Measuring the Osmotic Water Permeability Coefficient (P_f) of Spherical Cells: Isolated Plant Protoplasts as an Example

The overall goal of the following experiment is to learn about the function of aquaporins by determining the osmotic water permeability coefficient, or PF of plant protoplasts. Cut out a piece from a leaf without axial epidermis and put it on an isotonic solution. Transfer the pieces to an enzymatic solution and incubate for 20 minutes in 28 degrees Celsius, then to a fresh isotonic solution.

Next, collect the protoplasts containing solution with a clipped off tip. As a second step, prepare an experimental chamber. Attach a cover slip to the bottom of the slide using silicone grease.

Then coat the chamber bottom with a positive charge bearing glue and fill the chamber with an isotonic solution. Isolated protoplasts are then added to the chamber, and the chamber is placed delicately on the microscope stage. Subsequently, the protoplasts are exposed to a hypotonic solution while videos are recorded to capture their swelling time course.

Next, the swelling time course is analyzed numerically by software including cur fitting procedures in order to determine the PF of each protoplasts based on the numerical analysis results are obtained that show differences in PF induced, for example, by expression of different aquaporin genes. This method can help answer key questions in the field of cellular membrane physiology, such as the regulation of the function of aquaporin. Though this method can provide insight into matic water, permeability of plant water plant that are either native or genetically modified.

It can also be applied to other systems such as ide exposed origin of interest. Prior to starting this procedure, prepare the required isotonic and hypertonic solutions and verify their osmolarity. Using an OSM oter, the osmolarity of each solution must be within 3%of the target value.

Fill a 10 centimeter Petri dish with about six drops of isotonic solution. Peel the ABBA seal or lower a rabbit opsys leaf epidermis and cut the peeled leaf into squares of about four by four millimeters. Place the small pieces on the isotonic solution drops with the exposed a axial side face down and touching the solution.

Next, dissolve 5.7 milligrams of the enzyme mix in 165 microliters of isotonic solution in a 1.5 milliliter tube and mix gently by pipetting for a minute or so until dissolved. Place several similar drops of the enzymatic solution in the same Petri dish. Transfer the leaf pieces onto the enzymatic solution.Drops.

Close the dish and seal the lid with one round of perfil. Float the dish in a water bath set to 28 degrees Celsius for 20 minutes. After 20 minutes, add several more drops of the isotonic solution to the dish using forceps.

Lift each leaf piece by its edge to transfer it to a new isotonic solution Drop and then sequentially to a second drop. Shake the leaf piece in the second drop to release the protoplasts using a clipped off 100 microliter pipet tip. Collect the drops with the protoplasts into a 1.5 milliliter tube.

Prepare the profusion system by filling one column with the isotonic solution and another column with the hypotonic solution. Open the valve to let some hypotonic solution flow to fill the tubing all the way down to the inlet manifold. After ensuring there are no trapped air bubbles, close the valve.

Repeat for the isotonic solution. Seal a cover slip using silicone grease to make a bottom for the chamber within the plexiglass slide to make the chamber bottom sticky for protoplasts, coat it with positive charge bearing protamine sulfate or poly L lysine. Use a pipette tip to spread this glue over the cover slip.

Wait for one to two minutes. Rinse three to four times with the isotonic solution and shake away the remaining solution. Fill the chamber up with the isotonic solution.

Then use a clipped off pipette tip to add a drop of protoplasts containing solution to the chamber. And wait three to four minutes for the protoplasts to settle. Cover the chamber with a transparent cover touching the solution surface.

Avoid trapping air bubbles beneath. Place a slide gently on an inverted microscope table and connect connected to the perfusion system and the pump guarding against air bubbles in the tubing. Turn on the isotonic solution flow for constant perfusion at one milliliter per minute.

For recording volume changes, choose a view field with as many cells as possible that are spherical in shape, and with a well-focused cell contour at their largest perimeter. Record a 62nd video of selected immobile protoplasts at a rate of one image per second or one hertz. Start the recording with a 15 second wash of the isotonic solution and switch to the hypertonic solution for 45 seconds.

