Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The goal of this procedure is to induce and observe autophagy in a filamentous fungus. This is accomplished by first inoculating small volumes of nutrient containing medium with spores or small mycelium pieces. In the second step, starvation pads are prepared in the cavities of glass microscope slides, and then the inoculate is transferred onto the pads.

In the final step, the starvation pads are incubated in a wet chamber to induce autophagy. Ultimately, the autophagy can be tracked by monitoring the translocation of fluorescent marker proteins of interest to the vacuoles using fluorescent microscopy. This method can help to study key issues in the cell biology field, such as identification of new compounds that modulate the autophagy process.

To prepare for a starvation experiment, if the P cresa genum strain of interest is kept on green rice, first place, two to three rice grains covered with sating mycelia into a 1.5 micro centrifuge tube, and fill the tube with 500 microliters of yeast extract glucose growth or wide GG medium. Then vortex the tube for 30 seconds so that spores detach from the rice and incubate the tube for one day at room temperature. The next day dilute the spore suspension to a one to 50 concentration in sterile tap water and mix well if the p cryogen is kept on agar.

Use a pipette tip to transfer small pieces of the mycelium into a 1.5 milliliter micro centrifuge tube containing 400 microliters of sterile tap water and approximately 100 milligrams of glass beads. Then vortex the tube for two minutes so that the mycelia become fragmented. To cultivate the PCE genum on a starvation pad, first warmer heating plate to approximately 60 degrees Celsius.

Next, eat two grams of aros in 100 milliliters of tap water in a microwave until the resulting solution is completely clear while the aros is cooling to approximately 60 degrees Celsius. Bill one, 1.5 milliliter micro centri tube per sample with 400 microliters of sterile tap water or compound solution. Then warm the tubes and microscope slides on the heating plate after at least 60 seconds.

Transfer 400 microliters of 60 degrees Celsius aros to all of the tubes, bringing the total volume in each tube to 800 microliters and vortex the tubes briefly, then place the tubes back onto the heating plate to prevent premature solidification. Now pipette 140 microliters of the appropriate aros solution into the cavity of each microscope Slide. Taking care not to make bubbles immediately flatten the aros with cover slips, and then rest the slides for approximately four minutes.

At room temperature when the aros pads have solidified, use a thumb or index finger to carefully slide off the cover slips and place the microscope slides and pads into a wet chamber. Next, pipette five microliters of the previously prepared P er genum onto the center of the starvation pad, and wrap the wet chamber in a plastic bag for extra protection against desiccation, taking care not to move the box too much or disturb the cultures. Then store the inoculates at room temperature for at least 20 hours before analyzing the samples.

The next day, transfer the slides to dry tissue paper and then dispense 50 microliters of water onto each starvation pad. Use a cover slip to squeeze out the surplus water for removal with fresh tissue paper. Then for each slide, use a 20 x objective to acquire an overview of the mycelium growth.

To assess the localization of the GFP in the Hy-Fi. Open the images with the 63 x or 100 x objective. Finally, place the slides back into the wet chamber after each viewing until the next experimental time.

Point in these images, the G-F-P-S-K-L-P Creo Genum strain was used to assess peroxisome degradation at 20 hours of incubation. GFP was not observed in the vacuoles, nor were those organelles enlarged. However, at 40 hours of growth, GFP labeled VACUOLES could be visualized in some of the hyphy indicating the activation of autophagy with further observance of GFP fluorescence and enlarged vacuoles at 60 hours.

In this experiment, a delta 80 G one mutant of p caressa Genum was used as a negative control. Note, the lack of vacuole enlargement and GFP, even at 60 hours of growth as a positive control, the autophagy stimulating drug rapamycin was used as observed with the untreated G-F-P-S-K-L strain. GFP labeled vacuoles became visible after 40 hours of growth with the Hy-Fi becoming completely filled with huge GFP containing vacuoles by 60 hours.

After watching this video, you should have a good understanding of how to analyze autophagy in mentis fungi.