1
Q
What are the two sets of dynamic filaments that are involved in mobility and motility in eukaryotic cells?
A
- Microfilaments (Actin)
- Microtubules (Tubulin)
2
Q
What are the differences between mirofilaments and microtubules?
A
- Microfilaments: 8 nm and can be branched
- Microtubules: 25 nm and never branched
3
Q
What properties do both mirofilaments and microtubules have?
A
Polar and have associated motor proteins
4
Q
How similar are the actin’s in yeast and man?
A
Highly conserved
5
Q
How many actin isoforms are there in humans?
A
- Six actin genes
- 4 alpha actin isoforms found in various muscle types
- Non-muscle cells contain beta and gamma actin
6
Q
How many forms of actin are found in the cell?
A
2 forms: G-actin and F-actin (F-actin is the biologically active form)
7
Q
What is the structure of G-actin?
A
- Monomeric
- Globular shape
- Nucleotide binding site and bound divalent cation (Mg2+ in vivo)
- Pointed (-ve) and barbed (+ve) ends
8
Q
What is the structure of F-actin?
A
- Paired helical filament of actin monomers (complete turn=14 monomers)
- In the cell, filaments and monomers in equilibrium (In favour of filaments in normal conditions)
- Polar
9
Q
Why is an F-actin filament polar?
A
All its monomers point in the same direction as each myosin head binds to F-actin at a 45 degree angle
10
Q
Why is F-actin polarity important for movement?
A
- All actin monomers point in the same direction
- All myosins bind at same angle
- So myosin walks along the actin filament in one direction
- Basis for muscle contraction, cell motility and intracellular transport
11
Q
What is the pattern of growth of actin polymerisation?
A
Slow initial growth phase followed by a very fast growth
12
Q
Why does actin polymerisation follow its pattern of growth?
A
- Initial stages of polymerisation are energetically unfavourable
- Once trimer formed, polymerisation increases rapidly
- When G-actin becomes limiting, polymerisation slows
13
Q
From what directions are actin monomers added and lost?
A
Added at the barbed (+ve) end and lost from the pointed (-ve) end
14
Q
How does the nucleotide at the pointed end of an actin filament determine its stability?
A
- If actin-ATP located at pointed end, filament is stable
- If actin-ADP located at pointed end, filament unravels until another actin-ATP occupies the pointed end
15
Q
What are the properties of capping proteins for an F-actin filament?
A
- Essential for it to be stable
- CAPZ is the capping protein in skeletal muscle
- Deletion is lethal in Drosophila
- CAPZ is the capping protein in skeletal muscle
16
Q
What are the functions of actin-binding proteins?
A
Regulate the assembly of the F-actin filament and allow it to form 3D networks (gels) that can in turn be depolymerised back to G-actin via a gel-sol transition
17
Q
What are the different functions of actin-binding proteins and what are examples of them?
A
- Severing e.g. gelsolin
- Cofilamentous binding e.g. tropomyosin
- Bundling e.g. Fimbrin
- Gel-forming e.g. ABP-120, alpha-actinin
- Contraction e.g. myosin
- Monomer binding e.g. profilin
- Filament capping e.g. capping protein
18
Q
How does the cell preserve a pool of monomeric actin to build new filaments?
A
- Monomer binding proteins e.g. profilin and thymosin Beta4
- Thymosin B4 sequesters actin monomers and so reduces monomers available for polymerisation
- Profilin promotes nucleotide exchange and promotes filament formation and can be activated by binding PIP2
19
Q
What is the function of the actin binding protein Dystrophin?
A
Anchors F-actin to the cell membrane ( C-terminus bound to protein in cell membrane & N-terminus to actin)
20
Q
What is the main cause of Muscular Dystrophy?
A
Major cause is deletions in the dystrophin gene
21
Q
What are the properties of Duchenne MD?
A
- Severe muscular dystrophy
- Result of frameshifts in the dystrophin gene so protein does not have the binding site needed to make contact with the membrane
- Sever muscle weakness and early death, often in teens
22
Q
What are the properties o Becker MD?
A
- Less severe muscular dystrophy than Duchenne MD
- Deletions in the dystrophin gene that retain the reading frame
- Shorter protein but can still make contact with the membrane, so weakened muscle
- Life expectancy can be 50-60 years
23
Q
What are the properties of actin bundling proteins?
A
- E.g. Fimbrin
- In non-muscle tissues
- Allow generation of higher order structures such as actin cables and microvilli
24
Q
What is the core protein in microvilli?
A
Actin
25
What are the functions of gelation proteins?
Create networks of F-actin e.g. filamin
26
What are the functions of gelsolin?
- Severs actin filaments
| - Important component of serum as it prevents the formation of actin clots
27
How do actin filaments form branches?
- Forms branched actin networks at leading edge of migrating cells
- Branches grow from sides of existing filaments at 70 degrees
- Important in movement of cells
- Allows precise delivery of cargo within cell
28
What is the motor protein for actin filaments?
Myosin
29
What are the properties of myosin?
- Myosin motors interact with actin
- 20 subclasses of myosin
- Myosin heads walk along actin filaments in discrete steps
- Myosin isoforms cellular roles differ depending on their tail domains
- Myosin II found in sarcomeres in muscle cells and is important for the formation of actomyosin contractile bundles in non-muscle cells (essential for cell movement and division)
- Myosins I ad V are required for movement of vesicles
30
What is the processivity of myosin?