Save the movie in Tiff format at To analyze a series of images of a swelling cell, use the Image Explorer and protoplasts analyzer plugins in the image J software. Start image J.To open the movie, click file on the Image J panel, then consecutively on the dropdown menus as they unfold import. Then image explorer.

Highlight the chosen movie. Right click on it, and then left. Click on protoplasts analyzer.

On the first image, use the mouse to draw circles around the selected protoplasts. Then click okay. In the table of detection parameters that appears to launch the protoplasts detection algorithm, click local on the top panel of the protoplasts image.

Then process. In the dropdown menu, examine the green circles around the selected protoplasts throughout the movie. Using the bottom slider, save the results in an Excel file.

The analysis is done frame by frame. Therefore, in the case of more than one cell, sorting is needed to separate the lines belonging to each cell Here. The three time courses are shown in an Excel sheet before and after sorting with the area values converted from pixels to square micrometers.

Each time course is then exported to a separate text file. To begin this procedure, connect the chamber to the perfusion system and the pump and turn on the indicator die flow for a constant perfusion rate of one milliliter per minute, record a 62nd video at the rate of one hertz. Start the recording with 15 seconds of indicator die and then switch to the hypertonic solution For 45 seconds, stop the recording.

Flush with the indicator. Die again for at least 30 seconds. Before starting a new movie record and save five to six movies to analyze the movies.

To obtain an average time course of the changing indicator die transmittance, start image J click file. Then open and browse for the movie. For each movie, draw a 10 pixel wide vertical rectangle anywhere on the first image of the movie, click image on the Image J main panel.

Then click crop in the dropdown menu. To align the 60 frames in one row, click image and then click consecutively in the dropdown menus as they unfold stacks and make montage. Draw a one pixel high horizontal rectangle anywhere along the whole row of images and click analyze in the Image J main panel.

Next, click plot profile. In the dropdown menu, copy the lists of the transmittance data to an Excel file. Average the transmittance time courses obtained from the Sero movies of the indicator die flushes.

Generate a realtime base by multiplying the image sequential number by 0.1. Save the average time course to a text file to compute the various parameters of the osmolarity time course. Use the MATLAB fitting program PF fit, which is available for use.

Free of charge. Details on downloading and using this program are available in the protocol text. In the indicator fit panel.

Import the data of the mean time course of the indicator dye transmittance. Manually enter the current experiment parameters, the initial guesses of the parameters with ant H.Describing the time course of the indicator dye concentration also may be changed. Click run to view the plot of the time courses of the indicator DAI concentration and of the modeled bath osmolarity.

A good fit to the data is essential to determine the water permeability coefficient. Switch to the volume fit panel. Choose for import the area's data file.

Choose last indicator fitting as the parameter source. Choose the model class. Start with two.

The PF parameters pf slow PF and delay need to be initialized, for example, as shown, but other values are also possible. Mark checks for all three parameters to be fitted. Click run.

Then eyeball the interim figure and adjust the delay parameter and the length of the record if needed. Examine the results graph to evaluate the fit, quality, and record the fit error. Change the initializing parameters a few fold each and rerun.

Repeat this procedure several times, starting with different combinations of initialization parameters, aiming for the lowest value of the fit error in order to determine the water permeability coefficient or pf, and compare the activity of different aquaporins Mesil Protoplasts from a rabbit opsys leaf were isolated and separately transformed with three gene constructs with aquaporin genes from a rabbit, opsys and maze. The protoplasts were labeled by co-ran with a vector encoding enhanced green fluorescent protein. The time courses of the cell volume changes upon exposure to hypotonic challenge were obtained for each cell as well as the time course of the bath osmolarity change during the hypotonic challenge, the PF values were derived for each cell.

Using the PF FIT program, it was observed that the PF values of the PROTOPLASTS transformed with each of the three aquaporins were significantly higher than the PF of the control cell transformed with GFP alone. Once mastered this technique can deliver analysis of a few tens of cells in a few experimental days if it is performed properly. After watching this video, you should have a good understanding of how to determine the SMO water permeability in isolated protoplasts or others focal cells subjected to a hypotonic assay.

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