module 7 Flashcards
define: cytoskeleton
- network of prot. based filaments that provide scaffolding struc. to cell
- dynamic network
- allows growth, movement, differentiation
- made up of 3 diff. filaments
1. actin
2. microtubules
3. intermediate filaments
explain: cytoskeleton role in cell structure
- proves shape + struc.
⤷ esp. specialized struc. in differentiated cells - ex. struc.:
⤷ microtubules in cilia
⤷ actin filaments in microvilli of epi. cells - cell shape dep. on unique functions of filament
explain: dynamic property of cytoskeleton
- important for cells that move, undergo migration, or cell division
- ex. ovarian cancer cell migrating
⤷ expresses actin-GFP - ex. breast cancer cell undergoing division (mitosis)
⤷ expresses tubulin:GFP
explain: labelling for each fiber in cytoskeleton
ACTIN
- typically labelled w/ fluorescently-tagged phalloidin
⤷ derived from death cap mushroom
- binds w/ high affinity + specificity
- stabilizes filament
- can also be labelled w/ antibody or prot. fusion (ex. Actin:GFP)
MICROTUBULES
- labelled w/ antibodies specific to a tubulin subunit or prot. fusion (ex. Tubulin:GFP)
INTERMEDIATE FILAMENTS (IF)
- labelled w/ antibody specific to subunit of IF or GFP-fusion
explain: each type of fiber in cytoskeleton
ACTIN
- microfilaments
- thinnest
- made of monomeric actin asubunits
MICROTUBULES
- thickest
- made of dimeric subunits of alpha and beta tubulin
IF
- many types
- each assemebled from diff. prot.
explain: distribution + location of cytoskeleton fiber types for epithelial cell
- actin = forms shape of microvilli at apical side
- IF = span cell for struc. support
- microtubules = form networks for intracellular transport
explain: filament-specific motor prot.
- move along actin and microtubules
- no motor prot. for IF
- myosin move along actin
- kinesin and dynein move along microtubules
- generally: heads bind to actin/microtubules, tail attaches to cargo
- powered by ATP hydrolysis
explain: actin-based cell mvts. (location of actin, func.)
- highest density of actin = cell periphery
- func.:
⤷ establish microvilli
⤷ form contractile bundles that form sarcomeres (power musc. cell contraction)
⤷ form filopodia and lamellipodia for cell migration
⤷ form contractile ring (directs cytokinesis or division)
explain: struc. of actin
- actin = F-actin
- 2 strands of helical polymer
⤷ each strand = G-actin - actin filaments = polar
⤷ use myosin head binding to show which side is plus vs minus
⤷ plus = grows faster + barbed
⤷ minus = grows slower, may shrink, pointed
⤷ plus side = newer actin = not covered in myosin
explain: G-actin
- single actin monomer
- 4 structural domains w/ a large cleft between 2 and 4
- cleft forms ATP-nucleotide binding site
- each actin = polar, so microfilaments of these subunits = also polar
- ATP-binding pocket = pointed to minus-end of polymer
⤷ so binding pocket isn’t exposed, except for 1 pair right at minus-end
explain: polymerization and depolymerization of F-actin (+ role of ATP)
- F-actin = formed from polymerization of G-actin
- actin = constantly going polymerization and depolymerization
⤷ at both ends but more growth at plus and more shrinkage at minus - regulated by ATP binding
- ATP bound actin can join plus end when Actin-ATP is high enough
- acting has intrinsic ATPase -> hydrolyzes ATP
⤷ so most of actin is covered in ADP - ADP not released bc nucleotide binding sites = covered in actin
- rate of polymerization = faster than rate of depolymerization FOR PLUS END
- rate of depoly. = faster than rate of poly. FOR MINUS END
⤷ Actin-ADP comes of minus end more readily
question: what is critical concentration (actin)?
