microtubules 2

Question 1

what do tubulin heterodimers associate to form?

Answer 1

protofilaments

Question 2

what does the loss of the GTP cap result in?

Answer 2

results in a switch from growth to rapid shrinkage

Question 3

what does a chymograph do?

Answer 3

converts movie into 2 dimensional image

Question 4

what is being shown in this image

Answer 4

dynamic instability of microtubules

Question 5

give examples of things the cell uses to regulate the cytoskeleton (microtubule associated proteins) . what do each of them do?

Answer 5

-polymerases: accelerate growth
-depolymerases: destroy GTP cap and trigger collapse
-+TIP proteins: recognizes growing end
-severing enzymes: sever in the middle
-nucleation factors: initiate birth of new microtubules

Question 6

true or false, once microtubules “die”, they arent rebuilt

Answer 6

false, they are born and reborn continuously

Question 7

true or false, micortubules only born in cells undergoing specific processes (ex: mitosis)

Answer 7

false, nucleated in all cells always, even in those not doing anything specific like mitosis. cells are constantly nucleating microtubules

Question 8

what experiment allowed us to see that microtubules are predominantly nucleated by the centrosome?

Answer 8

experience where we take cells, shift from 37 degrees to 4 degrees, then put them in cold. all microtubules will collapse, but if we put back to 37 degrees, regrowth from centrosome only

Question 9

true or false, microtubules are temperature dependent

Answer 9

true. they hate the cold. like warm temp (ex: 37 degrees)

Question 10

what does the centrosome consist of?

Answer 10

2 centrioles, pericentriolar material and gamma-TURCs

Question 11

what angle is between the mother and the daughter centriole

Answer 11

90

Question 12

what is a centriole composed of?

Answer 12

microtubule triplets (3 microtubules smacked together)

Question 13

what is the pericentriol material? what is its major component?

Answer 13

surrounds centrioles. electron dense amphormous proteins (appear dense in electron microscopy)

major component: gamma tubulin

Question 14

what does the gamma tubulin ring complex allow for?

Answer 14

provides a template for microtubule nucleation

Question 15

why is gamma tubulin important, even though it isnt part of the tubulin heterodimer like alpha and beta?

Answer 15

cause accessory proteins can organize it into the gamma tubulin ring complex

Question 16

true or false, all micortubules are polymerized by the gamma tubulin ring complex

Answer 16

false. initially we thought this, but then it was discovered that some microtubules come from a place that doesnt have the ring complex

Question 17

true or false, microtubules elongate from gamma tubulin ring complex

Answer 17

true

Question 18

what is the analogy between the gamma tubulin ring complex for microtubules and the ARP 2-3 complex for actin?

Answer 18

-both provide a template
-both adopt a shape that looks similar to the polymer they’re trying to produce

Question 19

which end of the micortubule is capped by the gamma tubulin ring complex?

Answer 19

the negative end

Question 20

give an example of a place where there are microtubules, but no gamma tubulin ring complex

Answer 20

-in neurons (axons specifically)
-in the growth cone (tip of axon where it grows from), there are NO gamma tubulin rings, yet for the axon to elongate, lots of new microtubules need to be born in that region

Question 21

what is responsible for nucleation in the neurons (just a hypothesis, not proven yet)?

Answer 21

doublecortin protein (related to Gary’s research)

Question 22

what are the two domains of doublecortin?

Answer 22

-N-DC
-C-DC

Question 23

what is the hypothesis about doublecortin and its mutation?

Answer 23

hypothesis that doublecortin is responsible for the nucleation of microtubules in axons. when there is a single amino acid mutation, neurons are unable to migrate to form cerebral cortex, which leads to neurodevelopmental diseases

basically, neurons arent able to migrate out properly to form the cortex, because there is an issue with microtubule formation, which is a core component of the neuron

Question 24

true or false, double cortex syndrome is genetic when a female has it (she can pass it to her girls)

Answer 24

true

Question 25

what is type 1 lissencephaly?

Answer 25

-single amino acid substitution in doublecortin in men. -presents itself as smooth cerebral cortex. -symptoms include speech issues and development issues

Question 26

what is double cortex syndrome?

Answer 26

-single amino acid subsitution in doublecortin in females -secondary layer of gray matter -presents itself as epilespy

Question 27

what are the 2 ways in which cells can nucleate polymers? which way is doublecortin thought to work?

