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