- rate of actin monomer addition is equal to rate of removal
- no net growth
- if conc. of actin monomers > critical conc. = poly. > depoly. = growth (vv for shrinkage)
- critical conc. and working conc. are diff. at each end of the actin
question: what regulates the rates of actin growth in the cell (poly., depoly., critical conc.)?
- prot. assoc. w/ dynamics of actin poly./depoly/
- ex. profilin binds to Actin-ATP
⤷ promotes ATP binding
⤷ activates monomer
⤷ dimers accumulates at plus end -> increases conc. of actin monomers - ex. thymosin binds to actin monomers
⤷ inhibits polymerization
⤷ dimers accumulate at plus end -> create a buffer of stored actin monomers
⤷ caps prot. on ends of actin to inhibit poly. or depoly.
explain: treadmilling
- no net increase in length of actin filament bc poly. rate at plus end = depoly. rate at minus end
- no change in length but position changes
- filament moves forward
- important process for cell mvt. and migration
question: how does actin power the mvt. of cells?
- reorganizing actin filaments that can push out of cell mem.
- formation of filopodia and lamellipodia in migrating cell
STEPS
1. forming leading edge of cell
⤷ part closest in direction of mvt.
2. forming lamellipodia
⤷ fan-like expansions of mem.
3. forming filopodia
⤷ w/in region where lamellipodia formed
⤷ finger like projections
4. mvt. of cell forward
explain: myosin prot. (general types, func.)
- move along actin + power intracellular cargo trafficking
- myosin I, II, V in almost all euk.
- all have head domain at N-term.
⤷ has actin binding site
⤷ has site that binds + hydrolyzes ATP - heads = same but tails = diff.
⤷ bc carry diff. cargos at diff. rates - most move towards plus end of actin
explain: myosin II (struc., func.)
- 2 heavy chains form coiled-coil motif + 4 light chains
- phosphorylating light chains drive poly. of myosin prot. -> initiates extension of myosin tails + activates acting binding domains on head
⤷ gets phosphorylated by myosin light chain kinase (MLCK) - myosin II doesn’t carry cargo
- func. = generates contraction forces
- 15 - 20 myosin II filaments -> forms myosin II thick filament (a bipolar filament)
question: what is the struc. of a sarcomere (how are things attached together)?
- myosin II filaments + actin = sarcomeres
⤷ forms striated musc. - plus ends of actin on z-disks
- capping prot. cap the ends
⤷ tropomodulin at minus
⤷ CapZ at plus - nebulin binds the parallel actins
⤷ myosin thick filaments in between - myosin attached to z-disk by titin
question: how does a sarcomere do musc. contraction and relaxation?
- contraction = myosin heads bind to actin
⤷ pulls actin closer to mulled -> shortens sarcomere -> contraction - myosin heads cycle through ATP binding and ATP hydrolysis to power myosin mvt. along actin
- process = calcium dep.
- relaxation = myosin dissociates
⤷ elongates sarcomere - Ca dissociates from actin -> myosin releases actin
explain: energy used to power musc. contraction
- converting chem. E into mech. E
- conversion = mediated by myosin
⤷ myosin has conformational changes (mech. cycle) regulated by ATP binding and hydrolysis (chem. cycle)
explain: cycle of E in a musc. relaxation + contraction
- myosin attached to actin
- ATP binds to myosin -> releases actin
- ATP hydrolyzed by myosin head -> conformational change
- change in myosin = returns to relaxed conformation
- phosphate from break down of ATP increases affinity of myosin head for actin
⤷ allows binding again - release of ADP from myosin -> conformational change
- change pulls actin forward
- returns to step 1 again
explain: myosin V (struc., func.)
- powers intracellular trafficking of cargo along actin
- ex. mvt. of pigment filled vesicles (melanosomes, hold melanin)
⤷ melanocytes in epidermis connect to keratinocytes
⤷ distributes pigment to help protect cell DNA from UV damage - myosin V helps distribute melanosomes to cell mem.
