Microtubules

Question 1

purification of microtubules

Answer 1

1. brain - must be cold! decreases the hydrophobic interactions when low temp, hydrophobic interactions is what holds MT together - if you put them in ice, MT skeleton loses its round shape
2. spin - get rid of pellet (nuclei, mitochondria, membrane), keep supernatant
3. Add Glycerol and GTO - H2O sequestering! get out the water to increase the concentration of microtubules
4. incubate at 37 degrees - increase hydrophobic interactions again! form polymers
5. spin
6. keep pellet! protein comp of pellet no longer changes, get rid of supernatant
7. make cold again, spin, do a million times to purify microtubules

Question 2

molecular weight of alpha/beta tubulin

Answer 2

50 kDa each - 100 kDa total! 50% identical in AA

Question 3

microtubule protofilamnt

Answer 3

alpha and beta tubulin dimers polymerize end to end, 13 associate laterally around a hollow core to form microtubule, each row is the protofilament

Question 4

microtubule subunit

Answer 4

tubulin heterodimer - alpha and beta, stack head to tail (alpha, beta, alpha, beta)

Question 5

dyenin

Answer 5

moves toward negative end of microtubule (usually toward cell center), retrograde transport, converts ATP into mechanical energy to walk along microtubule, head domain on microtubule, head has ATP hydrolysis, intermediate and light chains attach to cargo, bigger head than kinesins so may walk farther?

Question 6

kinesins

Answer 6

walk toward positive end of microtubule (usually away from cell center - anterograde),
bind site for ATP on head - attached to MT, tails have light chain to bind cargo

Question 7

depolymerizing drugs

Answer 7

Colchicine
Nocodazole
if incubate with these, MT will disappear (will reform if restore)

Question 8

polymerizing drugs

Answer 8

Vinca Alkaloids (Vinblastine, Vincristine)
Taxol
cells can’t go through cell division! stabilizes MT and prevents from disassembly, blocks mitosis! no cell division - any division needs MT and this disrupts mitosis
very toxic! have to balance

Question 9

centrosome

Answer 9

main place of MT origin, regulate cell division
discrete structure in pair
2 centrioles next to each other = 1 centrosome
replicates during interphase of cell cycle! mitotic spindle forms between 2 centrosomes

Question 10

alpha tubulin

Answer 10

GTP/GDP bind pocket next to interface - locked in and not exchangable!
MINUS

Question 11

beta tubulin

Answer 11

GTP/GDP bind site open to solvent! E-site is exchangable, beta site is exposed
PLUS

Question 12

Microtubule C(c)

Answer 12

below this concentration of MT - nothing happens!
above Cc –> rise in absorbence (mass) of MT - looks like it reaches equilibrium
at certain point - MT number decreases but MT length increases! number of polymers decrease and length increases - mass is the same!

Question 13

Dynamic instability

Answer 13

GTP bound to alpha tubulin - for structure only!
GTP bound to beta tubulin - hydrolyzed when mount to MT
rescue and catastrophe
polymerization and depolymerization
equilibrium can’t describe!

Question 14

Rescue

Answer 14

from depolymerization to polymerization - GROW!

GTP –> GDP, microtubule grows!

Question 15

Catastrophe

Answer 15

from polymerization to depolymerization - SHRINK!

rate of tubulin dimers slows - GTP on GTP cap is hydrolyzed to GDP before new tubulin is added and it comes apart!

Question 16

polymerization of MT

Answer 16

when GTP is incorporated into plus side, GTP –> GDP, once it’s incorporated, rate of addition of GTP –> size

Question 17

depolymerization of MT

Answer 17

negative end shrinks back - exposed GDP subunits unravels and lose it!

Question 18

EB3

Answer 18

end binding protein - binds to positive tips, plus side of GTP MT cap, may stabilize MT?

Question 19

Incomplete GTP hydrolysis

Answer 19

along MT - GTP hydrolysis not complete! if lose cap –> catastrophe! if it’s all GDP it will shrink to nothing - local GTP areas so it can recover!