1. MOLECULAR
ARCHITECHTURE
OF FUNGI

2. 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

3. Molecular cytologist
 To describe a working model
of the fungi at the molecular
level so that predictions can
be made of their behaviour

4. Why are fungi as ideal
organisms among the
eukaryotic microbes for studies
of subcellular structure?

5. 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.

6. 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

7. 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

8. 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

9. 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

10. 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

11. DIFFERENTIAL CENTRIFUGATION
Separate organelles on the basis of
mass and shape
Larger organelles heavier
Small organelles require high
gravitational forces
Broken organelles e.g. nuclei and
mitochondria – do not sediment at the
same speeds as whole organelles
 contaminate preparations of
smaller structures.

14. 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

15. Density Gradient Centrifugation

16. 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.

17. 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

20. ISOPYCNIC CENTRIFUGATION
Forms bands of particles at different
levels in the gradient depending on
their buoyant densities

22. 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

23. Cell surface – 3 contiguous
interconnected matrices:
 An exocellular component
 A wall component
 Plasmalemma

24. 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

25. 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

26. 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

27. 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.

28. 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

29. GLYCOPROTEIN
Peptidopolysaccharides
 A Polypeptide backbone with
polysaccharide branches
Containing known sugar monomers:
mannoprotein contain mannose
Peptidogalactomannan contain
galactose and mannan

30. IMPORTANT FUNCTIONAL ELEMENTS
1. Enzymes
2. Structural components
3. Regulators of cell contact
interactions (mating)
4. Principal immunogenic materials of
the cell surface

31. 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.

32. 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.

33. 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.

34. the facilitation of protein folding and the
transport of synthesized proteins in sacs
called cisternae.

35. 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.

37. the sorting and processing of proteins
destined for secretion from eukaryotic
cells. In filamentous fungi, organization of
the Golgi apparatus reflects the unique
challenges brought about by the highly
polarized nature of hyphal growth.
a spatially organized and dynamic Golgi
apparatus represents an adaptation that is
as important for hyphal extension
Golgi apparatus

38. 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

39. VESICLES
Growing 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 system
Vesicles can fuse with plasma membrane
when they want to release their contents

41. 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

42. VACUOLES - store and recycle
cellular metabolites, e.g. enzymes and
nutrients.

45. 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)

46. MITOCHONDRIA
Highly variable organelles that
change in form, chemical
composition and functional
abilities
Location
developmental stage
conditions of growth

47. Doubled-
membraned
organelles with
a smooth outer
membrane and
a convoluted
inner
membrane that
produces
folded, platelik
e, or tubular
cristae within

48. Cristae – tubular or platelike
Platelike – chitinous walled
fungi
(Chytridiomycetes, Zygomycete
s, Ascomycetes and
Basidomycetes)
Tubular – cellulosic and wall-
less fungi
(Oomycetes and Myxomycetes)

49. 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

50. 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).

51. Enzymes of TCA
cycle, ribosomes, other protein
synthesis machinery, DNA, fatty
acid oxidation other than
succinic dehydrogenase are in
the matrix.

52. 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).

53. 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

54. • 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

55. CYTOSKELETON
A network of protein fibers
made of microtubule
(hollow tubes) and
microfilaments
(filamentous fibers).

56. Maintains cell shape
Anchors organelles and
proteins
Allows for organelle movement
and cellular movement in some
cell types

57. 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)

58. 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)

59. 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