Name: Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2015-02-01T15:00:00.000+00:00
Duration: 6 min 51 s
Description: Learn to induce and observe autophagy in Penicillium chrysogenum using starvation pads and fluorescence microscopy. This method tracks fluorescent marker translocation to vacuoles for cell biology research.

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5 minutes

CQS receives a fellowship from the LOEWE Excellence Cluster for Integrative Fungal Research (IPF). The author would like to thank Ida J. van der Klei for the P. chrysogenum strains used in this work and Andreas S. Reichert for the gift of rapamycin.

1. Preparation of P. chrysogenum for the Starvation Experiments
<ol>
	<li>If the P. chrysogenum strain of interest is kept on rice (&#8216;green rice&#8217;), place 2-3 rice grains covered with sporulating mycelium into a 1.5 ml microcentrifuge tube. Fill it with 500 &#181;l YGG (10 g/l KCl, 20 g/l glucose, 10 g/l yeast nitrogen base, 5 g/l K2HPO4, 20 g/l yeast extract).
	<ol>
		<li>Vortex the tube for 30 sec so that spores can detach from the rice efficiently.</li>
		<li>Incubate the tube for 1 day at room temperature (e.g., between 20 and 25 &#176;C).</li>
		<li>Prepare a 1/50 dilution of this spore suspension in sterile tap water, mix well.</li>
	</ol>
	</li>
	<li>If the P. chrysogenum strain of interest is kept on an agar plate (e.g., YGG agar plate), transfer small pieces of the mycelium into a 1.5 ml microcentrifuge tube containing 400 &#181;l sterile tap water and approximately 100 mg glass beads by using a sterile tooth pick or pipette tip.
	<ol>
		<li>Vortex the tube for 2 min so that the mycelium becomes fragmented. Use the contents of the tube immediately.</li>
	</ol>
	</li>
</ol>
2. Preparatory Steps for Cultivation of P. chrysogenum on Starvation Pads
<ol>
	<li>Switch on a heating plate and set it to approximately 60 &#176;C.</li>
	<li>Dissolve 2 g of agarose in 100 ml tap water by cooking the solution in a microwave oven. Take care that this solution is completely clear after heating. If it is not, cook it again until the agarose is dissolved completely. Let the agarose solution cool down to approximately 60 &#176;C before use.</li>
	<li>Fill 1.5 ml microcentrifuge tubes (2-4, depending on the number of samples) with 400 &#181;l sterile tap water each (no additional compounds added) or 400-x &#181;l (addition of x &#181;l of compound solution in step 2.5) and place them onto the heating plate for at least 1 min so that the water inside gets warm.</li>
	<li>Place microscope slides that contain a central cavity onto the heating plate for at least 1 min.</li>
	<li>Add 400 &#181;l 2 % agarose to the solution in the 1.5 ml microcentrifuge tubes and vortex briefly. After this step if desired, add additional compounds (i.e., 1 &#181;M rapamycin). If this is done, vortex briefly after each additional substance is pipetted to the tube. Put the tubes back onto the heating table to prevent premature solidification. 
	NOTE: The total volume in the microcentrifuge tube should be 800 &#181;l.</li>
</ol>
3. Preparation of Microscope Slides Containing Starvation Pads
<ol>
	<li>Pipette 140 &#181;l of the agarose solution into the cavity of the microscope slide (which is still located on the heating plate). Try to avoid making bubbles if possible.</li>
	<li>Immediately place a cover slip onto the agarose solution. 
	NOTE: This will result in a flat surface.</li>
	<li>Put the microscope slide onto the lab bench for approximately 4 min to allow the agarose pad to solidify.</li>
	<li>Carefully remove the coverslip from the agarose pad by sliding it off with the help of a thumb or index finger (wear gloves to avoid contamination!).</li>
	<li>Place the microscope slide with the pad into a wet chamber to prevent desiccation. Recommendation: Use an empty tip box that still has the insert for holding the pipette tips. Add sterile water to the box so that the bottom is covered. Place the microscope slides onto the insert and close the box.</li>
</ol>
4. Inoculating the Starvation Pads with P. chrysogenum and Incubation
<ol>
	<li>Pipette 5 &#181;l of the 1/50 spore dilution (if the cultures were grown on rice) or 5 &#181;l of the undiluted solution containing mycelium fragments (if the cultures were grown on an agar plate) onto the centre of the starvation pad.</li>
	<li>Close the wet chamber and wrap it in a plastic bag (this serves as additional protection against desiccation of the starvation pads). Be careful not to move the box too much because otherwise the inoculation drops will be disturbed.</li>
	<li>Store the wrapped box at RT for at least 20 h before analyzing the samples.</li>
</ol>
5. Microscopic Analysis of the Samples
<ol>
	<li>After 20 hr of incubation the samples are ready for microscopic analysis; put the microscope slide on dry tissue paper (the bottom tends to become wet).</li>
	<li>Pipette a small volume of water (ca. 50 &#181;l) onto the starvation pad. Put a coverslip onto the pad and squeeze out surplus water. Remove the surplus water with a tissue paper.</li>
	<li>Put the microscope slide onto the observation table of the fluorescence microscope. To get an overview of mycelium growth, use a 20X objective. Use 63X or 100X objectives for studying the localization of GFP in the hyphae. In order to prevent gradual desiccation of the sample do not analyze the samples for longer than 15 min before putting them back into the wet chamber.</li>
	<li>Repeat 5.3 at regular intervals (e.g., after 40 hr and 60 hr of growth). 
	NOTE: The sample can be put back into the wet chamber. It is not necessary to remove the coverslip as it does not affect mycelium growth.</li>
</ol>