- Myosin is involved not only in muscle contraction but also in transport within the cell
- Two types of interaction of myosin with actin:
- Contraction = transient interaction with actin (i.e. rowing)
- Transport = Must maintain contact with actin (i.e. high processivity, like DNA polymerase)
31
How do myosins differ from one another?
- Conserved motor domain at the N-terminus to bind actin & ATP
- Differ considerably in the C-terminal portion as defines specific functions
32
What is the tail structure of Myosin II?
Coiled-coil tail - Two double helices next to each other
33
What are the different structures of the different myosins?
- Only myosin II has an unbroken coiled-coil tail so is the only myosin that can assemble into a filament (Found in striated muscle thick filament)
- Some other myosins (e.g. myosin V) have regions of coiled-coil that are interspersed by non-helical regions (usually because of proline) and so form a dimer
- A few myosin are monomeric (e.g. myosin I)
34
What is the basic unit of the myofibril?
The sarcomere
35
What are the functions of nebulin and titin in skeletal and cardiac muscle?
Regulate the length of the actin and myosin filaments
36
How do myosin heads process along actin filaments?
By hydrolysing MgATP to MgADP in the presence of actin so the actin and myosin filaments slide together
37
Why is myosin an incompetent ATPase?
Can only complete the cycle of MgATP hydrolysis when bound to actin
38
How is muscle contraction regulated?
Via inhibition of the actin-activated ATPase - the regulatory proteins are tropomyosin and the troponin complex
-Release of calcium releases the inhibition
39
What can mutations in myosin I, VI and VII cause?
Deafness
40
What is the role of myosin in cell locomotion?
- Myosin I is localised at the leading edge of the cell, allowing the cell to send out lamelipodia so that the cell moves forward
- Myosin II is confined to the rear of the cell and pushes the back of the cell along the surface
41
What are the organisation and dynamics of microtubules?
- A microtubule os a polymer of tubulin subunits that are organised as a cylindrical hollow tube
- Some microtubules are stable - these are found in non-differentiating cells. Stable microtubules are an integral part of the neuronal axon, are essential for intracellular transport and are the backbone of cilia and flagella
- Transient microtubules are found in dividing cells and are essential for chromosome reorganisation in mitosis
42
What are microtubules assembled from?
- Assembled from and alpha, beta heterodimer of tubulin
| - Microtubules cannot form from homodimers or a monomer
43
How do aB tubulin dimers bind GTP?
- Each dimer binds 2 molecules of GTP
- One GTP binding site is in alpha-tubulin and this binds GTP irreversibly
- The second site is on beta-tubulin and this binds GTP reversibly and hydrolyses it to GDP
44
What does the anti-cancer drug Taxol bind to?
Beta-tubulin
45
What is the structure of the microtubule cylinder?
- Long, hollow cylinders
- 13 protofilaments in the microtubule
- All protofilaments in a microtubule have the same orientation so one end of the microtubule is ringed by alpha-tubulin and the other by beta-tubulin
- The beta-tubulin end is the fast-growing (+) end
46
What is the effect of GDP and GTP on microtubule stability?
GTP-tubulin stabilises the microtubule while GDP-tubulin causes disassembly
47
What are the key points of axonal transport?
- Model system for microtubules
- Protein synthesis carried out in nerve cell body
- Shows that intact organelles are transported
- At the end of its life, the organelles is transported back to cell body for re-use
- Motor must be unidirectional - so need two motors
- Kinesin (+, directed for delivery)
- Dynein (-, directed for return)
48
What are the properties of kinesin?
- Motor protein that uses microtubules as tracks in order to move its cargo within the cell
- Found in all cells
- Processive (+) end directed motor proteins
- All contain a globular head (motor domain) but differ in their tail domains
- Classified as cytosolic or mitotic - Cytosolic transport vesicles and organelles
49
What are the properties of dynein?
- Motor protein that uses microtubules as tracks in order to move its cargo within the cell
- Found in all cells
- -ve end directed motor protein involved i intracellular transport and in cell movement
50
What is the difference in direction of travel between Kinesin and Dynein?
- Kinesin moves organelles to the +ve end (Away from the nucleus)
- Dynein moves organelles to the -ve end (Towards the nucleus)
51
What is the role of myosin in the delivery of cargo with microtubules?
- Microtubules do not branch so cannot perform precise delivery
- Myosin V associates with a microtubule and waits for kinesin carrying a cargo
- The myosin V then picks up the cargo and delivers it to the target by moving along actin
52
What are the properties of stabilising MAPs (Microtubule associated proteins)?
- Two domains: MT binding domain and a projection domain
- Projection domain can bind to membranes, filaments...
- Examples: MAP1, MAP2 and Tau
- Aberrant polymerisation of Tau is linked to neurodegenerative disorders
53
What is the function of microtubules and motor proteins in mitosis?
The mitotic apparatus (Spindle fibres and microtubule organising centre) is a microtubule machine for separating chromosomes
54
What is the function of microtubules in motility?
- Free-living cells use microtubules for movement (Using cilia or flagella)
- Static cells also use cilia: beating of cilia propels fluid over the surface of the cell
- Highly-ordered structure=The Axoneme
- Motor protein is dynein
55
What is the structure of eukaryotic cilia and flagella?
A core of doublet microtubules studded with axonemal dyneins, around a central pair of microtubules
56
What are the possible methods of myosin movement along a filament?
- Inchworm: Same head leads
| - Hand-over hand: Similar movement to walking
57
How was it discovered myosin movement along a filament is hand-over-hand?
Measured distance a probe travels in a step: 72 nm not 36 nm so must be hand-over-hand as twice the distance at once
MBB161 - Biochemistry
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