Cytoskeleton summary Flashcards
Microtubule structure
Microtubules: POLAR: +end has exposed beta tubulin
-end has exposed alpha tubulin
= tubulin heterodimer
= x 13 = protofilament 25nm dia
Actin Structure
Actin: POLAR: +end BARBED, G actin added
-end POINTED, G actin lost
G actin monomer (globular)
Forms F actin (filamentous) = two filaments in a right handed helix 5-9nm dia
Intermediate filament structure
IF: NON POLAR: long protein in a-helix, N&C terminus
monomers twist to form a dimer ‘coiled coil’
x2 dimers coil to form a tetramer
x2 tetramers stagger to form protofilament
x8 tetramers form an intermediate filament
10nm dia
Three stages of dynamic instability
Growth
Catastrophe
Rescue
Which has dynamic instability?
Microtubules
At both ends
Faster at plus end
What nucleates the microtubule in the MTOC?
A ring of GAMMA TUBULIN
Alpha and beta tubulin bind GTP but only
Beta tubulin is a GTPase
Hydrolyses bound GTP to GDP initiating catastrophe
The rate of growth of a microtubule depends on
Free tubulin GTP concentration
What is the critical concentration in regards to microtubules?
The threshold concentration of free tubulin GTP that determines growth or shrinkage
Higher than critical conc = growth
Lower than critical conc = shrinkage
Is the critical concentration of the plus end higher or lower than the minus end?
Lower
This is why treadmilling can occur
What is the critical concentration in regards to microtubules?
The threshold concentration of free tubulin GTP that determines growth or shrinkage
Higher than critical conc = growth
Lower than critical conc = shrinkage
What is the MTOC made up of?
A pair of centrioles and the PCM (peri centriolar material)
What does gamma tubulin form in the centrosome?
TUSC and TURC
Form a ring that nucleates microtubules from the minus end
Cells use the cytoskeleton to
Maintain polarity
Between the apical surface and the basolateral membrane
What are the microtubule associated proteins?
Stathmin - binds tubulin dimers (prevents assembly)
Kinesin + - motor protein
Dynein - - motor protein
Katanin - severs microtubules
Tau - binds microtubules and stabilises them
g-TURC - nucleates MT assembly
+TIPs - links plus end to membranes
XMAP215 - accelerates and stabilises + end
Kinesin13 - enhances catastrophe at + end
Plectin - links to intermediate filaments
MAP2 - filament bundling and cross linking
The cytoskeleton is integral in differentiation by allowing
Asymmetric cell division
e.g. in budding yeast actin cables and patches allow for asymmetric cell division that gives rise to ‘budding’
What allows the cytoskeleton to be dynamic? 2
- Small subunits - Allows rapid structural reorganisations (smaller components are able to diffuse in the cytoplasm)
- Weak non covalent interactions between filaments
(allows rapid assembly and dissasembly)
What do accessory proteins do?
- convert signals into cytoskeletal action
- determine sites of assembly of filaments
- regulate construction of filaments
- change kinetics of filament assembly and disassembly
- harness energy to generate force and growth
- link filaments to organelles or the plasma membrane
- allow movement
What are the actin associated proteins?
Formin - nucleates assembly, remains at +end
ARP2/3 - forms branched actin, remains at -end
Thymosin - binds subunits (prevents assembly)
Profilin - binds subunits (speeds assembly)
Cofilin - binds ADP actin (accelerates disassembly)
Tropomyosin - stabilises filaments
Gelosin - severs filaments and binds to + end
Capping protein - prevents assembly and disassembly at +end
Bundling/cross linking proteins:
Fimbrin, a-actinin, filamin
Attachment to membrane proteins:
spectrin, ERM
What are the actin associated proteins?
Formin - nucleates assembly, remains at +end
ARP2/3 - nucleates branched actin, remains at -end
Thymosin - binds subunits (prevents assembly)
Profilin - binds subunits (speeds assembly)
Cofilin - binds ADP actin (accelerates disassembly)
Tropomyosin - stabilises filaments
Gelosin - severs filaments and binds to + end
Capping protein - prevents assembly and disassembly at +end
Bundling/cross linking proteins:
Fimbrin, a-actinin, filamin
Attachment to membrane proteins:
spectrin, ERM
Diseases of microtubules
Tauopathy:
(e.g. Alzheimer’s disease)
Tau is hyperphosphorylated and detaches from MTs:
1) MTs become less stable and depolymerise;
2) Tau becomes insoluble and aggregates into filaments called Neurofibrillary tangles (NFT)
Primary cilia dyskinesia:
e.g. Situs inversus
Respiratory disease, infertility - no beating at the embryonic node
What do actin filaments do?
- allow the cell to move, maintain or change the shape of the plasma membrane
- provide force to crawl along a substrate = lamellipodia
- form the contractile ring
- stable actin bundles form the microvilli on apical surface
- actin and myosin = muscle contraction
- sense environment = filopodia
G actin is an
ATPase
Hydrolyses ATP to ADP soon after it is added to the plus end of the polymer
Becomes unstable after hydrolysis
Actin lost from the minus end
What is the critical concentration with regards to actin?
The threshold concentration of free actin-ATP above which it will grow and below which it will shrink
Higher than critical conc = growth
Lower than critical conc = shrinkage
When the rate of loss = rate of addition - TREADMILLING
Actin treadmilling allows
Movement
Length is constant but polymer moves
Rate of loss = rate of addition
Compared to microtubules, actin filaments are
More flexible, but much shorter - accessory proteins have to link
What nucleates straight and branched actin?
