Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

The overall goal of this procedure is to visualize and quantitate the dynamics of individual clathrin coated pits in live cells. This is accomplished by first transecting fluorescently tagged endocytic and cargo proteins into an adherence cell line. The next step is to image the cells with total internal reflection, fluorescence or turf microscopy.

The lifetimes of small sets of clathrin coated pits are then quantitated manually using an image processing and analysis program. The final step is to quantify the lifetimes of clathrin coated pits by objective automated detection and analysis. Ultimately, turf microscopy is used to quantitate the dynamics of individual clathrin coated pits at single event resolution to investigate clathrin mediated endocytosis of G-protein coupled receptors.

This method can help answer key questions in the membrane trafficking field, such as how the assembly and the dynamics of endocytosis can change based on the different endocytic proteins and cargo proteins present. To begin transfect HEC 2 93 cells with plasmids encoding tag G protein coupled receptors or GPCRs and or components of the Claro machinery. In a 12 well plate as detailed in the text protocol the day after transfection add 0.5 milliliters of phosphate buffered saline or PBS with one millimolar EDTA to each.

Well then place three rounds sterile 25 millimeter cover slips into separate wells of a six. Well plate Fill each well with two milliliters of media. Gently push the cover slip down to the bottom of the well to prevent cells from growing on the underside of the cover slip.

Next, manually agitate a well of the 12 well plate to lift off the cells from the bottom of the well. Add one milliliter of DM EM with 10%FBS to resend. Then distribute the lifted cells from one well evenly among the three cover slips.

Allow the cells to grow on the cover slips for at least 48 hours before imaging. Also ensure that cells are spread out and flat for imaging of the clathrin coated pit and cargo dynamics. First, pre incubate the cells with antibodies as described in the text protocol.

Then prepare to transfer the cover slip to a live cell imaging chamber by using a pair of forceps and a 25 gauge needle bent at the tip into a hook. Hold the pair of forceps in the dominant hand. Hold the bent needle in the other.

Turn the needle until the hook curves downwards towards the glass. Gently drag the needle hook side down across the bottom of a six well plate until it hooks onto the cover slip. Take care not to scratch the surface of the cover slip as it will detach cells.

Gently lift the cover slip up from the bottom of the well. Balance the cover slip against the wall of the well for support. Use the forceps to grasp near the edge of the cover slip and move the cover slip cell side up to the imaging chamber.

Assemble the chamber and add 700 microliters of prewarm imaging medium. After transferring the chamber onto the microscope, bring cells into focus using a turf oil immersion objective image the cells in conventional epi, fluorescence, or confocal modes of illumination to identify cells expressing the appropriate constructs as an important safety precaution. Always be aware of the angle of the laser beam and always direct it away from the observer.

Next, focus down to the bottom surface of an expressing cell until the outline of plasma membrane around the cell disappears and the bottom surface of the cell appears. This is most apparent with a plasma membrane localized cargo protein such as the mu opioid receptor. If using the same lasers for imaging in conventional epi fluorescence increase the angle of incidence of the laser until outta focus.

Fluorescence in the interior of the cell disappears and no outline of plasma membrane around the cell is seen. Once in turf m, ensure the excitation laser reflects back through the objective and is not visible above the objective. By checking if the laser spot is visible, refine the focus to get a crisp image.

Once cells are identified, acquire images at least every three seconds for 10 minutes. Repeat all steps to image additional cells. To begin analysis of endocytic dynamics, open the file in image J.Images are stored as a single stack of TIFF files.

With interleaf channels. Convert the images to hyper stacks by selecting image hyper stacks and stack to hyper stack in the pop-up window. Enter the number of channels acquired.

Enter one for Zack and enter the number of movie frames. The hyper stack format yields a window with two scroll bars. Use the top bar to move through the channels and the bottom bar to move through time.

Scroll to the channel of the protein whose lifetimes will be assayed. Move beyond the first frame of the movie to reduce the inclusion of pre-existing clathrin coated pits or CCPs whose entire durations are not visible. Draw a region of interest or ROI around a spot chosen at random.

Count the number of frames a spot is visible. To begin analysis with object recognition, open the raw image file in the image analysis Software by default emerged. Color image appears.

Use the display adjustment window to adjust the brightness of a channel. Click on the name of a channel to change the displayed color. Uncheck the box next to the channel to prevent its display.

Create spots by clicking on the icon with orange spots. Select an ROI around the cell using it to exclude areas that will incorrectly detect bright leading edges and plaques. Measure the diameter of a spot to provide initial estimates for the algorithm.

Switch to slice mode and click on the polar ends of a spot to measure its diameter. Do this for four or five round spots that look indicative of A CCP at the height of its stability after formation before cession, use these measurements to calculate average diameter down to the nearest 10th of a micron. To detect spots return to surpass mode, select the appropriate channel for detection.

Enter the average measured diameter, usually 0.3 or 0.4 microns. Click the blue forward arrow to quantify the spots. Filter the spots by quality through adjusting the filter to capture as many spots as possible without background.

The quality filter is selected by default. Use the quality curve as a guide to set an appropriate threshold. Move the bottom threshold of the filter with the left mouse button to this first dip in the curve.

This is a good starting point for the filter. As this position usually refines detection to only visible spots. Remove arant spots in the edit spot Screen switch to select mode and click on the spot After it turns yellow, click delete.

Then click the blue arrow to continue building the algorithm. Measure tracks by setting tracks to brownie in motion. The CCP should not exhibit any directed movement, only minimal drifting across the plasma membrane.

This algorithm is most appropriate for that motion. Next, set the max distance to a small value such as 0.7 microns. Setting a small distance minimizes connection of adjacent spots and still allows for continued tracking of a cell spot through CCP or cell movement.

Then set max gap size to one. This allows the track to continue if only missing one frame. In succession gap sizes of more than three causes different tracks to be incorrectly linked together.

The next step is to switch track viewing to dragon tail. Scroll through the movie observing the detection quality. If adjacent spots are being connected, decrease the max distance below 0.7 microns.

If spots are being broken up too readily, increase the max gap size. Click the blue arrow to create tracks. This leads to the filter track screen to filter tracks.

Use an intensity filter to remove additional bright plaques. Then use a displacement filter to remove endosomes that enter the turf field. Use caution as longer spots will have longer displacement as well.

Click the green double arrow to complete the protocol. To export data, go to the statistics tab. Click on the single floppy disc icon to export the selected statistic.

Click on the icon that looks like a series of floppy discs to export all statistics. Focusing on the A adherent plasma membrane and confocal mode reveals a cell that is filled with dim fluorescence. In contrast, focusing on the center of the cell reveals the plasma membrane as a ring of fluorescence around the cell.

Outta focus fluorescence becomes apparent when switching to conventional epi, fluorescence or turf with an incorrect angle. When the turf angle is correct, the plasma membrane is very crisp, clear and bright, and outta focus. Fluorescence no longer disrupts the image.

When beta arrestin is in a cytosolic pool, very little is in the turf field and it appears hazy and outer focus. Once the MOR is activated by agonist, beta, arrestin is recruited to the plasma membrane and both beta arrestin and the receptor redistribute to CCPs. The lifetimes of these clusters are quantified manually.

Using the three parameters for completed endocytosis, the spots that denote CCPs can disappear completely blink on and off, or pinch off a larger structure. Shown here a CCPs detected using a MAs After filtering to remove non-dynamic plaque structures, overlaid white spheres, denote detected spots, dimmer spots that could not be detected for their whole lifetimes were also excluded with the quality filter. After you watch this video, you should have a really good understanding of how to visualize individual catheter coded pits using turf microscopy.