Molecular Architecture of Fungi

MOLECULAR

ARCHITECHTURE

OF FUNGI

MOLECULAR ARCHITECTURE

Describe subcellular structure

according to the kinds and

juxtaposition of molecules that

compose these structures.

Composition, functions and

interactions of the major organelles

Similarities of fungi to other organisms

and their differences

Molecular cytologist

To describe a working model

of the fungi at the molecular

level so that predictions can

be made of their behaviour

Why are fungi as ideal

organisms among the

eukaryotic microbes for studies

of subcellular structure?

METHOD OF ORGANELLE ISOLATION

Many techniques of special interest to particular organelles

Generally

Breakage of hyphae to release organelles

Differential centrifugation

Density centrifugation

To separate organelles into suitable quantities to study.

HOMOGENIZATION PROCEDURE

Breakage of hyphae is major problem that must be solved for each fungus on an individual basis.

Obtain a reasonable degree of breakage

Organelles released early in the homogenization may be destroyed very quickly

3 FACTORS TO PROTECT ORGANELLES

AFTER THEIR RELEASE:

1. Protection from mechanical

disruption by the cell breakage

process

2. Osmotic protection from

suspending buffer

3. Protection from enzymatic

degradation

Forces generated to shear, crush

or explode the hyphae

Generate large amounts of heat

Control temperature, prepare cold or

near 0°C.

Choice of breakage procedure

Reduce the time of

homogenization

Increase viscosity of the medium

CELL BREAKAGE APPARATUS:

1. Mortar and pestle – simplest

2. Blender – rotating blades

3. Rotating element within a static

element

Develop greater shear forces

With or without abrasives, sand or

glass beads

4. Ultrasonic generators

Use high frequency sound to create

vibrations within the solution

ENZYMATIC DEGRADATION

Involves attacking the wall with

digestive enzymes from other

microorganisms.

Results in wall-less protoplasts that

can be gently broken

Gentle but slow technique

Suitable for general descriptive work

DIFFERENTIAL CENTRIFUGATION

Separate organelles on the basis of mass and shape

Larger organelles heavierSmall organelles require high

gravitational forcesBroken organelles e.g. nuclei and

mitochondria – do not sediment at the same speeds as whole organelles contaminate preparations of smaller structures.

DENSITY GRADIENT

CENTRIFUGATION

May yield better results

Different structures sediment only part

way down the tube, forming bands or

zones

Collected by separating the contents

of the centrifuge tube into fractions

according to their level.

Step gradient and continuous gradient

Density Gradient Centrifugation

STEP GRADIENTS

Made from solutions of

sucrose, sorbitol or other solutes that

dissolve readily in water to make

solutions of different densities and

viscosities.

Consist of two or more layers of higher

concentrations at the bottom, next till

the tube is full.

CONTINUOUS GRADIENT

Provide finer separations

Density changes gradually form bottom

to top of the centrifuge tube

Because sucrose solutions are very

viscous, little mixing between adjacent

regions in the gradient occurs.

Sucrose concentration increases

accordingly with depth in the tube –

slowing the rate of particles sediment

ISOPYCNIC CENTRIFUGATION

Forms bands of particles at different

levels in the gradient depending on

their buoyant densities

CELL WALL

Contain recognition factors on its surface that are involve in protection, interaction and shape & rigidity

Synthases and hydrolases (e.g. lipases, cellulases)

Some wall components serve as storage reserves

Vital living components of the fungus

Cell surface – 3 contiguous

interconnected matrices:

