The overall goal of using a combination of micromanipulation and light microscopy techniques such as FRAP and photoactivation is to monitor the spaciotemporal dynamics of proteins involved in cytoskeletal regulation and migration in distinct conditions or a signaling pathway dependent manner. The micromanipulation methods discussed here are useful for direct delivery of any type of molecule into cells as well as for a light induced manipulation of protein activity within defined subcellular locations. So the main advantage of the techniques presented here is that they allow determining the instantaneous effects that proteins exert on cells together with tracking their mobility throughout the cell.
To begin this procedure, passage previously grown B16-F1 cells at a one to five ratio into a three centimeter plastic dish. Aspirate the cell culture medium. Wash the cells with PBS, aspirate the PBS and add Trypsin EDTA to detach the cells.
Add cell culture medium to the trypsinized cells. Re-suspend and transfer the cells to falcon tubes, after this centrifuge at 1000 rpm for three to five minutes. After seating the B16-F1 cells into a three centimeter dish and allowing them to spread for at least six hours, transfect the B16-F1 cells by adding a mixture of 500 nanograms of DNA construct and one microliter of transfection reagent in a solution containing 150 millimolar sodium chloride.
For the B16-F1 cells coat 15 millimeter cover slips with 150 microliters of laminin solution and incubate for one hour at room temperature. For the NIH 3T3 cells coat the cover slips with fibronectin solution and incubate for one hour at room temperature. After incubation wash the laminin and fibronectin incubated cover slips with PBS then aspirate the PBS.
Seed two milliliters of the NIH 3T3 fibroblasts in one to 20 ratio from a confluent dish onto the fibronectin coated cover slips. Pipet two milliliters of the transfected B16-F1 cells in a one to 30 ratio from a confluent dish onto the laminin coated cover slips. Following this allow the cells to spread on laminin and fibronectin coated cover slips overnight in a tissue culture incubator at 37 degrees Celsius prior to microscopy.
Place the cover glass with the cells side up on a heat conductive RC-26 aluminum imaging chamber. Next place a plastic sealer on top of the cover glass to make a secure seal between the cover slip and the chamber, fix by screwing the plastic sealer with the sliding clamps of the chamber to avoid the medium leaking. Now, pipet 37 degrees Celsius pre-heated microscopy medium into the central area.
Insert the heat detector into the designated slot of the chamber and link the electrodes of the chamber to a TC-324B automatic temperature controller maintaining a constant temperature of 37 degrees Celsius. Centrifuge a previously thawed protein solution at 10, 000G for at least 30 minutes to remove protein aggregates that can lead to needle clogging if present in the microinjection capillary. Using a flexible pipet tip load a microinjection needle with one microliter of protein solution from the back side.
If air bubbles are present in the needle tip, gently tap the needle base in order to remove them. Then carefully adjust the needle holder on the micromanipulation device. Upon screwing the microinjection needle into the needle holder apply pressure to the needle using a microinjection pressure device before trans-locating the needle tip into the cell culture medium.
Next, position the needle in the field of view using a low magnification objective then find a cell of interest and gradually lower the needle above the cell. For microinjection gently touch the plasma membrane of the cell which may suffice to penetrate the cell or aid transient membrane rupture by a very gentle tap on the microscope setup. Stop the injection process as soon as flow into the cell is visible by moving the needle tip up into the medium.
Before triggering the laser, switch to the GFP channel and initiate image time lapse acquisition. Following this, manually draw the region to be photobleached on the GFP channel while viewing the display. Initiate photobleaching by a manual trigger of the 405 nanometer laser at least three to four frames after initiation of image acquisition.
Set the GFP 488 nanometer image acquisition in the software to 500 millisecond exposure and 1500 millisecond time interval. Next adjust the software settings for acquiring either dual channel or triple channel time lapse movies by marking the wavelength series square and selecting the desired number of channels in the acquire wavelength menu. Before triggering the laser, initiate image time lapse acquisition and manually draw the region to be photoactivated on the phase contrast channel while viewing the display.
