lec 10- microtubules 3

Question 1

was it once thought that acetylation only happens on a-tubulin?

Answer 1

yes, but a single beta-tubulin sight has been found on the inside of microtubules

Question 2

if MAPS and motors can’t access the internal site of acetylation, how does it occur?

Answer 2

it occurs by the tubulin acetyltransferase called aTAT1

Question 3

how does aTAT1 get into the microtubule?

Answer 3

-by entering from the free open ends of the microtubule
-by entering from the breaks in the microtubule

Question 4

does aTAT1 bind to the surface of microtubules?

Answer 4

yes

Question 5

where does acetylated tubulin occur?

Answer 5

-in patches called hot spots in the microtubule in areas where there are bends
-also in the open ends of the microtubule

Question 6

what does aTAT1 do once bound to the surface of the microtubule? what does sit do?

Answer 6

-when a break in the microtubule occurs, aTAT1 enters the break and acetylated the localized tubulin
-it helps provide increased strength in the region of the microtubule (like repairing a bone)

Question 7

do aTAT KO mice live?

Answer 7

yes, and have no major abnormalities

Question 8

do long lived microtubules have more acetylated tubulin? what does acetylation protect long lived microtubules from?

Answer 8

-yes, acetylated tubulin in the centrioles and basal bodies
-aging

Question 9

what percentage of acetylated tubulin do cilia have?

Answer 9

100%

Question 10

what does acetylation do?

Answer 10

-strenghtens and enhances microtubule flexibility and provides resistance against mechanical stress in those areas of acetylation

Question 11

what 2 tubulin deacetylases remove the acetylation?

Answer 11

HDAC6 and SIRT2

Question 12

how does HDAC6 enter the microtubule?

Answer 12

not fully known, but it interacts with EB1 at the + end, indicating it as a potential way of entrance

Question 13

why does pure acetylated tubulin (in vitro) assemble microtubules much slower than deacetylated tubulin? do they form at similar rates once nucleated?

Answer 13

-because it decreases the spontaneous nucleation rate of the microtubules
-yes, the microtubules form at the same rate

Question 14

does detyrosination only happen on a-tubulin?

Answer 14

yes

Question 15

what is detyrosination?

Answer 15

the removal of the terminal tyrosine residue from the C-terminus of the tubulin

Question 16

what causes detyrosination?

Answer 16

vasohibin 1 and 2

Question 17

what regulates vasohibin?

Answer 17

SVBP (small vasohibin binding proteins)

Question 18

are vasohibins involved in blood vessel formation?

Answer 18

yes

Question 19

what do Tubulin Tyrosine Ligase (TTL) do?

Answer 19

rapidly re-tyrosinates all soluble tubulin dimers, essentially brings the tubulin back to its original state

Question 20

when does polyglutamylation or polyglycylation occur?

Answer 20

-when glutamic acid (or glycine) chains are attached to glutamic acid residues towards the C-terminues of tubulin

Question 21

can tubulin be polyglutamylated and polyglycylated at the same time?

Answer 21

no

Question 22

what does polyglutamylation/polyglycylation do? what type of tubulin does it occur on?

Answer 22

-increases microtubule stability
-occurs on both a and B-tubulin

Question 23

what do microtubule severing proteins do?

Answer 23

-they cut microtubules, causing their rapid disassembly

Question 24

how do microtubule severing proteins function?

Answer 24

act as inactive monomers in the cell normally, then oligomerize at the microtubules, cut the microtubules, and then dissociate back to the cytoplasm as inactive monomers

Question 25

what are the three types of microtubule severing proteins?

Answer 25

katanin, spastin, fidgetin

Question 26

of what family are the microtubule severing proteins

Answer 26

ATPase superfamily

Question 27

what do the severing proteins use as energy?

Answer 27

ATP hydrolysis energy to sever microtubules to create new ends

Question 28

where are all the severing proteins localized?

Answer 28

at the mitotic pole

Question 29

can severing proteins also regulate the direction and speed of cell motility?

Answer 29

yes

Question 30

are severing proteins monomeric in the presence of ADP?

Answer 30

yes

Question 31

what form do katanin and spastin take in the presence of ATP?

Answer 31

hexamer

Question 32

do severing proteins start out as monomers then turn into hexamers when bound to microtubules?

Answer 32

yes

Question 33

what is the 2nm pore present at the center of the enzyme when bound to the microtubule?

Answer 33

the active site

Question 34

what does the active site pore bind to?