Answer 27

-template by base-plate: ARP 2-3, gamma tubulin complex -arrange monomers: bring subunits together ex: spire -doublecortin hypothesis is that it acts like a spire

Question 28

how does a spire work?

Answer 28

-for actin -4 domains that bind to actin subunits to make nucleus (brings them together)

Question 29

what are MAPS? what do they do? give some examples

Answer 29

-microtubule associated proteins regulate the cytoskeleton in many diff. ways - some can help control nucleation process by interacting with the template (such as gamma tubulin complex) -some inhibit and some promote nucleation

Question 30

what are severing enzymes

Answer 30

ATPases that cut microtubules in the middle. another way for the cell to control/remodel their microtubules

Question 31

true or false, some MAPs interact directly with the nucleation template (ex: gamma tubulin complex)

Answer 31

true. some also dont though. in the end, whether they interact with them directly or not, they all lead to same thing (either promoting or inhibiting polymerization)

Question 32

how does the severing enzyme work?

Answer 32

form is a hexameric ring with a pore on the inside. the pore catches the c terminal of the tubulin protein's tail, and yanks it though, which creates holes and defects (cutting it)

Question 33

what is the purpose of the severing enzyme?

Answer 33

not actually to destroy microtubules. their role is rather to remodel the microtubules, by multiplying them. for example, severing enzymes would make 100 long microtubules and turn them into 500 short ones

Question 34

how do severing enzymes not detroy the microtubule, since severring them in their middle exposes their GDP lattice and gets rid of the cap?

Answer 34

saving factors come in after to stabilize the cut microtubule. if it wasnt for them, the microtubule would fall apart

Question 35

what type of ATPase are severing enzymes?

Answer 35

AAA ATPases

Question 36

what do +TIP proteins do?

Answer 36

track growing of microtubule ends. always beehive of activiity happening as microtubule is growing

Question 37

where do the +TIP proteins bind?

Answer 37

GTP cap

Question 38

which +TIP recognizes the GTP cap? what does this allow for?

Answer 38

-EB family proteins (specifically EB1) -allows other +TIP proteins to hitch a ride

Question 39

what do depolymerases do?

Answer 39

remove tubulin from end and trigger catastrophe (removes the GTP cap)

Question 40

give an example of a depolymerase and explain what it does

Answer 40

kinesin 13, which is an ATPase. it hydrolyzes ATP and uses that energy to get on the + end of the microtubule and tear it apart

Question 41

specific example of kinesin 13 that prof did experiment on? what was the experiment?

Answer 41

MCAK. put microtubules on glass cover, and then added MCAK. then the polymer shrinked, and protofilament forced into a curved structure (unfolding=catastrophe)

Question 42

what happens during a catastrophe?

Answer 42

-The microtubule starts peeling apart -Tubulin dimers rapidly fall off, leading to fast shrinkage of the microtubule.

Question 43

give an example of a polymerase microtubule associated protein

Answer 43

XMAP215

Question 44

what does XMAP215 do?

Answer 44

-accelerate microtubule growth

Question 45

true or false, XMAP215 only lowers activation energy for the Kon

Answer 45

false, also for Koff, and the reaction favoured depends on the concentration of reactants -this is because it acts as a catalyst for microtubule polymerization, therefore it lowers delta G for both the forward and reverse reaction

Question 46

what happens to microtubules based on the concentration of tubulin in the presence of XMAP215?

Answer 46

no tubulin in solution: inverse reaction favoured, cause its reactants (microtubules) have a greater concentration than its products (tubulin). leads to shrinkage a little bit of tubulin: equilibrium. no net growth or shrinkage lots of tubulin: forward reaction accelerated. net growth reaction with highest reactant concentration is favoured

Question 47

what exactly does XMAP215 do to be a catalyst? what were the theories on the way it does this?

Answer 47

-stabilizes weakly attached tubulin dimers at the ends two hypothesis: -at first, was thought that tubulin binding domains (TOG domains) wrapped around a single tubulin domain to form a complex -that theory was then disproven, as scientists realized that only 1 domain fit onto the heterodimer. what actually happens is that one domain reaches out, grabs a heterodimer and brings it to the microtubule end (like a formin)

Question 48

what does XMAP215 act like?

Answer 48

like a forming

Question 49

compare proteins involved in actin with proteins involved in microtubules

Answer 49

Question 50

give an example of a specific severing enzyme

Answer 50

katanin