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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
Introduction fungi
Katrin Krause, Elke-Martina Jung IMPRS-gBGC workshop: Analytical Techniques in Microbiology
Mycena chlorophos, wikipedia.org Panellus stipticus, wikipedia.org
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
Hyphae , hyphal network=mycelium
apical & invasive growth
Yeast (single cells)
Candida albicans
Form of cells
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
Neurospora crassa, Deacon. 2006. Fungal biology
- aging of hyphae
- 2-30 mm diameter, extension zone 40 mm/min
- Spitzenkörper: actin microfilament cluster in AVC center
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
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 Müller & Löffler, 1982
Diversity in living strategies
Xanthoria parietina Ustilago maydis
symbionts plant parasites
saprobionts
Cyathus striatus Cryptococcus neoformans
human pathogens
http://www.bact.wisc.edu/themicrobialworld/Whittaker.jpg
Fungal kingdom
Slime molds
True fungi
Higher fungi
Dikarya
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
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
• haploid genome
• cell wall: chitin/chitosan, b-1,3-glucan
• no mobile cells (zoospores)
Rhizopus stolonifer (Mucorales)
on tomato
Entomophthora aphidis (Entomophthorales)
with aphids
Rhizopus nigricans
Zygospore (sexual spore)
Glomeromycota
• relationship with other fungi unclear
• AM fungi (arbuscular mycorrhiza)
• associated with 70 % of plants
• haustoria-like structures vesicle/arbuscules
Ascomycota
• yeast - filamentous fungi
• 75 % of all described fungi
• septated hyphae, septae with pores
• cell wall: β-1,3-glucan and chitin
Antheridium
Trichogyne
Ascogonium Ascogenous hyphae Hook formation
Ascus
Development from a hook to an ascus with ascospores
Mitrophora semilibera
Basidiomycota
• ca. 30,000 species (37 %)
• mainly terrestrial lifestyle
• cell wall: chitin, glucan
Agaricus campestris
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
Classification of Basidiomycota
Sexual development of basidiomycetes
(Kothe et al. 2003)
Schizophyllum commune
In the main stage of life the mycelium of basidiomycota is a dikaryon.
Model system Schizophyllum commune (Gemeiner Spaltblättling)
A B
0.5 cm
C D E
Schubert et al. 2004
Initials Primordia Fruiting bodies
Fruiting bodies Lamellae with
lengthwise split
gills
Life cycle of ectomycorrhizal fungi
http://ecomicro.univ-lyon1.fr
Schwantes, 1996
Types of mycorrhiza
Glomeromycota
ww.hinti.ch Basidiomycota
www.museums.org Ascomycota www.
Legambientearcipelagosca
ne.it
Basidiomycota
www.bio.vu.nl Basidiomycota
www.lh-lawe.de Basidio- & Ascomycota
Ectomycorrhiza
Kothe et al. 2013
DAPI stained nuclei
Ectomycorrhiza formation
Wagner et al. 2016
Hydrophobins
Wessels, J. G. H. (1996) Fungal Hydrophobins:proteins that function at an interface,
1, 9-15Trends in Plant Science Wessels, J. G. H. (1999) Fungi in Their Own Right, 27, 134-145Fungal Genetics and Biology
soluble monomers & stable aggregates in 2 % SDS bei 100 °C
c cc c c cc c
N-terminus
C-terminus
• protection (aerial mycelium, conidiospores)
• pseudoparenchyme formation (fruiting bodies, air channels)
• surface contact (pathogenesis, appressoria)
Function of hydrophobins
Host specific
mycorrhiza
“right”
“false”
tree Tricholoma & pine: rhodamine-labeled hydrophobin Tr. & spruce: rhodamine-labeled hydrophobin
Function of hydrophobins
Wettability
Sc3
wild type
“knock-out”
BASF
Production of hydrophobic surfaces
Decontamination of crude oil
Metals, (secondary) plant metabolites, xenobiotics
vacuole
2015
The ectomycorrhizal MATE transporter mte1 in yeast
bar = 1 µm
bright field DAPI Anti His merge
mte1
wt
wood
www.chmltech.com
Brown rot Loss of stability and weight
cube breaks
Dark-brown color
White rot Structure is preserved
Bleached, lighter, fibrous and moldy
Demarcation lines
Brown rot - Cellulose degradation
Fritsche, 1990
Endo-b-1,4-glucanase
Exo-b-1,4-glucanase
b-1,4-glucosidase
Schwefelporling (Laetiporus sulphureus)
Lärchenporling (Laricifomes officinalis)
Echter Hausschwamm (Serpula lacrymans)
http://www.pilzarbeitskreis.de
Trametes versicolor (Schmetterlingsporling)
Phellinus (Feuerschwamm)
Phanerochaete chrysosporium
Fomes (Zunderschwamm)
Schizophyllum commune
Pleurotus ostreatus (Austernseitling)
- Mushroom production
Polyporus species
- Pencil production
3dimensional macro molecule from phenylpropanoid
units
A) Ether bonds, B) Biphenyl bonds, C)
Phenylcoumarin structure
D) Lignin degrading enzyme system
after Fritsche, 1990; http://www.gymbsb.de
D
White rot – Lignin degradation
White rot by Schizophyllum commune
Propidium jodide stained hyphae on apple tree wood
Contamination of environment = Use of bioremediation
-
(Gianfreda & Rao, 2004)
Fritsche, 1990
Persistence time of bioactive xenobiotics
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
Sudini et al. 2015
- 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)
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.
