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 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 ‘self-eating’) 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 ‘starvation pads’ 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.
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 ‘starvation pad’ 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’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., Δatg1 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 ‘SKL’ 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 ‘starvation pad’ method also for other filamentous fungi (e.g., Neurospora crassa, Sordaria macrospora, Aspergillus species, etc.).
1. Preparation of P. chrysogenum for the Starvation Experiments
2. Preparatory Steps for Cultivation of P. chrysogenum on Starvation Pads
3. Preparation of Microscope Slides Containing Starvation Pads
4. Inoculating the Starvation Pads with P. chrysogenum and Incubation
5. Microscopic Analysis of the Samples
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 it labels these organelles 10. Therefore the two parameters for observing autophagy (pexophagy) in this strain, vacuole enlargement and localization of GFP-SKL to vacuoles, are conveniently monitored by fluorescence microscopy.
At 20 hr of incubation, GFP is never observed in vacuoles (Figure 1). Also, vacuole enlargement is not taking place. However, at 40 hr of growth GFP-labelled vacuoles become visible in some of the hyphae, indicating autophagy (pexophagy) to become active (Figure 1). At 60 hr of growth the amount of GFP-labelled vacuoles has increased further. As a negative control a Δatg1 mutant of P. chrysogenum was utilized (Δatg1 (GFP-SKL)) (Figure 2). Neither enlarged nor GFP-labelled vacuoles can be observed in this strain. As a positive control, the autophagy-stimulating drug rapamycin is used (Figure 3). After 40 hr of growth GFP-labelled vacuoles become visible. At 60 hr, the hyphae are completely filled with huge GFP-containing vacuoles (Figure 3). This finding indicates that, as expected, massive autophagy takes place here. Collectively, these results demonstrate the validity of the experimental set-up.
Figure 1: Localization of GFP-SKL in Ws54-1255 (GFP-SKL) during cultivation on starvation pads. At the indicated times, cultures grown on starvation pads were analyzed using fluorescence microscopy. Representative images are shown for each time point. Corresponding bright field areas are shown below each fluorescence channel image. White “v”: GFP-SKL localized to vacuoles indicating peroxisome degradation. Scale bars: 10 µm.
Figure 2: Localization of GFP-SKL in Δatg1 (GFP-SKL) during cultivation on starvation pads. At the indicated times, cultures grown on starvation pads were analyzed using fluorescence microscopy. Representative images are shown for each time point. Corresponding bright field areas are shown below each fluorescence channel image. Scale bars: 10 µm.
Figure 3: Localization of GFP-SKL in Ws54-1255 (GFP-SKL) treated with rapamycin for the induction of autophagy. At the indicated times cultures grown on starvation pads supplemented with 1 µM rapamycin were analyzed using fluorescence microscopy. Representative images are shown for each time point. Corresponding bright field areas are shown below each fluorescence channel image. White “v”: GFP-SKL localized to vacuoles indicating peroxisome degradation. Scale bars: 10 µm.
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 possible to use mycelia grown on starvation pads for analysis in more sophisticated microscopes (e. g., confocal laser scanning microscopes). It does not matter whether the sample is put below the objective of above the objective (inverted microscope). Various fluorescent proteins targeted to certain compartments can be used to monitor the fate of different organelles during autophagy (e.g., mitochondria -> mitophagy; endoplasmic reticulum -> ER-phagy, etc.). In summary, the technique described here extends the spectrum of methods for microscopically studying fungi 12.
The protocol is generally straightforward and easy to use. However, it is very important that the starvation pads never desiccate as this will result in artefacts and cell death. Therefore it is strongly suggested that (i) the microscope slides containing the still liquid agarose solution are removed immediately from the heating plate (step 3.3), (ii) that the cover slip for making a flat surface is removed from the starvation pad after a maximum of 5 min (steps 3.3 and 3.4) , (iii) that the microscope slide is always kept in the wet chamber when not analyzed (steps 3.5 and 5.4) and (iv) that microscopic analysis does not take more than 15 min (step 5.3). Another important aspect is that the starting amount of spores or mycelial fragments pipetted onto the starvation pad should be not too high or too low. This can be easily controlled by dilution of the stock by using sterile tap water. The 1/50 dilution described in 1.1.3 works well for P. chrysogenum spore suspensions. It is the recommended dilution for use with our fungi but might require some adjustments.
A limitation of the method is that it is not advisable for time-lapse imaging experiments for more than 15 min. This is due to the gradual desiccation of the starvation pad once it is removed from the wet chamber. If longer observation times are needed it is possible to add small amounts of sterile tap water to the fringe of the starvation pad although this does not prevent some desiccation in central regions of the pad. Another limitation is that the method is not strictly quantitative. In unicellular organisms it is possible to score events per cell (e.g., amount of cells containing GFP in the vacuole). In contrast, P. chrysogenum as a filamentous fungus is characterized by a multicellular architecture. Cells are divided by septae which contain a central pore that allows the passage of cytoplasm and organelles. Therefore samples can be only analyzed ‘semi quantitatively’ (e.g., number of hyphae containing GFP localized to vacuoles). To achieve significance it is very important that the number of hyphae analyzed is high enough (at least 80 per time point and condition tested but more is definitively preferable).
The utilization of starvation pads for studying autophagy can be easily adapted for use in other filamentous fungi. Therefore the protocol presented here is not only limited to P. chrysogenum. It should also be possible to adapt it for with single-celled fungi, i.e. yeasts. Transfer of the protocol to higher organisms (e.g., nematodes) probably requires more specialized adaptations. It will be interesting to see whether the method described in this work also finds its use in other fields.
The authors have nothing to disclose.
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.
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Microscope slides with central cavity | Carl Roth GmbH, Karlsruhe, Germany | H884.1 | These can be used multiple times after cleaning. |
Glass beads | Carl Roth GmbH, Karlsruhe, Germany | A553.1 | Diameter: 0.25 – 0.50 mm |