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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+quality+control:+an+integrated+network+of+pathways.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+filamentous+fungi.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina:+a+model+organism+to+study+mechanisms+of+healthy+ageing.">Podospora anserina: a model organism to study mechanisms of healthy ageing.</a> Mol. Genet. Genomics. 280 (5), 365-374 (2008).</li><li>Pinan-Lucarr&#233;, B., Iraqui, I., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina+target+of+rapamycin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+ATG8+homologue+Aoatg8+and+role+of+autophagy+in+differentiation+and+germination+in+Aspergillus+oryzae.">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-Lucarr&#233;, B., Balguerie, A., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+cell+death+in+Podospora+autophagy+mutants.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+fungal+cell+death+is+necessary+for+infection+by+the+rice+blast+fungus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+deficiency+promotes+beta-lactam+production+in+Penicillium+chrysogenum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomes+are+required+for+efficient+penicillin+biosynthesis+in+Penicillium+chrysogenum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding,+improving+and+using+green+fluorescent+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+regulation+of+autophagy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+living+cells+of+Aspergillus+in+vitro.">Imaging living cells of Aspergillus in vitro.</a> Med. Mycol. 47, 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 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 -&#62; mitophagy; endoplasmic reticulum -&#62; 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 &#8216;semi quantitatively&#8217; (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.

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

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 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 &#916;atg1 mutant of P. chrysogenum was utilized (&#916;atg1 (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.
<img alt="Figure 1" src="/files/ftp_upload/52577/52577fig1highres.jpg" /> 
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 &#8220;v&#8221;: GFP-SKL localized to vacuoles indicating peroxisome degradation. Scale bars: 10 &#181;m.
<img alt="Figure 2" src="/files/ftp_upload/52577/52577fig2highres.jpg" /> 
Figure 2: Localization of GFP-SKL in &#916;atg1 (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 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/52577/52577fig3highres.jpg" /> 
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 &#181;M 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 &#8220;v&#8221;: GFP-SKL localized to vacuoles indicating peroxisome degradation. Scale bars: 10 &#181;m.

Watch this Scientific Journal Video about Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy at JoVE.com

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

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.

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 ...

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Biology

8.5K Views.  Senckenberg Research Institute. 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.

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

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Immunology and Infection

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Neuroscience

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

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

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Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Study of Protein-protein Interactions in Autophagy Research

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body