Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

The overall goal of this procedure is to follow the intracellular trafficking of amyloid precursor protein or a PP in live cells. This is accomplished by first transecting cells with a PP tagged with photo activat, GFP and compartment markers for lysosomes and the gold G.Next, the cells are photo activated in specific regions of interest in the gold G.Then photo activation is stopped and the clearance of a PP from downstream compartments is imaged. Finally, trafficking of a PP is analyzed using analysis software such as emus.

Ultimately, photo activation and confocal imaging are used to show the clearance of a PP protein in live cells. Demonstrating this technique will be Joshua Tam, a PhD student from my laboratory After plating SN 56 cells and allowing them to proliferate according to the text protocol. In a cell culture hood, use plasmids expressing a fluorescent protein tagged galacto cell transferase, a fluorescent protein tagged compartment marker, and a protein of interest tagged with P-A-G-F-P to transfect.

The cells incubate the transfected cells at 37 degrees Celsius in a 5%carbon dioxide incubator for 24 hours. To prepare the microscope stage prewarm PBS and HBSS to 37 degrees Celsius, warm up the microscope stage to 37 degrees Celsius. Remove the differentiation medium by using the PBS to wash the cells twice and add two milliliters of prewarm HBSS.

Then place the cells on the warmed microscope stage and allow five to 10 minutes for the confocal plate temperature to equilibrate with the microscope stage. With the microscope eyepiece, find cells transfected with GAL TCFP lamp one MRFP, and beta A PP PA GFP. Then set the confocal slice for each channel to one micrometer and take a low resolution image crop to image only the cell of interest using the adjustment knob.

Manually set the focal plane to the middle of the cell, then select the edit ROI button in the command console, and draw three to five circular ROIs within the trans GOI network or TGN. Select the circular ROI button and click and drag over GAL TCFP positive regions of the cell. Then take an image of the cell with the overlaid ROIs and save a copy of the ROIs for later reference.

Next to set up the microscope for a bleach time course. Set the laser diode to maximum power. Then set the microscope for 120 cycles of imaging.

Set the microscope to bleach only the ROIs for 20 to 30 iterations. After each image, the time to image and bleach an image will vary. Set a time delay between images, so each cycle is approximately 30 seconds.

Then start the time course after 15 minutes, turn off the bleaching, but continue capturing images of the cell. Continue capturing images for 45 minutes to follow the clearance of beta A-P-P-P-A-G-F-P to determine if lysosomes are the final compartment. For beta A PP, add membrane permeable protease inhibitors such as L 6 85, 4 58, or chloroquine to cause proteins to accumulate in the lysosome before imaging.

Then find cells and photo activate is just demonstrated. Image the cells for an additional 45 minutes to determine where beta A-P-P-P-A-G-F-P accumulates in the absence of cleavage. Analyze the delivery of protein to lysosomes or another downstream compartment and subsequent clearance according to the method outlined in the text protocol.

To ensure accuracy of photo activation within the Golgi. Warm up HBSS to 37 degrees Celsius repair 66 micromolar NOOL and DMSO for one confocal plate pipette two milliliters of 37 degrees Celsius HBSS and add 7.96 microliters of no conazole from 16.60 millimolar, no conazole stock as a control solution. Pipette two milliliters of 37 degrees Celsius HBSS into a two milliliter tube, and at 7.96 microliters of DMSO incubate the cells and the solutions for five minutes before imaging.

Next, find the cells in the Zack panel. Press the Z sectioning button and set the interval to one micrometer or less for higher spatial resolution. Then before photo activation to prepare the microscope to take a ZS stack.

After the photo activation period, click on the ZS stack button and use fast XY to start scanning. Adjust the focal plane to the top of the cell and press mark first. To set the first position in the stack, adjust the focal plane to the bottom of the cells and press mark.

Last to set the position of the stack bleach the cells as described earlier in this video. Then photo, activate an image from zero to 15 minutes. Stop imaging after 30 frames and immediately save an image of the video.

After saving the video, immediately acquire a Zack using the parameters just described, filter and carry out colocalization according to the text protocol, as demonstrated here, beta a PP leaves the TGN and it appears to traffic rapidly to lamp one. During the photo activation period, vesicles are seen leaving the Golgi destined for lysosomes after photo activation. The beta A-P-P-P-A-G-F-P is rapidly cleared from the lysosome treatment with nool leads to the accumulation of a PP within the GAL, TCFP labeled compartments and prevents trafficking to lysosomes zones after delivery of beta a PP to the lysosome.

The P-A-G-F-P fluorescence is cleared rapidly, which could be the result of trafficking from the lysosome to another compartment. Conversely, cleavage of beta A PP by gamma secretase would be expected to lead to diffusion of P-A-G-F-P fluorescence from the lysosomal membrane to determine whether beta a PP is delivered to another compartment or cleared. SN 56 cells were treated with a specific inhibitor against gamma secretase or an alkalinizing agent.

The addition of either L 6 85, 4 58, or chloroquine resulted in a PP accumulation in the lysosome. A PP is delivered to the cell surface before being endo, atos and processed in endosomes and lysosomes. However, in untreated cells cell surface beta a PP could not be detected by confocal microscopy.

Interestingly, gamma secretase inhibitor treatment leads to detectable beta A-P-P-P-A-G-F-P at the cell surface, suggesting that gamma secretase inhibitor treatment may root more beta a PP to the cell surface After its development. This technique paved the way for researchers in the field of Alzheimer's disease to explore the trafficking of a PP in neuronal cell lines. After watching this video, you should be able to label and follow the intracellular trafficking of proteins using live cell microscopy.