conflicting reports. To address these questions, we use Caenorhabditis elegans as a model undulatory swimmer and examine, in experiment and theory, the orientation of C. elegans in the presence of flow. We find that when close to a stationary surface the animal aligns itself against the direction of the flow. We elucidate for the first time to our knowledge the mechanisms of rheotaxis in worms and show that rheotaxis can be explained solely by mechanical forces and does not require sensory input or deliberate action. The interaction between the flow field induced by the swimmer and a nearby surface causes the swimmer to tilt toward the surface and the velocity gradient associated with the flow rotates the animal to face upstream. Fluid mechanical computer simulations faithfully mimic the behavior observed in experiments, supporting the notion that rheotaxis behavior can be fully explained by hydrodynamics. Our study highlights the important role of hydrodynamics in the behavior of small undulating swimmers and may assist in developing control strategies to affect the animals' life cycles. |
Gait synchronization in Caenorhabditis elegans
Yuan, J., Raizen, D. M. & Bau, H. H. Proc. Natl Acad. Sci. USA 111, 6865-6870 (2014). Collective motion is observed in swarms of swimmers of various sizes, ranging from self-propelled nanoparticles to fish. The mechanisms that govern interactions among individuals are debated, and vary from one species to another. Although the interactions among relatively large animals, such as fish, are controlled by their nervous systems, the interactions among microorganisms, which lack nervous systems, are controlled through physical and chemical pathways. Little is known, however, regarding the mechanism of collective movements in microscopic organisms with nervous systems. To attempt to remedy this, we studied collective swimming behavior in the nematode Caenorhabditis elegans, a microorganism with a compact nervous system. We evaluated the contributions of hydrodynamic forces, contact forces, and mechanosensory input to the interactions among individuals. We devised an experiment to examine pair interactions as a function of the distance between the animals and observed that gait synchronization occurred only when the animals were in close proximity, independent of genes required for mechanosensation. Our measurements and simulations indicate that steric hindrance is the dominant factor responsible for motion synchronization in C. elegans, and that hydrodynamic interactions and genotype do not play a significant role. We infer that a similar mechanism may apply to other microscopic swimming organisms and self-propelled particles. |
Orienting actin filaments for directional motility of processive myosin motors
Yuan, J., Pillarisetti, A., Goldman, Y. E. & Bau, H. H. Nano Lett. 13, 79-84 (2013). To utilize molecular motors in manmade systems, it is necessary to control the motors’ motion. We describe a technique to orient actin filaments so that their barbed ends point in the same direction, enabling same-type motors to travel unidirectionally. Myosin-V and myosin-VI were observed to travel, respectively, toward and away from the filaments’ barbed ends. When both motors were present, they occasionally passed each other while “walking” in opposite directions along single actin filaments. |