Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops

This research focuses on soft biological materials and aims to explore the underlying physical mechanisms that regulate shape and organization and materials inspired by biological cells.

Biological and in vitro model systems are inherently complex with many pitfalls, and sample preparation is critical to their reproducibility. This protocol is designed to improve reproducibility in these systems.

These results pave the way to detect, characterize mechanisms, and understand the function of physical forces and biomaterials. This may be generalizable to various biopolymers and organelles.

[Narrator] To begin, prepare the sample to be loaded by adding five microliters of 10XF buffer in a micro centrifuge tube and add a volume of deionized distilled water such that the final sample volume will be 50 microliters after the remaining components are added. Pipette one microliter of 25% beta-mercaptoethanol, one microliter of 225 milligrams per milliliter glucose, one microliter of a mixture of glucose oxidase and 85,000 units per milliliter catalyst into the solution. Then add one microliter of 25 millimolar ATP and 10 microliters of 2%, 15 centipoise methylcellulose, and mix thoroughly by pipetting. To begin actin polymerization, mix unlabeled actin with labeled actin and add the actin mix to the prepared F buffer solution. Pipette the solution up and down to ensure proper mixing. Leave the actin to polymerize at room temperature in the micro centrifuge tube. Next, to prepare a flow cell, place two pieces of double-sided tape parallel to each other across the width of the microscope slide to form the boundaries of the flow cell channel. Position the cover slip over the tape channel ensuring it is perpendicular to the microscope slide. Press down on the area of the cover slip that is in contact with the double-sided tape to create a good seal and eliminate air pockets. Using a razor blade, trim any excess tape off the microscope slide and cover slip such that the only visible tape is within the sample channel. To prepare a cylinder sample chamber for two deplaner samples, first rinse the cover slip with ethanol, water, and ethanol. Dry it with filtered air. Apply a thin layer of five minute epoxy to adhere the glass cloning cylinder to the cover slip. Add 3.5 microliters of oil surfactant solution to a transparent or light colored micro centrifuge tube. Without mixing, pipette five microliters of the actin solution onto the top of the oil surfactant layer. Close the tube. Hold the top of the micro centrifuge tube and flick the bottom edge to form an initial foam with visible emulsion and continue flicking until it forms uniform micro emulsions. Prior to loading the flow cell, mix a small amount of five minute epoxy. To load a flow cell, pipette one to three microliters of oil surfactant solution into the sample channel of the flow cell to create a small plug that wets the channel. Tilt the flow cell to remove any air gap that forms due to the receding oil surfactant meniscus before adding the sample. Immediately pipette the sample solution emulsion suspension into the entrance of the flow cell to fill the channel. Seal each side of the flow cell channel with five minute epoxy. To load the cylinder sample chamber for non emulsion samples, pipette five microliters of oil surfactant solution into the bottom of the glass cylinder chamber. Tilt and slowly rotate the cover slip to coat the surface and lower cylinder with the surfactant solution. Remove excess oil surfactant solution using a pipette to obtain a thin layer while preventing complete evaporation of the oil. Immediately add the sample solution to the chamber. Cover the chamber with a small piece of polytetrafluoroethylene, or PTFE tape, to prevent evaporation and flow. Mount the sample onto the microscope stage. Start time-lapse imaging of actin using a 20x, 40x, 60x, or 100x objective with oil or water immersion to ensure a sufficiently high numerical aperture for high resolution imaging. If polymerizing in a sample chamber, allow polymerization for approximately 30 minutes or until there is no visible filament lengthening or motion. Add a cross linker to the sample, then add myosin as pre polymerized filaments by slowly pipetting approximately half of the total volume into the sample. Finally, start the time lapse imaging. The representative confocal fluorescence image of the entangled actin network is shown here. The entangled actin network is uniformly distributed across the surface. Bright spots in this image represent filament overlaps, whereas dark regions indicate minimal actin presence. Thermal fluctuations lead to local intensity changes and visible bending of actin filaments. The addition of myosin II causes bending and reorganization of the actin network with visible accumulation of myosin puncta at long times. Network contraction depends on the myosin concentration and ATP concentration. In cross-linked actin networks, myosin induced contraction leads to coordinated motion over longer length scales compared to entangled networks.