IMPRS-gBGC workshop: Analytical Techniques in Microscopy Supervisors: Katrin Krause & Elke-Martina Jung September 13th, 2016 09.00-10.00 L Introduction fungi and fluorescence microscopy 10.00-12.00 P (Immuno)fluorescence staining 13.00-16.00 D, P Fluorescence and laserscanning microscopy Mycena chlorophos, wikipedia.org Panellus stipticus, wikipedia.org Introduction fungi Katrin Krause, Elke-Martina Jung IMPRS-gBGC workshop: Analytical Techniques in Microbiology Characteristics • ca 1.5 million species, only 75,000 species have been described • eukaryotes: one or more nuclei • no chlorophyll • C heterotroph (chemo-organotroph) • absorb soluble nutrients through cell wall and plasma membrane • aerob (soil living fungi) • mainly immobile • storage compounds: glycogen, mannitol, trehalose and lipids (spores!) • structural cell wall polymers: mostly chitin and β-1,3-glucan • spores Form of cells Hyphae , hyphal network=mycelium Yeast (single cells) apical & invasive growth Candida albicans Structure of fungal hypha Aut=autolysis AVC=apical vesicle cluster Chlam=chlamydospore ER=endoplasmatic reticulum G=Golgi (equivalent) Gl=glycogen L=lipid M=mitochondria MT=microtubules MW=melanized wall N=nucleus P=plasmalemma R=ribosomes S=septum SP=septal plug V=vacuole W=wall Wo=Woronin body - aging of hyphae - 2-30 mm diameter, extension zone 40 mm/min - Spitzenkörper: actin microfilament cluster in AVC center Neurospora crassa, Deacon. 2006. Fungal biology Structure of a budding yeast BS=bud scar ER=endoplasmatc reticulum G=Golgi L=lipid body M=mitochondrion N=nucleus SPB=spindle pool body V=vesicle Vac=vacuole W=wall Saccharomyces cerevisiae, Deacon. 2006. Fungal biology Components of the wall & its architechture Distingt layers in subapical region of N. crassa (a) b-1,3-glucan & b-1,6-glucan (b) Glucoprotein reticulum in protein (c) Discrete protein layer (d) Chitin microfibrills in protein (e) Plasma membrane Deacon. 2006. Fungal biology Müller & Löffler, 1982 Diversity in living strategies symbionts plant parasites Xanthoria parietina Ustilago maydis saprobionts human pathogens Cyathus striatus Cryptococcus neoformans Fungal kingdom Dikarya Higher fungi True fungi Slime molds Tree of life - based on rRNA (18S, 5.8S, 28S) - true fungi and fungus-like organisms - Phylum Glomeromycota - Mitosporic fungi (deuteromycotina, „Fungi imperfecti) not known sexual stage http://www.bact.wisc.edu/themicrobialworld/Whittaker.jpg Slime molds – social amoebas Dictyostelium discoideum • single amoeba assembling in hunger signals • formation of a mobile „snail stage“, which is forming a sorokarp (fruiting body) • single cells, no plasmodia • in focus of research in development of multi-cellular organisms Zygomycota • non-septated, multi-nuclear hyphae Entomophthora aphidis (Entomophthorales) • haploid genome with aphids • cell wall: chitin/chitosan, b-1,3-glucan • no mobile cells (zoospores) Rhizopus stolonifer (Mucorales) on tomato Zygospore (sexual spore) Rhizopus nigricans Glomeromycota • relationship with other fungi unclear • AM fungi (arbuscular mycorrhiza) • associated with 70 % of plants • haustoria-like structures vesicle/arbuscules Mitrophora semilibera Ascomycota • yeast - filamentous fungi • 75 % of all described fungi • septated hyphae, septae with pores • cell wall: β-1,3-glucan and chitin Hook formation Ascogonium Ascogenous hyphae Trichogyne Antheridium Ascus Development from a hook to an ascus with ascospores Basidiomycota • ca. 30,000 species (37 %) • mainly terrestrial lifestyle • cell wall: chitin, glucan Agaricus campestris Classification of Basidiomycota 3 major subgroups Uredinomycetes with rust fungi e.g. Puccinia graminis Ustilaginomycetes with smut fungi e.g. Ustilago maydis Hymenomycetes with mushrooms, puff balls, jelly fungi Sexual development of basidiomycetes Schizophyllum commune (Kothe et al. 2003) In the main stage of life the mycelium of basidiomycota is a dikaryon. Model system Schizophyllum commune (Gemeiner Spaltblättling) A B C D E 0.5 cm Initials Primordia Fruiting bodies Fruiting bodies Lamellae with lengthwise split gills Schubert et al. 2004 Life cycle of ectomycorrhizal fungi http://ecomicro.univ-lyon1.fr Types of mycorrhiza Basidiowww.lh-lawe.de- & Ascomycota Glomeromycota www.bio.vu.nlBasidiomycota Basidiomycotaww.hinti.ch www. www.museums.orgAscomycota BasidiomycotaLegambientearcipelagosca Schwantes, 1996 ne.it Ectomycorrhiza DAPI stained nuclei Kothe et al. 2013 Ectomycorrhiza formation Wagner et al. 2016 N-terminus Hydrophobins c cc c c cc c C-terminus Wessels, J. G. H. (1996) Fungal Hydrophobins: proteins that function at an interface, Wessels, J. G. H. (1999) Fungi in Their Own Right, Fungal Genetics and Biology 27, 134-145 Trends in Plant Science 1, 9-15 soluble monomers & stable aggregates