We have developed an in vitro approach to characterize the mechanics of poroelastic actomyosin gels as a model system of the cell cytoskeleton, and more generally, the cytoplasm, which have been shown to behave as a poroelastic active material. This method provides insights into how myosin contractility contributes to the emergence of a fluid flow and how network and cytosol velocities are correlated in time and space. This simple approach allows us to extract the mechanical properties of dynamically evolving systems without using any sophisticated equipment or techniques where conventional tools like AFIM cannot be employed.
This method can provide insights into the rheology of poroelastic active materials regardless of the specificity of the active gel and the embedded fluid. To begin, place 10 to 12 number 1.5 glass coverslips in a homemade polytetrafluoroethylene holder and prepare Piranha solution in a 400 milliliter beaker. Prepare the solution on ice in a chemical hood.
Transfer the holder into the Piranha filled beaker. After three hours, transfer the holder into a clean beaker filled with fresh double distilled water to wash the excess Piranha from the coverslips and transfer the beaker into a sonication bath at 80 hertz and full power for 10 minutes at 25 degrees Celsius. Repeat this step two more times with fresh double distilled water.
For salinization, transfer the holder into a clean beaker filled with 300 milliliters of pure methanol, then into a sonication bath for 30 minutes. Then transfer the holder into a silane solution. Seal the beaker with parafilm and keep it in the refrigerator at four degrees Celsius overnight.
On the next day, transfer the holder into a clean beaker filled with 300 milliliters of pure methanol for 15 seconds. Dry the bottom of the holder to remove the excess methanol and transfer it into a clean beaker. Then transfer the beaker to the oven to dry the glass coverslips for five minutes at 120 degrees Celsius.
To achieve surface passivation, take two coverslips for each experiment and place them in a Petri dish coated with a parafilm layer initially cleaned with ethanol. Incubate each coverslip with one milliliter of methoxypolyethylene glycol maleimide in 1X PBS for one hour at 22 degrees Celsius. At the end of the incubation process, tilt the Petri dish to remove and peg.
Rinse each coverslip with five milliliters of double distilled water and dry with a flow of nitrogen gas. Since the coverslips must be kept wet after passivation, immediately put one milliliter of 10 millimolar Tris on the pegylated surfaces and use the coverslips with two hours. To form large myosin aggregates, dilute the myosin stock solution with 10 millimolar Tris to reach a final concentration of 25 millimolar potassium chloride.
Keep the diluted myosin solution for 10 minutes at room temperature and then transfer it to ice until use. The motors are fully active for up to two hours. to track the solvent flow across the gel pores, prepare passivated fluorescent beads and reduce the interaction between the beads and the actomyosin network by incubating one microliter of beads with five micromolar G-actin for 20 minutes.
Remove the excess G-actin by centrifugation. Repeat this step with 10 milligrams per milliliter of bovine serum albumin to block the remaining uncoated surface and use the beads at a final dilution of one to 10, 000 in the experiments. To prepare the actomyosin solution, thaw the protein aliquots stored at minus 80 degrees Celsius and place them on ice.
Then prepare the solution to reach a final concentration of 10 millimolar Tris, five micromolar G-actin, 280 nanomolar GST Fascin, and 1.67 nanomolar myosin II.Take one of the polyethylene glycol passivated coverslips. Place a greased paraform spacer on top of it and place it in the sample holder. Add 1.1 microliters of the actomyosin solution to the holder mounted coverslip and place the second coverslip on top.
Screw the holder to seal the sample. Place the sample holder on the microscope and start the acquisition. Prepare the microscope in advance to reduce the initial acquisition time.
To image the samples with an inverted fluorescent microscope, visualize the whole gel area by using low magnifications of 2.5X objective and follow its macroscopic contraction with time. Use a 10X objective to characterize the network porosity and structure and to follow the network self-organization and contraction with time. For the simultaneous two-color imaging, excite the actin at 488 nanometers and myosin II motors at 561 nanometers and record the images simultaneously on an EMCCD camera using a dual emission apparatus.
Use the same imaging system to simultaneously image the actin gel and the fluorescent myosin II motors. For simultaneous two-color imaging, excite the actin at 488 nanometers and 2, 300 nanometer beads at 561 nanometers. And record the images simultaneously on an EMCCD camera using a dual emission apparatus.
Use the same imaging system to simultaneously image the actin gel and the fluorescent beads. The first day involves demonstrating that the actomyosin network behaves as an elastic material. A schematic representation of an actomyosin network is depicted here.
Fluorescence microscopy images demonstrate that the actin filaments spontaneously nucleate and polymerize into an isotropic interconnected network that coarsens with time and eventually contracts macroscopically. Network attraction initiates at the gel periphery and propagates inward into the gel bulk. The gel center is marked with a C.The actin filament bundles remain straight during network contraction.
The distribution of the ratio between the contour length and the end-to-end distance at T316 seconds and T327 seconds is shown here. The second aim involves demonstrating that an outward solvent flow is generated by myosin contractility. The image of the gel at low magnification at intermediate stages of contraction is shown here.
These circles mark the positions of four beads. The arrows mark the global direction of the bead motion. The trajectories of the nine selected beads are depicted here.
Resolving the network porosity and the movement of the solvent across the gel pores at advanced stages of contraction are depicted here. The third aim involves demonstrating that stress relaxation is characterized by an effective poroelastic diffusion constant. Top view fluorescence images of a contracting actomyosin gel at low magnification from the mixing time up to the steady state are shown here.
The actomyosin networks behave as a poroelastic active material. While attempting this procedure, work fast and accurately and keep in mind the importance of surface preservation.