A=B= A≠B≠ A=B≠
Nuclear migration: control via B and pheromone response
A≠B=
nuclear migration with
clamp formation,
dikaryon, spores
no morphological
changes
„flat“ swallen hyphae,
no aerial mycelium,
20>nuclei
pseudoclamps, Barrage
reaction
compatible incompatible semicompatible semicompatible
DAPI Anti-tub Anti-tub
DAPI
Intact microtubules are nessessary & speed of 11 mm/sec
In the main stage of life the mycelium of Basidiomycetes is
a dikaryon with clamps.
cone hook
clamp
1
2
3
4
5
6 Schubert et al. 2004
Anti-tub chitin
http://images.encarta.msn.com/xrefmedia/dencmed/targets/illus/ilt/T012705A.gif,
http://cgdc3.igmors.u-psud.fr/microbiologie/basidiomycetes_fichiers/image003.jpg
Septated mycelium with complicatedly structured
pores
Dolipore
Septa in fungal hyphae
Simple septa Dolipore-septa
parenthosome
Woronin bodies
Wide of pores 15-900 nm – nucleus can pass
Vesicular
Non-perforated
Perforated
Weber. 1993. Allgemeine Mykologie
Paxillus involutus, chitin staining
Fluorescent staining
cell structure reagents emission colour Filter Axioplan
DNA (nucleus)
Chitin (cell wall)
Mitochondria
DAPI
Chalcofluor White
MitoTracker
450 nm
435 nm
525 nm
blue
blue
green
green
02
02
10
24
Actin
RLP (Rhodamine
labelled phalloidin)
570 nm
bright red
dark red
15
24
Fluorescent staining
DAPI
Calcofluor
DAPI + MitoTracker
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
structure
antibody 1 antibody 2 labelled
with
colour
tubulin
actin
T9026
mouse
A2066
rabbit
F4018
goat
C2306
sheep
FITC
Cy3
green
red
12-43
LSM 780-Elyra
Tubulin
• tubulin forms long bundles along longitudinal axis during the cell
• microtubules are components of tip growth
• pathway for intracellular movement of organelles and proteins
Alberts et al., 2002
Localisation of actin
[Geitmann and Emons 2000]
• as plaques in filamentous cells
• aggregation on cell tips and septa
• part of the cytoskeleton, which allows motility
Immunofluorescent staining: anti-tub, anti-act & DAPI
Model of the cytosceleton in filamentous fungi
[G.Steinberg 2005]
Immunofluorescent staining: anti-tub, anti-act & DAPI
Autofluorescence
Spontaneous emission of light after excitation:
• porphyrins (red)
• lipids (lemon – dark yellow)
• chalk (white)
• dermoxanthon (Cortinarius: yellow green)
• aesculin (blue)
• protoberberin alcaloids (yellow)
• keratin (blue white)
• collagen (weak blue)
• elastin (white)
• cartilage (light blue)
http://www.chemie.uni-jena.de/institute/oc/weiss/naturstoffe.htm#
http://www.chemie.uni-jena.de/institute/oc/weiss/naturstoffe.htm#
Autofluorescence
• often emission in different wavelengths with lower intensity
• use negative controls (without 1st or 2nd antibody)
• use other filter sets (fluorescence microscopy) or
specific wavelengths (laser scanning microscopy)
DAPI staining of DNA/RNA
FITC stained hydrophobin
autofluorescence
bright field
Bioluminescent fungi
• 75 species of Agaricales (Basidiomycota) with one
exceptional ascomycete
• luciferase: production of light by the oxidation of
fungal luciferin sesquiterpene panal
• greenish light at 520–530 nm
Mycena chlorophos, wikipedia.org Panellus stipticus, wikipedia.org
Thank you for your attention!
Questions?
Practical