تحليل الالتهام الذاتي في<em> البنسليوم العسجدية</em> باستخدام التجويع وسادات في تركيبة مع المجهر مضان

Summary

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.

Abstract

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.

Introduction

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 starvation^1,2,3.

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.).

Protocol

1. Preparation of P. chrysogenum for the Starvation Experiments If the P. chrysogenum strain of interest is kept on rice (‘green rice’), place 2-3 rice grains covered with sporulating mycelium into a 1.5 ml microcentrifuge tube. Fill it with 500 µl YGG (10 g/l KCl, 20 g/l glucose, 10 g/l yeast nitrogen base, 5 g/l K2HPO4, 20 g/l yeast extract). Vortex the tube for 30 sec so that spores can detach from the rice efficiently. Incubat…

Representative Results

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 …

Discussion

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…

Materials

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