Following this initiate photoactivation by a manual trigger of the 405 nanometer laser at least three to four frames after initiation of image acquisition. For analysis of the FRAP results, open the time lapse movies derived from VisiView on the MetaMorph software. Derive intensity values of the photobleached regions for each time point of fluorescence recovery by manually drawing respective regions using MetaMorph.
Draw a shape at the tip of the lamellipodium that covers the entire or part of the photobleached area and manually adjust it's position on subsequent frames if needed in order to track changes in the lamellipodial intensities of respective region over time during displacement of the advancing tip. For correction of background and photobleaching acquisition analyze regions outside and inside of the cells respectively. Once a region of interest is selected, extract it's intensity values on MetaMorph by using the menu measure region measurements.
Ensure a lapsed time and average intensity options are selected in the configure menu. Click open log and select dynamic data exchange. Then click okay to open an excel spreadsheet and click the open log button again to paste the MetaMorph values into excel.
Use the intensity values derived from MetaMorph to calculate FRAP fluorescence recovery curves by pasting the intensity values into an excel spreadsheet containing appropriate formulas. The fluorescence recovery of the photobleached region is normalized to background and inside regions. For calculating the half time of fluorescence recovery paste the values of fluorescence recovery curves with corresponding times into SigmaPlot then perform a curve fit using the dynamic fit wizard exponential rise to maximum tool, selecting mono or bi-exponential function depending on the best curve fit.
The parameters obtained by solving selected function can be pasted into an excel spreadsheet containing the appropriate formula which allows calculating respective half time of recovery in seconds. For measuring the diffusion of photoactivatable actin upon activation as well as it's accumulation within a specific region such as the lamellipodium use MetaMorph to determine the intensities over time in respective regions as well as outside of the cell in order to determine background fluorescence for normalization. For examining the precise rate of displacement of photoactivatable actin away from the activation region or it's rate of incorporation within the lamellipodium generate fluorescence curves from data previously derived from MetaMorph.
Paste the intensity values for the background region used for normalization the photoactivated cytosolic region or the lamellipodial region to be investigated into an excel spreadsheet containing appropriate formulas. Face contrast images of an NIH 3T3 fibroblast cell before and after microinjection of the small GTPase Rac1 are shown here. At approximately 12 minutes post-microinjection the cell has changed it's morphology which indicates successful injection.
Recycling of bleached EGFP VASP by non-bleached molecules at lamellipodium tip is show here. In this particular example recovery fluorescence plateaus at roughly 80%of the fluorescence before bleaching. Upon photoactivation, photoactivated GFP actin rapidly diffuses out of the cytosolic region.
A fraction of photoactivated actin monomers translocates to the front creating a rapid burst of fluorescence in lamellipodia. Incorporation of fluorescence is delectably lower in lamellipodia distant to the photoactivated region as the fraction of photoactivated actin monomers becomes diluted with nonactivated monomers on their journey through the cytosol. As photoactived GFP actin diffuses away from the photoactivated region over time it is continuously replaced by nonphotoactivated nonfluorescent actin.
As a consequence a gradual decrease in fluorescence intensity of this region is observed. Over time lamellipodia close to the cytosolic activation region rapidly incorporate fluorescent actin. The following drop in fluorescence is simply due to actin depolymerization and monomer diffusion away from the lamellipodium.
So when experiments are performed on a single microscopy system the combination of microinjection and either FRAP or photoactivation techniques can realistically be performed in a matter of a few minutes depending on the nature of the proteins investigated. Always remember to keep yourselves happy before, during and after micromanipulation or following exposure to laser light. For FRAP and photoactivation it is particularly important that the region selected maintain their structural integrity throughout the duration of the movie.
The microinjection procedure can be complimented by local application of plasma membrane permeable drugs or cytoskeletal inhibitors in order to address immediate consequences of given treatments at a subcellular level. Micromanipulation techniques have paved the way to explore diffusion properties and subcellular dynamics of cytoskeletal components and complexes and to understand secondhand pathways regulating cellular morphogenesis. After watching this video you should have a good understanding of how to track and monitor the spatiotemporal dynamics of cytosolic proteins, of proteins incorporated into the act inside the cytoskeleton or residing in different subcellular organelles, ultimately unraveling the kinetics of cell regulation.