Answer 34

carboxy-terminal tail (CTT) of tubulin to begin severing

Question 35

what happens if CTT is removed?

Answer 35

severing doesn't occur

Question 36

what was the first identified microtubule severing protein?

Answer 36

katanin

Question 37

what can katanin cut?

Answer 37

-random lengths round the microtubule -can cut taxol stabilized microtubules

Question 38

what are the characteristics of katanin?

Answer 38

-a severing protein with an enzymatic ability -60kDa and 80kDa -prefers to bind to and sever acetylated lysine microtubules -named after the katana

Question 39

what are the characteristics of spastin?

Answer 39

-cuts equal lengths along the microtubules -named after hereditary spastic paraplegia (causes stiffness in lower limbs) -can also sever taxol stabilized microtubules -upregulated by polyglutamylation -down-regulated by detyrosination

Question 40

what are the characteristics of fidgetin?

Answer 40

-cuts faster at microtubule - ends -named after gene mutation in fidget mouse -can also sever taxol stabilized microtubules

Question 41

what is colchicine?

Answer 41

-an old microtubule drug

Question 42

what is colchicine from?

Answer 42

meadow saffron

Question 43

what does colchicine do?

Answer 43

-binds to all tubulin dimers, preventing new microtubules to form -microtubules disassemble via dynamic instability and no-elongation occurs

Question 44

do any microtubules remain in the cell when colchicine is added? what thing with microtubules remains?

Answer 44

no, only the centrosome remains (stable microtubules)

Question 45

can the centrosome nucleate microtubules?

Answer 45

yes, when the colchicine is removed, they come back

Question 46

what is nocadazol? what are its chatacteristics?

Answer 46

-a synthetic microtubule drug -it binds to all tubulin dimers -doesnt all the formation of microtubules, causes overall disassembly

Question 47

what is Taxol and what is it from? what does it do? what is it used to treat?

Answer 47

-a microtubule drug -from the Pacific yew tree -binds to microtubules and stabilizes them, microtubules cant depolymerize, stops mitosis -been used for cancer treatment

Question 48

what are vinca alkaloids?

Answer 48

-a drug from Madagascar periwrinkle plants

Question 49

what are the characteristics of vinca alkaloids?

Answer 49

-bind to the interface of tubulin heterodimers -stop microtubule assembly at low concentrations -at high concentrations they preferentially bind to unpolymerized B-tubulin, thus blocking its incorporation into polymerized microtubules -causes disassembly of mcirotubules -used for cancer treatments

Question 50

what do MAP 1, MAP1A and MAP1B bind to? how many microtubule binding sites do MAP1A and B have?

Answer 50

-actin -3 for A and 1 for B

Question 51

what does anti-MAP1 antibodies do?

Answer 51

strains stress fibers

Question 52

what does MAP1 do?

Answer 52

-bind and cross-link microtubules and actin filaments

Question 53

what does WHAMM (WASp Homologue Associated with Actin, Membranes, and Microtubules) do? what region binds to microtubules?

Answer 53

promotes actin filament nucleation by activating Arp 2/3 complex -has a coiled coil region that binds to microtubules

Question 54

where is WHAMM located?

Answer 54

at membranes (at the Golgi complex) with actin filaments and with microtubules

Question 55

how do polymerizing microtubules move material?

Answer 55

they push intracellular material

Question 56

how do depolymerizing microtubules move material?

Answer 56

they pull intracellular material

Question 57

what happens to microtubules that are 10 microns in length?

Answer 57

they buckle

Question 58

what else do microtubules do?

Answer 58

-position the nucleus in fission yeast -move chromosomes -move the ER

Question 59

what are the two ways ER movement happens?

Answer 59

1. microtubule molecular motors transport the ER along microtubules 2. the ER docks on the microtubules and rides the polymerization( and depol.) of microtubule towards the microtubule + ends, happens by direct binding of EB1 to the ER calcium protein STIM.1

Question 60

does profilin co-distribute with microtubules?

Answer 60

yes

Question 61

how do they know formin links profilin to microtubules?

Answer 61

-when a formin inhibitor like SMIFH2 or Dia2 is used, a decrease in profilin association with microtubules occurs

Question 62

when you increase lamellipodia formation, does a decrease in microtubule-profilin interactions occur?

Answer 62

yes

Question 63

does siRNA of profilin cause an increase in acetylated tubulin?

Answer 63

yes