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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