in 2 % SDS bei 100 °C Function of hydrophobins • protection (aerial mycelium, conidiospores) • pseudoparenchyme formation (fruiting bodies, air channels) • surface contact (pathogenesis, appressoria) Host specific mycorrhiza “right” “false” Tricholoma & pine: rhodamine-labeled hydrophobin tree Tr. & spruce: rhodamine-labeled hydrophobin Function of hydrophobins Wettability Sc3 wild type “knock-out” BASF Production of hydrophobic surfaces Decontamination of crude oil 2015 Metals, (secondary) plant metabolites, xenobiotics vacuole The ectomycorrhizal MATE transporter mte1 in yeast bright field DAPI Anti His merge mte1 wt bar = 1 µm wood Brown rot White rot Loss of stability and weight Structure is preserved cube breaks Bleached, lighter, fibrous and moldy Dark-brown color Demarcation lines www.chmltech.com Brown rot - Cellulose degradation Endo-b-1,4-glucanase Exo-b-1,4-glucanase http://www.pilzarbeitskreis.de Schwefelporling (Laetiporus sulphureus) Lärchenporling (Laricifomes officinalis) Echter Hausschwamm (Serpula lacrymans) b-1,4-glucosidase Fritsche, 1990 White rot – Lignin degradation D Trametes versicolor (Schmetterlingsporling) Phellinus (Feuerschwamm) Phanerochaete chrysosporium Fomes (Zunderschwamm) 3dimensional macro molecule from phenylpropanoid Schizophyllum commune units Pleurotus ostreatus (Austernseitling) A) Ether bonds, B) Biphenyl bonds, C) - Mushroom production Phenylcoumarin structure D) Lignin degrading enzyme system Polyporus species after Fritsche, 1990; http://www.gymbsb.de - Pencil production White rot by Schizophyllum commune Propidium jodide stained hyphae on apple tree wood Contamination of environment = Use of bioremediation - (Gianfreda & Rao, 2004) Persistence time of bioactive xenobiotics Fritsche, 1990 Bioremediation by basidiomycetes • ubiquitously distributed • cheap substrates e.g. agricultural waste • filamentous growth allows good penetration into the soil • constitutive enzymes (expression in nutrient limitation) (Gianfreda & Rao, 2004) Accumulation of polycyclic aromatic hydrocarbons (PAHs) Wang et al. 2009 10 m M benzene (Ben), naphthalene (NP), anthracene (Ant), pyrene (Py), benzo[ a ]pyrene (BaP), and indeno[1,2,3-cd]pyrene (IND) for 3 h. Fungal toxins - N containing secundary metabolites ( N-heterocycles, oxolanes, peptides, N-N compounds), sesqui- and triterpenes, anthrachinones - poisonings & magical religous ceremonies - 95 % of lethal fungal poisonings in ME - death cap (Amanita phalloides) Sudini et al. 2015 Aflatoxin of Aspergillus flavus on coconut medium Aflatoxin B1 Tetrapolar mating system A und B together: Clamp fusion, Fruiting body development, Spore formation A genes: Clamp formation conjugated nuclear division B genes: Nuclear migration Homogenous incompatibility: identical i. allele is inducing termination of karyogamy. Nuclear migration: control via B and pheromone response A≠B≠ A=B= A=B≠ A≠B= nuclear migration with no morphological „flat“ swallen hyphae, pseudoclamps, Barrage clamp formation, changes no aerial mycelium, reaction dikaryon, spores 20>nuclei compatible incompatible semicompatible semicompatible DAPI DAPI Anti-tub Anti-tub Intact microtubules are nessessary & speed of 11 mm/sec In the main stage of life the mycelium of Basidiomycetes is a dikaryon with clamps. 1 2 cone hook 3 4 5 clamp Schubert et al. 2004 6 chitin Anti-tub Septated mycelium with complicatedly structured pores Dolipore http://images.encarta.msn.com/xrefmedia/dencmed/targets/illus/ilt/T012705A.gif, http://cgdc3.igmors.u-psud.fr/microbiologie/basidiomycetes_fichiers/image003.jpg Septa in fungal hyphae Simple septa Dolipore-septa parenthosome Vesicular Woronin bodies Non-perforated Perforated Paxillus involutus, chitin staining Weber. 1993. Allgemeine Mykologie Wide of pores 15-900 nm – nucleus can pass Fluorescent staining cell structure reagents emission colour Filter Axioplan DNA (nucleus) DAPI 450 nm blue 02 Chitin (cell wall) Chalcofluor White 435 nm blue 02 Mitochondria MitoTracker 525 nm green 10 green 24 Actin RLP (Rhodamine 570 nm bright red 15 labelled phalloidin) dark red 24 Fluorescent staining Calcofluor DAPI + MitoTracker DAPI Immunofluorescent staining Direct: more speed (only one incubation) and simplicity several antibodies labelled with different fluorochromes reagents are more expensive and less … Indirect: primary non-conjugated antibody second antibody conjugated to a fluorochrome (FITC-goat anti-mouse, ...) more fluorochrome molecules per antigen & higher specificity more difficult with more than one antibody (cross-reaction) Antibodies for Indirect immunfluorescence staining (IIF) cell antibody 1 antibody 2 labelled colour structure with tubulin T9026 F4018 FITC green mouse goat actin A2066 C2306 Cy3 red sheep rabbit 12-43 LSM 780-Elyra Tubulin • tubulin