Moir (Molecular Biomechanics) Flashcards

1
Q

What are the 2 types of dynamic filaments involved in mobility and motility?

A
  • microfilaments and microtubules
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2
Q

What are the differences between microfilaments and microtubules?

A
  • microfilaments 8nm and microtubules 25nm

- microfilaments can be branched but microtubules never branched

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3
Q

What are the similarities between microfilaments and microtubules?

A
  • polar
  • have assoc motor proteins, eg. myosin
  • varying motor protein only way to change function
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4
Q

What are the characteristics of actin genes?

A
  • abundant
  • highly conserved from yeast to man
  • 6 genes in humans, 4α isoforms found in various muscle types, non-muscle contains β and γ actin
  • vary in only 4/5 AAs as mutations would impair function
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5
Q

What are the types of actin?

A
  • monomeric / G-actin

- filamentous / F-actin

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6
Q

What is the structure of G-actin?

A
  • globular shape w/ nucleotide binding site and bound divalent cation in vivo (Mg+2)
  • ATP binding cleft
  • pointed (-) and barbed (+) end
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7
Q

What are the characteristics of F-actin? (structure, equilibrium)

A
  • biologically active form
  • paired helical filament of actin monomers
  • 14 monomers = 1 complete turn
  • eq in cell of filaments and monomers
  • under normal conditions, eq v in favour of filaments, so spontaneous polymerisation (G –> F)
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8
Q

What is the polarity of F-actin and why?

A
  • all monomers point same direction
  • proof is each myosin binds at identical 45° angle
  • polarity essential for movement
  • walks along actin filaments in 1 direction
  • basis for muscle contraction, cell mobility and intracellular transport
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9
Q

How are monomers added and removed from actin?

A
  • new monomers added to barbed end and lost from pointed end
  • nucleotide at pointed end defines stability
  • if ATP then stable
  • if ADP then unravels until another ATP reached
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10
Q

What is the role of capping proteins?

A
  • essential for F-actin filament stability
  • CAPZ at barbed end in skeletal muscle
  • tropomodulin at pointed end
  • deletion lethal in Drosophila
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11
Q

What is the role of actin binding proteins?

A
  • regulate assembly of F-actin
  • allow formation of 3D networks (gels)
  • depolymerised back to G-actin via gel-sol transition
  • eg. myosin
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12
Q

Why do cells preserve pool of monomeric actin?

A
  • to build new filaments
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13
Q

What is the muscular dystrophy gene and how big is it?

A
  • dystrophin gene
  • 1 of longest in genome (0.1%)
  • 79 exons
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14
Q

What causes duchenne MD (severe) and what are the effects of it?

A
  • deletions cause frame shifts
  • mutant protein binds actin but doesn’t have binding site to make contact with membrane
  • severe muscle weakness
  • req wheelchair and leads to early death (teens)
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15
Q

What causes Becker MD (milder) and what are the effects of it?

A
  • deletions that retain ORF
  • mutant protein shorter but still makes contact with membrane
  • muscle weakened but functions reasonably well
  • life exp = 50-60 yrs
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16
Q

What are the characteristics of actin in non-muscle tissues?

A
  • often in highly ordered structures
  • actin bundling proteins, eg fimbrin
  • allow gen of higher order structures, eg. actin cables and microvilli
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17
Q

How does actin affect surface area on microvilli?

A
  • increases it
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18
Q

How does dystrophin anchor F-actin?

A
  • C-terminus anchored to cell membrane
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19
Q

Is actin in the blood?

A
  • leaks from muscle cells due to normal wear and tear

- so present in blood

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20
Q

What is the importance of gelation proteins?

A
  • create F-actin networks
  • eg. filamen
  • v important in moving cells
  • gives strength
  • form higher level of structure
  • disassembled to add new monomers
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21
Q

How does gelsolin sever actin filament?

A
  • high levels prevents actin clots forming
  • stays attached to filament after breakage
  • blocks barbed end, can’t add more monomers
  • disassembled to add new monomers
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22
Q

What is the effect of a heart attack on actin in serum?

A
  • cardiac muscle actin always in serum due to normal wear and tear
  • increases after HA and can form actin clots
  • cardiac muscle troponin I in serum increases (part of regulatory process in striated muscle)
  • troponin test used to clinically diagnose HA , as cardiac troponin I specific to heart
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23
Q

How do actin filaments form branches?

A
  • at leading edge of migrating cells
  • branches grow from sides of existing filaments at 70°
  • v important in cell movement
  • allows precise delivery of cargo within cell
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24
Q

What are the myosin subclasses and their role?

