Moir (Molecular Biomechanics) Flashcards
What are the 2 types of dynamic filaments involved in mobility and motility?
- microfilaments and microtubules
What are the differences between microfilaments and microtubules?
- microfilaments 8nm and microtubules 25nm
- microfilaments can be branched but microtubules never branched
What are the similarities between microfilaments and microtubules?
- polar
- have assoc motor proteins, eg. myosin
- varying motor protein only way to change function
What are the characteristics of actin genes?
- 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
What are the types of actin?
- monomeric / G-actin
- filamentous / F-actin
What is the structure of G-actin?
- globular shape w/ nucleotide binding site and bound divalent cation in vivo (Mg+2)
- ATP binding cleft
- pointed (-) and barbed (+) end
What are the characteristics of F-actin? (structure, equilibrium)
- 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)
What is the polarity of F-actin and why?
- 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
How are monomers added and removed from actin?
- 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
What is the role of capping proteins?
- essential for F-actin filament stability
- CAPZ at barbed end in skeletal muscle
- tropomodulin at pointed end
- deletion lethal in Drosophila
What is the role of actin binding proteins?
- regulate assembly of F-actin
- allow formation of 3D networks (gels)
- depolymerised back to G-actin via gel-sol transition
- eg. myosin
Why do cells preserve pool of monomeric actin?
- to build new filaments
What is the muscular dystrophy gene and how big is it?
- dystrophin gene
- 1 of longest in genome (0.1%)
- 79 exons
What causes duchenne MD (severe) and what are the effects of it?
- 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)
What causes Becker MD (milder) and what are the effects of it?
- deletions that retain ORF
- mutant protein shorter but still makes contact with membrane
- muscle weakened but functions reasonably well
- life exp = 50-60 yrs
What are the characteristics of actin in non-muscle tissues?
- often in highly ordered structures
- actin bundling proteins, eg fimbrin
- allow gen of higher order structures, eg. actin cables and microvilli
How does actin affect surface area on microvilli?
- increases it
How does dystrophin anchor F-actin?
- C-terminus anchored to cell membrane
Is actin in the blood?
- leaks from muscle cells due to normal wear and tear
- so present in blood
What is the importance of gelation proteins?
- create F-actin networks
- eg. filamen
- v important in moving cells
- gives strength
- form higher level of structure
- disassembled to add new monomers
How does gelsolin sever actin filament?
- 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
What is the effect of a heart attack on actin in serum?
- 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
How do actin filaments form branches?
- 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
What are the myosin subclasses and their role?
- approx 20 subclasses
- all isoforms can interact w/ actin through head domains
- cellular roles differ depending on tail domains
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
What are myosin I and V needed for?
- vesicle movement
2 different types of interaction required between actin and myosin
- contraction, transient interaction w/ actin
- transport, must maintain contact with actin (high processivity)
Where is myosin II found?
- dimer that forms filament in skeletal and cardiac muscle
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
How is myosin II spontaneously formed?
- dimers assoc together
- reversal of direction heads point in middle of filament
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
What is the structure of myosin II in skeletal and cardiac muscle?
- unique C-terminal coiled-coil seq that forms filament
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
What is unique about myosin II?
- can assemble into filament so found in striated muscle
- “thick filament” in skeletal and cardiac muscle
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)
What are sarcomeres?
- basic unit of myofibril
- actin and myosin filaments slide together as muscle contracts
What are giant proteins?
- nebulin and titin “giant proteins”
- regulate length of actin and myosin filaments in skeletal and cardiac muscle
- titin elastic
What mutations can cause deafness?
- mutations in myosin I, VI, VII
How is contraction regulated?
- in cardiac and skeletal operates via inhibition of actin activated ATPase
- regulatory proteins are tropomyosin and troponin complex
How does Ca conc change during contraction?
- resting muscle = 10^-8
- contracting muscle = 10^-5
- release of Ca releases inhibition, “instant response”
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
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
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
What are the characteristics of transient MTs?
- dividing cells
- essential for reorganisation of chromosomes at mitosis
- target for chemo
How are MTs organised?
- MT is polymer of tubulin subunits organised as hollow tube, 25nm diameter
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
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
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
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
What are the requirements of motor proteins?
- unidirectional
- cell needs 2
What is the role of kinesin and dynein?
- motor proteins that use MTs as tracks to move cargo w/in cell
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
What are the characteristics of dynein?
- (-) end directed
- involved in intracellular transport and cell movement
- directed for return, material for re-use
What can happen to cargo not delivered?
- picked up by myosin
- synthesised and delivered
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
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
What are the domains of MT stabilising proteins?
- MT binding domain
- projection domain (can bind to other MTs)
What is the effect of MT stabilising proteins arm length?
- controls spacing of adjacent MTS
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