Straight = formin Branched = Arp2/3
Range of transport
Microtubules = long range Actin = short range
The rate limiting step in filament formation is
Nucleation (lag phase)
Proteins catalyse
Tubulin and actin have been
Highly conserved during eukaryotic evolution
Whereas accessory proteins have not
Why are actin and tubulin highly conserved in eukaryotes?
The filaments interact with so many proteins that structure variability is not possible
Mutations in the shape of the filament might be beneficial for one protein but not for another
So proteins are varied instead
Treadmilling of branched actin at the leading edge causes
Cell migration - lamellipodium
Integrins couple the intracellular actin to
The extracellular matrix to provide traction
Intermediate filaments withstand
Mechanical stress - shorter than mt and actin but very tough
In the nucleus: nuclear lamins protect DNA
In the nerve cells: neurofilaments
In the connective tissues: vimentin
Epithelia: keratins
Mutations in intermediate filaments can cause
Severeal human genetic diseases
e.g. epidermolysis bullosa simplex (keratin mutation)
e. g. Lou Gehrig’s disease (neurofilament mutation - abnormal assembly of neurofilaments in motor neuron cell bodies and axons results in muscle atrophy as neurofilament gene expression directly affects axonal diameter, and how fast a signal can travel)
e. g. Desmin (vimentin family) lacking mice have misaligned muscle fibres
all diseases characterised by cell rupture as a consequence of mechanical trauma
Many toxins target the
Cytoskeleton
Microtubule drugs:
Taxol - binds MTs, stabilises
Colchicine - binds subs, prevents assembly
Vinbastine - binds subs, prevents assembly
Nocodazole - binds subs, prevents assembly
Actin drugs: Phalloidin - binds subs, stabilises Cytochalasin - caps +ends Swinholide - severs filaments Lantruculin - binds subs, prevents assembly
Profilin
Binds to the face of the actin monomer opposite the ATP binding cleft
This blocks the side of the monomer that associates with the minus end of actin and leaves exposed only the plus end binding site
Profilin-actin complex adds to the filament
Addition causes a conformational change in actin that causes profilin to dissociate
Profilin activity is heavily regulated - profilin phosphorylation and profilin binding to inositol phospholipids
Profilin is localised at the cytosolic face of the plasma membrane because it binds to acidic phospholipids there - this enables extracellular signals to activate profilin quickly - like lamellipodia and filopodia
Profilin competes with Thymosin, which binds actin and stops it from polymerizing
Stathmin
Binds to tubulin heterodimers and prevents their addition to a microtubule
Stathmin both decreases the effective concentration of available tubulin subunits and makes a catastrophe more likely
Phosphorylation of stathmin inhibits its binding to tubulin and increases microtubule growth and decreases likelihood of catastrophe
Cancer cells frequently over express stathmin - the increased microtubule growth produces the characteristic malignant cell shape
Katanin
Japanese word for ‘sword’
Severs the 13 bonds of each protofilament
Katanin is made of two subuntis - one severs by hydrolysing ATP, the other directs Katanin to the centrosome
Katanin releases microtubules from the MTOC and causes rapid microtubule depolymerisation of the spindle during mitosis and meiosis
Gelsolin
Actin severing proteins
Requires high levels of cytosolic Ca2+ NOT ATP
Gelsolin has two subunits that bind to two different sites on actin - one on the filament surface and one between one actin subunit and the next
To do this, gelsolin waits for a thermal fluctuation that creates a small gap between neighbouring actin subunits in the filament, then places its subdomain into the gap, breaking the filament
MAPs
Microtubule associated proteins
Can bind along the sides of microtubules and stabilize or destabilize them
They do this by either raising or lowering the free energy of the polymer state by binding
Tau and MAP2 bundle microtubules - 2 domains: one binds along the microtubule and the other sticks out to contact other MAPs
MAPs are controlled by phosphorylation - e.g. protein kinases control MAPs during the formation of the mitotic spindle
Tau - binds to filaments and regulates transport of organelles, at very high concentrations Tau forms helices with itself (neurofibrillary tangles, Alzheimers)
Tropomyosin and Cofilin
Tropomyosin:
Stabilises actin
Binds along the sides of 7 actin filaments simultaneously
Prevents the actin filament from interacting with other proteins - e.g. binding of Ca2+ regulates tropomyosin and is integral to muscle contraction
Cofilin:
Destabilises actin
Binds to actin in both filament and monomeric forms
Cofilin binds along the filament, causing tight twisting and weakening the filament making it more easily broken by thermal motions
Also causes ADP actin to be more easily lost from the minus end
Cofilin binds preferentially to ADP actin so newer filaments (with more ATP bound actin) are resistant to cofilin, so older filaments preferentially dismantled
Cofilin is essential for polarised growth of the actin network associated with crawling
Catastrophe factors
Influence rates of growth and catastrophe
Kinesin13 family
Bind to microtubule ends and pry protofilaments apart, lowering the Ea
MAPs oppose catastrophe factors - e.g XMAP215 stabilize free microtubule ends and inhibit shrinking
Phosphorylation of XMAP during mitosis inhibits activity and essential for the formation of the mitotic spindle
Intermediate Filament proteins
Some IFs can bundle by self association (NF-M and NF-H bind with their C terminus)
Filaggrin bundles Keratin filaments
Plectrin cross links IFs to microtubules, actin and the plasma membrane
Mutations in plectrin cause epidermolysis bullosa, muscular dystrophy and neurodegeneration - very essential protein!
