Cytoskeleton Lectures Flashcards
What are characteristics of Actin?
- ATP
- Polarity
- (+) end is growth
- (-) end is depolymerization
-beneath plasma membrane - ATPase
- Myosins
- actin self assembles
What are characteristics of MT?
- Hallow tubes
- GTP (important for dynamics)
- GTPase -> B-tubulins (exchangeable GTP site, hydrolysis happens here)
- dyneins (- end) / kinesins (+ end)
1) Dynamic - mitotic spindle
2) Stable - axomeres - tubulin self-assembles
- a-tubulin: non-exchangable GTP site, locked in place, structural role.
What are characteristics of IF?
- made from a-helical monomers
- bundled
- apolar
- form nuclear lamina, meshwork on inner surface of nuclei, structural integrity to nucleus.
- dissasembles during mitosis, when nucleus dissasembles.
- no motors.
- amino and carboxyterminal domains.
- coiled coil
- anti parallel staggered
- nuclear lamins -> animal cells.
1) Keratin IFS
- mechanical strength for epidermis
- truncation mutation weakens IFs epidermal detachment from basal lamin
2) NFs (neuronal filaments)
Rank Actin, IF, and MT in terms of persistance length.
(1) MT -> 1mm
(2) Actin -> 10-17 uM
(3) IF -> 0.5-2 uM
What are cytoskeletal filaments made from?
- Built from asymmetric subunits, different number of protofilaments and display polarity.
What happens to actin vs MT at steady state?
- MT = dynamic instability
- Actin - treadmilling
In terms of filament stability, what happens when there is hydrolysis of NTP?
- nucleotide caps
- NTP assemble
- NDP dissasemble
- in filaments fast
- in monomers slow
Describe the assembly interactions in MT
- Lateral contacts -> weaker = flexibility
- Longitudinal contacts -> Stronger
Lattice destabilized by GTP hydrolysis
NTP cap lost, catastrophic (shrinking)
When there is a NTP cap, means it is crowing, delay between subunit addition and GTP hydrolysis.
GTP bound -> MT is straight.
GDP bound -> MT curved. (lattice force wants straight, strain)
What proteins are associated with MT?
1) Structural MAPs
2) Motor Proteins (kinesin, dynein - determines direction of movement)
3) Centrosomes - MOCs.
How is MT nucleated?
- Y-tubulin
- Stays after, achoring MT
- all eukaryotes
What are good and bad MAPs for MT?
bad:
- Ketanin
- Spastin
Binds to side of MT and sever them increasing depolarization and MT breakdown.
good:
- Map
- TAU
binds to MT in neurons crosslinking stabilize organize
What is the difference between Kinesin and Dynein?
(1) Kinesin
- +
- walking
(2) Dynein
- -
- AAAATPase family
- ATP hydrolysis for movement
- ATPase ring rotates, linker ring contracts, power stroke along MT, dimerization.
What are different types of MTs?
(1) Astral MTs - position spindle within cell.
(2) Kinetochore MTs - attach to chromosomes.
(3) Interpolar MTs - maintain spindle stability.
Cilia, flgella
mitotic spindl
Explain chromosome seperation in anaphase.
(1) Anaphase A
- chromosomes -> poles (MT depolarization)
(2) Anaphase B
- spindle poles move apart, driven by motor proteins.
What is actin filament limited by?
- nucleation
- rate limiting step cause early assembly intermediates are unstable
in actin polymerization what does the lag phase represent
aggregation
time required for nucleation before visible filament growth starts
how does actin get nucleated
Arp2/3 complex
- branched actin -> 70 degrees most stable for pushing force
what are formins
+ end
continue elongation
nucleate actin filaments
teater totter
new binding sites
essential
what regulates actin concentration and what can displace this
thymosin regulates it binds free actin subunits
Profillin can displace this allowing actin assembly , it still stops it at - end but allows it at + end.
formins cooperate with profillin
what are 3 actin crosslinking structures
1) stress fibres
2) cell cortex
3) Filopodia
what is function of a -actinin
allows myosin to fit between filaments contraction
what is function of fimbrin
- actin
- tightly packs no contractibility
what is function of filamin and spectrin
- gel like mesh strong and flexible
ERM-proteins
link actin to membrane proteins (CD44)
myosin
short steps
binding and unbinding
myosin ATP state - unbound
kinesin ATP state - bound
what do u use to study myosin
gliding assays
muscle contraction process vs non muscle cell
muscle cell: troposonin binds Ca+ , tropsomyosin moves, exposing myosin
non-muscle cell:
Rac-GTP makes lamelipodia formation, decrease in SF and decrease in myosin.
Rho-GTP, increase in myosin and increase in stress fibres
Cdc42 filopodia formation, parallel actin formation
What are the steps of cell motility
(1) Protrusion (actin polymerization)
forward extension of membrane at leading edge
2 main structures
a) Filopodia (cdc42)
b) lamellipodia (Rac)
WASP -> Arp2/3 -> bind AF
(2) Adhesion (integrins)
stabilize protrusion so cell can move forward.
(3) Traction (Myosin II contraction)
(4) Deadhesion and tail retraction.
rear adhesion must be released for cell to move efficiently.
MTs -> direct movement.
Actin -> drives forward movement.