A
  • approx 20 subclasses
  • all isoforms can interact w/ actin through head domains
  • cellular roles differ depending on tail domains
25
Where is myosin II present in highly ordered structures?
- sarcomeres | - in muscle cells and important for formation of actomyosin contractile bundles in non-muscle cells
26
What are myosin I and V needed for?
- vesicle movement
27
2 different types of interaction required between actin and myosin
- contraction, transient interaction w/ actin | - transport, must maintain contact with actin (high processivity)
28
Where is myosin II found?
- dimer that forms filament in skeletal and cardiac muscle
29
How is myosin II stabilised?
- residues A and D always hydrophobic and located on same side of helix - single chains wind around each other w/ hydrophobic surfaces contacting along length to min disruption from water
30
How is myosin II spontaneously formed?
- dimers assoc together | - reversal of direction heads point in middle of filament
31
What are the similarities and differences in the myosin superfamily?
- identified by seq comparisons, as all have conserved motor domain in N-terminal region that binds actin and ATP - vary lot in C-terminal region, defines specific function
32
What is the structure of myosin II in skeletal and cardiac muscle?
- unique C-terminal coiled-coil seq that forms filament
33
How does myosin move along actin?
- myosin heads process along actin filaments by hydrolysing MgATP to MgADP in actin presence - so filaments slide together - myosin not bound to actin, can't complete cycle of MgATP hydrolysis - myosin is incomplete ATPase
34
What is unique about myosin II?
- can assemble into filament so found in striated muscle | - "thick filament" in skeletal and cardiac muscle
35
What are the properties of other myosins? (not II)
- some dimeric but not filamentous - coiled-coil regions interspersed w/ non helical regions - few monomeric (eg. I)
36
What are sarcomeres?
- basic unit of myofibril | - actin and myosin filaments slide together as muscle contracts
37
What are giant proteins?
- nebulin and titin "giant proteins" - regulate length of actin and myosin filaments in skeletal and cardiac muscle - titin elastic
38
What mutations can cause deafness?
- mutations in myosin I, VI, VII
39
How is contraction regulated?
- in cardiac and skeletal operates via inhibition of actin activated ATPase - regulatory proteins are tropomyosin and troponin complex
40
How does Ca conc change during contraction?
- resting muscle = 10^-8 - contracting muscle = 10^-5 - release of Ca releases inhibition, "instant response"
41
What is the risk of having a myosin mutation?
- can cause inherited heart disease - major killer in healthy young adults (incidence 1:500) - impairs cardiac function, left ventricle enlarged
42
How do different myosins cause cell to move?
- can localise diff isoforms of myosin w/ cell using specific myosin antibodies - myosin I localised at leading edge, allowing cell to send out lamellipodia so moves forward - myosin II in rear and pushes back along surface
43
What are the characteristics of stable MTs?
- in non differentiating cells - integral part of neuronal axon - essential for intracellular transport - backbone of cilia and flagella
44
What are the characteristics of transient MTs?
- dividing cells - essential for reorganisation of chromosomes at mitosis - target for chemo
45
How are MTs organised?
- MT is polymer of tubulin subunits organised as hollow tube, 25nm diameter
46
How are MTs assembled?
- assembled from αβ heterodimer of tubulin - can't form homodimers or monomers - each αβ tubulin dimer binds 2GTP (1 binds in α-tubulin and binds GTP irreversibly, other on β-tubulin and reversible and hydrolyses it to GDP - anti-cancer drug Taxol binds to β-tubulin
47
What are the characteristics of MT protofilaments?
- 13 protofilaments - all same orientation - 1 end ringed by α-tubulin and other by β-tubulin - 2 ends differ in growth rates, β-tubulin fast growing (+) end
48
What is the role of GTP/GDP caps?
- GTP-tubulin stabilises MT - GDP-tubulin causes disassembly - if barbed end capped w/ GDP, causes instability and rapid disassembly - MTs grow by addition of tubulin-GTP to barbed end - GTP cap formed is relatively stable, if tubule loses cap, tubulin-GDP exposed and tubule retracts rapidly w/ dissociation of tubulin-GDP heterodimers
49
How can axonal transport by visualised as a model MT system?
- protein synthesis carried out in nerve cell body - can visualise products as transported along MT - shows intact organelles transported - at end of life organelle transported back to cell body for re-use
50
What are the requirements of motor proteins?
- unidirectional | - cell needs 2
51
What is the role of kinesin and dynein?
- motor proteins that use MTs as tracks to move cargo w/in cell
52
What are the characteristics of kinesin?
- processive (+) end directed - contain globular head - differ in tail domains - classified as cytosolic or mitotic (cytosolic transport vesicles and organelles - delivery, takes away from cell body
53
What are the characteristics of dynein?
- (-) end directed - involved in intracellular transport and cell movement - directed for return, material for re-use
54
What can happen to cargo not delivered?
- picked up by myosin | - synthesised and delivered
55
How is cargo delivered?
- MTs good for distribution, but now precise delivery, as don't branch - myosin V assoc w/ MT and waits for kinesin carrying cargo - cargo transferred to myosin V which delivers cargo precisely to target by moving along actin
56
What are the differences between myosin and kinesin and why?
- diverged from common ancestor - myosin retained ability to diffuse along tubulin in "non-productive" way - myosin has bigger head, so bigger stride
57
What are the domains of MT stabilising proteins?
- MT binding domain | - projection domain (can bind to other MTs)
58
What is the effect of MT stabilising proteins arm length?
- controls spacing of adjacent MTS
59
How can Tau be an Alzheimer's target?
- aberrant polymerisation of Tau linked to Alzheimer's | - evidence of pot efficacy of Tau aggregation inhibitor therapy, effective in early stages