- cargo carrying -> move in hand-over-hand fashion
⤷ trailing myosin head detaches and moved in front of leading (like it’s walking)
question: what would happen w/ a loss of func. mutation in myosin V?
- leads to dilute phenotype
- in animals
- pigment isn’t distributed into fur -> diluted colour
question: what methods can be used to study myosin mvt.?
- studied in vitro
- fluorescently labelled actin + myosin + ATP
- see chem. cycle + mech. cycle of myosin mvt. (involving hydrolysis of ATP)
explain: rates of myosin motor prot.
- from 0.2 - 60 um/sec
- dep. on cycle of ATP nucleotide binding + hydrolysis
- varies w/ rate of ATP hydrolysis and proportion of time myosin is bound to actin
- myosin V = 90% of cycle bound to actin
- myosin II = 5% of cycle bound to actin
- myosin V moves more slowly along actin
explain: step-size (w/ comparison of V vs II)
- distance power stroke propels myosin forward
- dep. on lever arm length
- V step-size = 3x longer than II
explain: microtubule struc.
- 13 protofilaments arrang. in circular pattern to form tube wall
- each protofilament = made of alpha and beta tubulin prot.
- looks like a spiraled ring under a microscope
question: how are alpha and beta tubulin bound to form microtubule?
- both bound to GTP
⤷ alpha bound tighter - GTP bound to alpha = never hydrolyzed
- GTP bound to beta = cyclically hydrolyzed
- GDP -> GTP for beta
- GTP = higher affinity for microtubule than GDP
explain: dynamic property of microtubules
- microtubules = polar
⤷ ends have diff. charac. + dynamics - plus end = fast growing
- minus end = slow growing
- beta subunit closer to plus (vv)
- rescue phase = dimers w/ alpha-beta-GTP = added to plus end
- catastrophe = dimers w/ alpha-beta GDP = removed from shrinking filament
- microtubule mostly has alpha-beta-GDP
- GTP cap/alpha-beta-GTP at plus end = favours growth over shrinkage
⤷ bc GTP dimers have slower rate of dissociation than GDP dimer bc GTP has higher affinity
explain: dynamic instability of microtubules
- plus end
- oscillating behaviour between growth and shortening
- poly. vs depoly.
- growth vs shrinkage
- rescue vs catastrophe
- generally, GTP dimers conc. lvls = at a lvl that allows poly.
question: what controls assembly + disassembly of microtubules and how?
- MAP = microtubule associated proteins
- interconnect microtubules to help for cross-bridges, increase stability, alter rigidity, influence assembly rate
- 2 groups
⤷ stabilize vs destabilize filament - ex. for stabilize:
⤷ Tau
⤷ EB1 - ex. for destabilize:
⤷ catastrophin
explain: microtubule nucleation
- gamma-tubulin involved in nucleation
⤷ starting off the growth - gamma-tubulin + prot. form gamma-tubulin ring complex (gamma-TuRC)
- ring nucleates minus end of microtubule
- acts like cap for minus end while growth happens at plus end
question: where do microtubules start forming in the cell?
- microtubule organizing center (MTOC)
- in animals: MTOC = centrosome
⤷ has centrioles and cloud of pericentriolar material (PCM) that has many gamma-TuRC - minus ends start from gamma-TuRC in MTOC
- plus ends directed towards periphery of cell
explain: the importance of MTOC in mitosis
- microtubules of mitotic spindle attach to chromo. in mitosis
- mitotic spindles = very dynamic
⤷ dep. on instability of microtubules - centrosomes duplicate in mitosis -> replicates MTOC
- when replicated MTOC separate -> microtubules are nucleated
- plus ends that grow help anchor spindle
⤷ other grow to make spindle and attached to the newly replicated chromo.
explain: microtubules in interphase, metaphase, and anaphase of mitosis
INTERPHASE
- microtubules fill cell
METAPHASE
- chromo. = associated w/ mitotic spindle
- microtubules hold chromo. at equator of spindle
⤷ microtubules stretch from poles of spindle to centromere of chromo.
