This protocol demonstrates how to investigate cell secretion using live imaging by TOF microscopy and provides tools for detecting clustering events or regions of high activity. Using this protocol, we can generate cell cultures on micropattern surfaces that normalize cell division and thus facilitate the special ranges of secretory events. Cells secrete a variety of macromolecules that play important roles in immune regulation.
In cancer, deregulated secretion affects the release of proteolytic enzymes that facilitate invasion. The quantification of secretion will enable us to better understand degraded secretion in cancer and other dynamic processes such has antigen presentation. Although our imaging and analysis protocol is straightforward, it requires the generation of single cells that are well spread on a piece of micropattern.
In this video, we will demonstrate how to culture cells on micropattern coverslips and how to visualize and analyze secretory events using spacial statistical tools. To prepare coverslips for micropatterning, activate ethanol sterilized 25 millimeter diameter glass coverslips under deep ultraviolet light for five minutes. While the coverslips are being activated, add one 30 microliter drop of polylysine graft polyethylene glycol per coverslip onto a piece of paraffin film within a humid chamber.
At the end of the activation, place the coverslips activated surface side down onto the drops and cover the humid chamber for a one-hour incubation at room temperature. At the end of the incubation, wash the coverslips two times in distilled water. While the coverslips are drying, wash a quartz photo mask one time with distilled water and one time with alcohol.
Dry the photo mask with filtered air flow and expose the photo mask chrome coated side up to deep ultraviolet light for five minutes. At the end of the illumination, add a 10 microliter drop of water onto the chrome coated side of each photo mask and place the polyethylene glycol side of each coverslip onto each drop of water. After removing any excess water, place a film cover over the slides to avoid dehydration.
Expose the non-chrome coated sides of the photo masks to deep ultraviolet light for an additional five minutes before adding excess water to the photo masks to remove the coverslips. This step will produce the micropattern imprints on the surface. Then incubate the coverslips in an extracellular matrix protein solution on paraffin film in a humid chamber for one hour under a laminar flow hood.
For cell seeding onto the prepared micropattern surface, at the end of the incubation, place the coverslips into a magnetic coverslip holder micropattern side up and immediately add pattern medium to the coverslips. After adding the seal, immobilize the coverslips with the magnetic device before filling the coverslip holder with additional pattern medium. Then close the holder with a glass lid.
When the cells are ready, add 5 times 10 to the 5th of the transfected immortalized retinal pigment epithelial cells to the coverslips and incubate the cells in the holder for 10 minutes in the cell culture incubator. At the end of the incubation, use one pipette to aspirate the old medium while using a second pipette to wash the cells five times with fresh medium per wash to remove the non-attached cells and any residual fetal bovine serum. After the last wash, return the holder to the incubator for three hours to allow full cell spreading.
To image exocytosis events, place the coverslip holder under a total internal reflection fluorescence microscope, and search for a cell expressing VAMP7-pHluorin that is fully spread. Change the angle of the laser until a total internal reflection fluorescence angle that allows the visualization of VAMP7-pHluorin exocytosis events is reached and perform a five-minute acquisition at a frequency compatible with the exocytosis rate and timescale. To obtain the exocytosis coordinates, open the acquired exocytosis event movie in Fiji and manually identify the exocytosis events by the appearance of a bright signal that spreads outward.
Use the point tool to mark the center of the exocytosis event and use analyze and measure to measure the x and y-coordinates as well as the temporal coordinate. Save the measurements of one cell in one text file. Open text file in a spreadsheet and remove all but the slice x and y-coordinate columns.
Next, use the oval tool and measure to measure the center and diameter and to obtain the x and y-coordinates and Feret's diameter of each cell. Save the measurements of all cells in one text file. Open the text file in a spreadsheet and remove all but the x and y-coordinate and Feret's diameter columns.
Then add the radius measurement for each cell, then obtain the radius from the Feret's diameter for each cell. Use the straight line tool to measure the thickness of the micropattern ring of each cell. Save this measurement for all cells in one text file, then divide the adhesion length by the cell radius to calculate the normalized adhesion length.
For single cell spatial analysis, first install the package of RStudio and load the package in RStudio. Run the package with the ESA function. A user interface will open, then select the directory for the dataset TXT files and a directory for the output plots.
The script will automatically start and perform the analysis, providing PDF files of the corresponding plots and TXT files containing the numerical results. Inside the cell, the floor in probe is quenched by the low pH of the lysosome, but during exocytosis, pHluorin begins to emit a signal as the pH increases due to proton release. The pHluorin signal exhibits a peak during exocytosis that represents the fast release of lysosomal protons, followed by an exponential signal decay that represents the 2D diffusion of the probe at the plasma membrane.
Typically, transfect immortalized human retinal pigmented epithelial cells demonstrate an important lysosomal secretion activity with an average exocytosis rate of 0.28 Hertz. It is possible to visualize the 2D distribution of exocytosis by kernel density estimation to reveal differences in local densities. There are three possible deviations from complete spatial randomness:clustering, dispersion, or a mixture of clustering and dispersion.
Ripley's K function can be used to assess these deviations. Of note, exocytosis events in the non-adhesive area are also clustered indicating that adhesion molecules are not the only structures that induce secretory hotspots at plasma membranes. Plotting the histogram of exocytosis events according to the modulus for a representative cell reveals a peak around the border between the adhesive and non-adhesive areas.
Paired analysis demonstrates that the surface density is lower in the adhesion area than in the non-adhesion area, potentially due to the strong decrease of exocytosis at the cell periphery. Combining cell adhesion on micropatterns with statistical analysis facilitates the ability to pinpoint how complex cellular processes such as secretion are regulated in space. More future work is needed to determine which molecular mechanism that underlie the clustering of secretory activity.