<ol><li>Fischer, F., Hamann, A., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+quality+control%3A+an+integrated+network+of+pathways%5BTitle%5D)+AND+%22Trends+Biochem.+Sci%22%5BJournal%5D)">Mitochondrial quality control: an integrated network of pathways.</a> Trends Biochem. Sci. 37 (7), 284-292 (2012).</li>
<li>Pollack, J. K., Harris, S. D., Marten, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy+in+filamentous+fungi%5BTitle%5D)+AND+%22Fungal+Genet.+Biol%22%5BJournal%5D)">Autophagy in filamentous fungi.</a> Fungal Genet. Biol. 46 (1), 1-8 (2009).</li>
<li>Scheckhuber, C. Q., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Podospora+anserina%3A+a+model+organism+to+study+mechanisms+of+healthy+ageing%5BTitle%5D)+AND+%22Mol.+Genet.+Genomics%22%5BJournal%5D)">Podospora anserina: a model organism to study mechanisms of healthy ageing.</a> Mol. Genet. Genomics. 280 (5), 365-374 (2008).</li>
<li>Pinan-Lucarré, B., Iraqui, I., Clavé, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Podospora+anserina+target+of+rapamycin%5BTitle%5D)+AND+%22Curr.+Genet%22%5BJournal%5D)">Podospora anserina target of rapamycin.</a> Curr. Genet. 50 (1), 23-31 (2006).</li>
<li>Kikuma, T., Ohneda, M., Arioka, M., Kitamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+analysis+of+the+ATG8+homologue+Aoatg8+and+role+of+autophagy+in+differentiation+and+germination+in+Aspergillus+oryzae%5BTitle%5D)+AND+%22Eukaryot.+Cell%22%5BJournal%5D)">Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae.</a> Eukaryot. Cell. 5 (8), 1328-1336 (2006).</li>
<li>Pinan-Lucarré, B., Balguerie, A., Clavé, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accelerated+cell+death+in+Podospora+autophagy+mutants%5BTitle%5D)+AND+%22Eukaryot.+Cell%22%5BJournal%5D)">Accelerated cell death in Podospora autophagy mutants.</a> Eukaryot. Cell. 4 (11), 1765-1774 (2005).</li>
<li>Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagic+fungal+cell+death+is+necessary+for+infection+by+the+rice+blast+fungus%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Autophagic fungal cell death is necessary for infection by the rice blast fungus.</a> Science. 312 (5773), 580-583 (2006).</li>
<li>Bartoszewska, M., Kiel, J. A., Bovenberg, R. A., Veenhuis, M., van der Klei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy+deficiency+promotes+beta-lactam+production+in+Penicillium+chrysogenum%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 77 (4), 1413-1422 (2011).</li>
<li>Meijer, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peroxisomes+are+required+for+efficient+penicillin+biosynthesis+in+Penicillium+chrysogenum%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 76 (17), 5702-5709 (2010).</li>
<li>Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding%2C+improving+and+using+green+fluorescent+proteins%5BTitle%5D)+AND+%22Trends+Biochem.+Sci%22%5BJournal%5D)">Understanding, improving and using green fluorescent proteins.</a> Trends Biochem. Sci. 20 (11), 448-455 (1995).</li>
<li>Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., Kim, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTOR+regulation+of+autophagy%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">mTOR regulation of autophagy.</a> FEBS Lett. 584 (7), 1287-1295 (2010).</li>
<li>Hickey, P. C., Read, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+living+cells+of+Aspergillus+in+vitro%5BTitle%5D)+AND+%22Med.+Mycol%22%5BJournal%5D)">Imaging living cells of Aspergillus in vitro.</a> Med. Mycol. 47, Suppl 1. S110-S119 (2009).</li>
</ol>

The author has nothing to disclose.