An exocellular component

A wall component

Plasmalemma

CELL WALL COMPOSITION

Polysaccharide

May be measured as specific compounds

Eg. Chitin, cellulose

Cell wall substance can be fractionated

by a series of selective extractions and

precipitations

Alkali-soluble fraction

Glycan, heteroglycans and glycoprotein

Alkali-insoluble fraction

chitin and/or cellulose and insoluble

glucan

Chitin, cellulose and β-glucan form

fibrillar networks

strength and forms of fungi

Rhizidiomyces and members of

Ceratocystis

cell wall contains chitin and

cellulose

Distribution of various other wall

polysaccharides – taxonomic

significance

Useful phylogenetic markers

SKELETAL POLYSACCHARIDES

Inner wall layers of hyphae and yeast cells contain the materials that remain after extractions with alkali and acid

Microcrystalline fibrils

All β-linked polysaccharides: cellulose, mannose and xylose

This allows the molecules to form straight fibers

Others – helical molecules

EXTRACTABLE GLYCANS

Extraction of the purified wall fraction

with alkali (eg. KOH) yields a mixture

of polysaccharide and proteinaceous

materials

Glycoprotein and minor sugar

monomers that are associated with

glycoprotein :

Mannose, fructose, galactose etc.

Acidic polysaccharides – major

components of hyphal walls of

zygomycetes fungi and characteristic

components of exocellular secretions and

fruiting bodies of Basidiomycetes

Mucor: glucuronic acid, fucose and

mannose

Tremella and Cyrptococcus: glucuronic

acid, xylose and mannose

GLYCOPROTEIN

Peptidopolysaccharides

A Polypeptide backbone with

polysaccharide branches

Containing known sugar monomers:

mannoprotein contain mannose

Peptidogalactomannan contain

galactose and mannan

IMPORTANT FUNCTIONAL ELEMENTS

1. Enzymes

2. Structural components

3. Regulators of cell contact

interactions (mating)

4. Principal immunogenic materials of

the cell surface

LIPID

Lipid content varied considerably

up to 19% of the dry weight of the wall

fractions

Triglyceride, phospholipid and sterol

as wall components.

May act to conserve water in aerially

dispersed spores.

PLASMALEMMA

Fungal membranes had the same general

structures as other biological membranes

Fluid mosaic

The enzymatic component of the membrane was

consistent with its role as the mediator.

include the H+ - ATPase (important in transport

process) and chitin synthetase

Most components of fungal membranes were

similar to those of animals.

ENDOPLASMIC RETICULUM

Membranes of the ER may have ribosomes associated with them RER or lack ribosomes SER.

ER receives proteins destined both for secretion and for vacuoles and carries out the first step in their glycosylation.

Function include facilitate protein folding and transport of synthesized proteins in sacs called cisternae.

the facilitation of protein folding and the

transport of synthesized proteins in sacs

called cisternae.

GOLGI APPARATUS

The GA of Oomycete fungi is

recognizable as stacks of flattened

cisternae with vesicular margins –

dictyosomes or Golgi bodies.

But in Zygomycetes, Ascomycetes and

Basidiomycetes dictyosomes are

reduced to one or a few element of

membrane related to the ER and

associated vesicles.

the sorting and processing of proteinsdestined for secretion from eukaryoticcells. In filamentous fungi, organization ofthe Golgi apparatus reflects the uniquechallenges brought about by the highlypolarized nature of hyphal growth.

a spatially organized and dynamic Golgiapparatus represents an adaptation that isas important for hyphal extension

Golgi apparatus

GOLGI APPARATUS FUNCTION

1. Molecules come in vesicles

2. Vesicles fuse with Golgi membrane

3. Molecules may be modified by

Golgi

4. Molecules pinched-off in separate

vesicle

5. Vesicle leaves Golgi apparatus

6. Vesicles may combine with plasma

membrane to secrete contents

VESICLESGrowing hyphal tips of all fungi have a

system of vesicles concentrated in the tips – vesicles that originate from GA.

Have been shown by ultra-structural localization techniques to contain protein, polysaccharides and phosphatases similar to those in the Golgi cisternae.

