lecture 3+4: molecular architecture of cells and hyphae

why is hyphae tough?

interlaced chitin microfibrils provide strength to withstand 7x pressure of car tires

* can be utilized as an environmentally sustainable material

hyphae and fungal nutrition

* strict heterotrophs through passive/active absorption
* secretion of digestion enzymes to hydrolyze complex sugars
* proteases, lipases, CAZymes
* secretion of secondary metabolites (like toxins) to defend food

zones of the hyphal tips (4)

1. __**secretion/apical growth zone**__: where enzymes are manufactured and excreted through the plasma membrane
2. __**absorption zone**__: one/two cell(s) after secretion zone where nutrient uptake occurs
3. __**storage zone**__: where excess sugar is stored as glycogen
4. __**senescence zone**__: old parts of the mycelium void of cytoplasm and organelles (usually separated by a septum)

why is exploration and invasion important for fungal growth?

compartmentalization of the hyphal tip (absorption zone after secretion zone) means apical extension must occur in order for the fungus to survive

* enzymes secreted at the apex
* secondary metabolites excretion and absorption occur sub-apically
* formation of lateral hyphae branches to maximize exploration
* nutrient-poor: go further out (exploration)
* nutrient-rich: more lateral/radial growth (invasion)

radial growth

movement out and away from erosion (nutrient depleted) zones is what gives the typical colony shape

fungal colony differentiation

distribution of biomass / growth patterns vary with age

* center = aging zone: where asexual spores are formed
* edges = peripheral zone: where nutrients are absorbed and growth occurs

structural features in the fungal hypha components (8)

* cell wall
* septa (in some)
* plasma membrane + associated proteins and pathways
* cytoskeleton
* secretory pathway
* cytoskeleton
* cytoplasmic organelles
* nucleus + nuclear content

functions of the fungal cell wall (6)

* structural barrier
* porous and permeable
* acts as a binding site for enzymes and glycoproteins
* enables interactions with other organisms
* determines growth patterns
* can be targetted by natural host defenses

components of the fungal cell wall (2, 3)

inner layer: for shape and strength

* glucans → β-1,3 glucans (dominant wall polymer), β-1,6 glucans and α-1,3 glucans (for crosslinking for yeasts and filamentous fungi respectively)
* chitin microfibrils: β-1,4 linked monomers of N-acetylglucosamine
* link to β-1,3 glucan to scaffold for glycoproteins

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outer layer: key properties of the cell wall

* melanin: provides rigidity and UV protection
* mannans (yeast) and/or galactomannans (filaments)
* short mannans decorating glycoproteins
* long mannan chains
* cell wall proteins: mannoproteins, glycoproteins, and non-glycoproteins

cell wall composition among taxonomic groups

dynamic → can vary greatly among fungal species

* Candida: chitin, β-1,3 glucan, β-1,6 glucan, and mannan
* C. neoformans: chitin, β-1,3 glucan, β-1,6 glucan, and a thick capsule

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can also vary among different life stages

* conidia have a lot of melanin for protection
* dimorphic fungi with yeast and filamentous forms

why do we care about the fungal cell wall?

1. **defines morphogenesis and is key to dimorphic switching**


1. dimorphic switches usually determine pathogenicity in humans and plants
2. remodeling also facilitates changes in other types of cell morphologies (i.e. sexual/asexual spores)
2. **important for stress adaptation and virulence**


1. provides mechanical strength to fungal cell
2. rapid changes in the cell wall lead to rapid adaptation
3. mediated by the cell wall integrity pathway (conserved in Fungi)
4. dihydroxy naphthalene melanin found appressorium cell wall
3. **important target for antifungal compounds**


1. many enzymes target fungal cell wall synthesis
4. **activator of innate immunity in plants and animals (target for plant defense response)**


1. oligomers of β-1,3 glucans induce innate immune defenses
2. development of mechanisms to avoid host response


1. secretion of CW-associated effector proteins to hide polymers or protect from host enzymes
2. deacetylation of chitin to chitosan

three major classes of antifungals that target cell wall synthesis

1. inhibitors of β-1,3 glucan synthesis: trigger osmotic lysis by inhibiting function of glucan synthase


1. echinocandins, enfumafungin, papulacandins
2. inhibitors of chitin synthesis: binds active site of chitin synthase


1. nikkomycins, polyoxins
3. inhibitors of melanin bio-synthesis


1. MBI-R, MBI-D, MBI-P

fungal septa

hyphae are divided by cross-walls = septae

* no septae = coenocytic
* formed as a process of hyphal extension
* have pores to exchange cytoplasm, organelles, and nuclei
* simple septa: extends inward
* dolipore septa: thickened at the cap to form a swelling “doliolum”

functions of septa

* differentiation and compartmentalization
* prevent cytoplasm loss
* during cell damage, a woronin body serves as a plug in the septa pore
* define position of hyphal growth
* septation → conidiation
* separates mother and daughter cells in budding yeast

why is the fungal secretory system important?

1. model system to study protein secretion and modification
2. crucial for virulence in pathogenic fungi
3. protein production at an industrial scale

conventional secretory pathway components(3)

CSP directs proteins, enzymes, and lipids to the plasma membrane and consists of


1. endoplasmic reticulum (ER): protein modification


1. rough ER for protein synthesis
2. smooth ER for lipid and steroid synthesis
2. golgi apparatus: addition protein modification and peptide processing
3. membrane-bound secretory vesicles: carry enzymes and other proteins to be secreted

CSP steps (1+4)

mRNA exits nucleolus and is captured by ribosomes for translation


1. polypeptide transfer from ribosome to ER to folding/modification
2. transportation in vesicles of the folded protein to the golgi appartus
3. passing through the cisternae for further modification/quality control
4. packaging of protein into vesicles for transportation to plasma membrane (secretion) or lysosomes (degradation)

fungal cytoskeleton composition

1. microtubules and motor protein
2. actin
3. apical vesicular complex and the spitzenkorpor

what are the motor proteins of microtubules?

* kinesin: transports secretory vesicles on microtubules toward hyphal tip (anterograde \[+\] direction)
* dynein: involved in endosome transport further into the cell (retrograde \[-\] direction)
* class V myosin: F-actin → functions as linkers between membranes and actin cytoskeleton

microtubules

1. polymers of α and β tubulin
2. orientation of the polymers determines the polarity of microtubules (+ and - ends)
3. provides tracks for intracellular transport of vesicles, RNA, protein, and organelles

actin

* F-actin is made of 2 globular (G) actin assembled into a double helix
* polymerization of F actin is mediated by forming
* crucial role in exocytosis, endocytosis, organelle movement, and cytokinesis

apical vesicular complex and the spitzenkorpor

* present only in hyphal tips of growing hyphae and sites of spore germination and branch formation
* mainly consists of dense cluster of vesicles which accumulate at the spitzenkorpor
* location where actin filaments receive vesicles and transport from microtubules to class V myosin motors)
* responsible for growth directionality
* present in Asco, Basidio, and Oomycetes

how are vesicle contents excreted from the cell?

fusion of vesicles in the membrane regulated by __s__oluble __N__-ethylalemide-sensitive factor __a__ttachment protein __re__ceptors (SNARE) proteins

* present in all eukaryotes that form membrane fusion machinery
* regular fusion of vesicles from a donor organelle to an acceptor compartment
* two types
* v(esicle)-SNARES: incorporated into membranes of transported vesicles
* t(arget)-SNARES: associated with membranes of a target organelle

how is polarity maintained?

both microtubule and actin cytoskeleton through formation of spitzenkorpor