The method presented here allows the convenient and reproducible study of autophagy in P. chrysogenum. For example, it can be used for screening the efficacy of various compounds whether they are capable of modulating the autophagy response of this fungus or not. The results with rapamycin demonstrate that inhibition of TOR signaling leads to a pronounced induction of autophagy in P. chrysogenum which has also been demonstrated for other organisms 11.
It is possibl...

Filamentous fungi are excellent model systems for the study of developmental processes. They offer several experimental benefits like cheap cultivation, high numbers of progeny and genetic accessibility. The last point is of particular relevance for the construction of transformants that allow investigating the importance of so-far uncharacterized genes for various cellular mechanisms. Filamentous fungi have been instrumental for the elucidation of several elements and mechanisms of cellular quality control pathways like protease activity for the degradation of aberrant proteins, mitochondrial dynamics for maintaining mitochondrial integrity and autophagy for the removal of surplus and/or dysfunctional cell components and for maintaining cell viability in times of starvation1,2,3.
There are several experimental techniques available for the study of autophagy in filamentous fungi 2: (i) investigation of vacuoles if they contain dense autophagic bodies when proteases are inhibited by transmission electron microscopy 4, (ii) visualization of autophagosomes by monitoring GFP-Atg8 foci via fluorescence microscopy 5,6 and (iii) detection of acidified autophagosomal structures by using the fluorescent dye monodansyl cadaverine 7.
Here, a novel method of growing Penicillium chrysogenum for autophagy studies is presented. The main element is the &#8216;starvation pad&#8217; which simply consists of 1 % agarose dissolved in sterilized tap water. Additional compounds (e. g., stressors, scavengers, autophagy modulators) can be added to the pad as long as they don&#8217;t display auto-fluorescence. The pad is located in microscope slides that contain a shallow central cavity. This pad in inoculated either with a spore suspension or with small mycelium fragments. The latter is advisable if the strain of interest fails to sporulate efficiently (e.g., &#916;atg1 strains 8). The slides are positioned in wet chambers (these can be easily constructed by using empty pipette tip boxes) to prevent desiccation of the sample and incubated at room temperature. P. chrysogenum is able to grow for a few days under these conditions. Autophagy can be observed microscopically by vacuolar enlargement which is a positive marker for fungal autophagy. In this contribution, a P. chrysogenum strain (Wisconsin 54-1255) is used that forms green fluorescent protein that is targeted to peroxisomes by its C-terminal &#8216;SKL&#8217; sequence 9. Therefore it is possible to monitor the degradation of peroxisomes. It is feasible to label also other compartments of the cell (e.g., mitochondria) by using appropriate localization signals and to analyze their degradation. Although data from P. chrysogenum Ws54-1255 (GFP-SKL) is presented here, it is certainly possible to use the &#8216;starvation pad&#8217; method also for other filamentous fungi (e.g., Neurospora crassa, Sordaria macrospora, Aspergillus species, etc.).

<table><tbody><tr><td>Microscope slides with central cavity</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>H884.1</td><td>These can be used multiple times after cleaning.</td></tr><tr><td>Glass beads</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>A553.1</td><td>Diameter: 0.25 - 0.50 mm</td></tr></tbody></table>

analysis of autophagy in penicillium chrysogenum by using starvation pads in combination with fluorescence microscopy

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

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

To demonstrate the utility of the protocol detailed above peroxisome degradation in the P. chrysogenum strain Ws54-1255 (GFP-SKL) was analyzed. In this strain GFP-SKL is usually imported into peroxisomes 9. This results in the appearance of multiple spherical shapes when the sample is analyzed via fluorescence microscopy. If autophagy occurs, vacuoles enlarge. GFP-SKL becomes incorporated into vacuoles by autophagy (pexophagy). Due to the fact that GFP is resistant to degradation by vacuole proteases ...

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

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.

analysis-autophagy-penicillium-chrysogenum-using-starvation-pads

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Learn to induce and observe autophagy in Penicillium chrysogenum using starvation pads and fluorescence microscopy. This method tracks fluorescent marker translocation to vacuoles for cell biology research.

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

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