ANAPHASE
- microtubules attached from chromo. shorten
- pull chromatids apart
question: what can inhibit microtubule dynamics?
microtubule toxins
- ex. colchicine
⤷ derived from plants (saffron and crocus)
⤷ inhibits poly.
⤷ binds and stabilizes free alpha-beta-tubulin dimers -> prevents subsequent addition or loss of other dimers
⤷ cells in mitosis arrest in metaphase when treated w/ colchicine - ex. taxol
⤷ binds to beta-tubulin + increases affinity for plus end
⤷ prevents depoly. + stabilizes microtubules
⤷ prevents assembly of mitotic spindle -> inhibits mitosis
**taxol = effective cancer treatment (paclitaxel)
⤷ derived from yew tree, hard to synthesize
- ex. vinblastine and nocodazole
⤷ cause fast depoly. of microtubules
name: motor prot. that move along microtubules
- kinesin and dynein
explain: kinesin (struc., func.)
- tetrameric
⤷ 2 heavy chains + 2 light chains - globular heads on heavy = moto domains
- heads bind to microtubules + generate mct. through ATP hydrolysis
- kinesins move cargo towards plus ends
⤷ move towards periphery of cell (away from MTOC) - tail determines specificity of cargo binding
- hand-over-hand motion
explain: mechanochemical cycle of kinesin
- 2 motor domains
⤷ always has 1 attached to microtubule
⤷ coordinated so that one is always present in complementary stage of chem. cycle
CYCLE
1. lagging head bound to ATP, leading bound to ATP
⤷ ATP = higher affinity for microtubule
2. ATPase from lagging hydrolyzes ATP
⤷ reduces affinity for lagging head
3. in leading head: ADP exchanged for ATP
⤷ increase affinity for leading head
4. binding ATP induces conformational change cause lagging head to swing forward
5. cycle resets
question: what are ways to measure kinesin mvt.?
NOMARSKI MICROSCOPE
- show mvt. along microtubule track anchored to dish
⤷ made from purified tubulin
FLUORESCENT MICROSCOPE
- gliding mobility assay
- motor prot. = anchored to glass slide
- prot. then move fluorescently labeled microtubules
explain: dynein (struc., func.)
- 2 heavy chains + variety of light chains
- minus end directed
- moves away from periphery + towards MTOC
- 2 forms
⤷ cytoplasmic
⤷ axonemal - cytoplasmic
⤷ direct mvt. of organells + vesicles in cyto. - axonemal
⤷ in struc. that power the mvt. of whole cells
explain: dynein power stroke
- driven by power stroke of linker
⤷ happens when phosphate release from ADP
- ATP releases motor head group from microtubule
- ATP hydrolysis
- released phosphate powers power-stroke of linker
- each power stroke pulls cargo towards minus side
explain: bidirectional mvt. of vesicles by microtubule transport
- cargo can move back and forth
⤷ dep. on the motor port. - ex. axons
⤷ minus ends at MTOC
⤷ plus ends extend along axons towards cell mem. of synapse
⤷ vesicles carrying neurotransmitters carried along microtubules
explain: tug of war concept for microtubules
**dynein towards minus, kinesin towards plus
- tug of war battle between dynein and kinesin
- regulatory prot. control direction in resp. to sig. from cell
explain: example of bidirectional mvt. in fish
- transport of melanosomes in skin cells of fish
- molecular motors carry melanosomes to periphery of cell or conc. them in the middle
- dynein = concentrate in middle
⤷ move melanosomes towards minus ends at MTOC - kinesin = disperse melanosomes
⤷ move towards plus ends at periphery - dispersing melanosomes -> cell appears darker
- conc. melanosomes -> cell appears lighter
- sig. the alternating control of motor prot. = sig. that use cAMP in sig. transduction pathway