Delivery systemVesicles can fuse with plasma membrane

when they want to release their contents

VACUOLES

observed in older parts of hyphae

in filamentous fungi

Three distinct functions

Storage of N and P

Packaging and secretion of

hydrolytic enzymes

Synthesis and secretion of

extracellular polysaccharides

VACUOLES - store and recycle

cellular metabolites, e.g. enzymes and

nutrients.

MICROBODIES

Several similar organelles range in size

from 0.1 – 1.7 μm with diverse functions

Peroxixomes - Microbodies containing

H2O2-producing oxidases and catalases

Glyoxysomes - fatty acid β-oxidation

enzymes and enzymes of the glyoxylate

cycle

Hydrogenosomes - Containing

hydrogenase and associated enzymes

(anaerobic obligate)

MITOCHONDRIA

Highly variable organelles that

change in form, chemical

composition and functional

abilities

Location

developmental stage

conditions of growth

Doubled-

membraned

organelles with

a smooth outer

membrane and

a convoluted

inner

membrane that

produces

folded, platelik

e, or tubular

cristae within

Cristae – tubular or platelike

Platelike – chitinous walledfungi(Chytridiomycetes, Zygomycetes, Ascomycetes andBasidomycetes)

Tubular – cellulosic and wall-less fungi

(Oomycetes and Myxomycetes)

DNA containing organelle

mtDNA located in a central fibrous region of the matrix

Coded for the mitochondrial ribosomal RNA and for several tRNA molecules.

Mitochondrial ribosomes were distinct from the cytosolic ribosomes

Smaller, smaller RNA molecules different base compositions.

Insensitive to cycloheximide but sensitive to chloramphenicol

Mitochondria serve as the powerhouse

of the cell

located outside the nucleus

generate most of the cell's supply of

adenosine triphosphate (ATP)

The production of ATP is accomplished

by oxidizing the major products of

glucose, pyruvate, and NADH, which

are produced in the cytosol

(Akao et al., 2001; Dahout-Gonzalez et al., 2006;

Garlid et al., 2003; Herrmann and Neupert 2000).

Enzymes of TCA

cycle, ribosomes, other protein

synthesis machinery, DNA, fatty

acid oxidation other than

succinic dehydrogenase are in

the matrix.

play a significant role in

signaling, cellular

differentiation, cell death, as

well as the control of the cell

cycle and cell growth (Anderson et al., 1981; Chipuk et al., 2006;

Mannella 2006; Rappaport et al., 1998).

RIBOSOMES

• about 60% RNA and 40%

• protein.

• They have a sedimentation rate of

80S, and their subunits have

sedimentation rates of 60S and 40S.

• assembled in the nucleoli of the

nucleus.

• All ribosomes provide sites for protein

synthesis

• some are arranged in chains as

polyribosomes

• Attached-endoplasmic reticulum

usually make proteins for

secretion from the cell

• free in the cytoplasm usually make

proteins for use in the cell

CYTOSKELETON

A network of protein fibers

made of microtubule

(hollow tubes) and

microfilaments

(filamentous fibers).

Maintains cell shape

Anchors organelles and

proteins

Allows for organelle movement

and cellular movement in some

cell types

Fungal NUCLEUS•1--5 m diameter

•> 1 pg DNA

•Up to 13--40 Mb (million base

pairs) DNA coding for 6,000 to

13,000 genes

•Intra-nuclear division--nuclear

envelope remains intact during

mitosis (unlike plants and

animals)

Isolation of fungal nuclei - difficult task

that generally results in low yield (10%

or less intact nuclei

Because of the difficulties to break hyphal

wall gently enough to preserve nuclear

structure

Nuclear structure partially stabilized by

sucrose, divalent ions and slightly basic

buffer (tris at pH 7.5-8)

1. Carrier of genetic material

DNA + protein = chromatin

2. Governs cell activities

3. Directs cell reproduction

4. Surrounded by Membrane =

nuclear envelope

5. Contains nucleolus—produces

ribosomes

which synthesize proteins