5: Cytoskeleton Overview

Question 1

what is the cytoskeleton?

Answer 1

intricate network of protein filaments that extend throughout the cytoplasm

Question 2

How do MTs have polarity?

Answer 2

one side is alpha (-) end
one side is beta (+) end

Question 2

MT subunits are

Answer 2

alpha and beta tubulin which are 55kDa each. these form a stable dimer of 8nm

Question 3

diameter of MT?
length of MT?

Answer 3

25nm
up to 100s of µm long (project out of cell - flagella)

Question 4

alpha-beta dimers form

Answer 4

protofilaments when B subunit is in GTP subunit

Question 5

are MTs perfectly circular?

Answer 5

no
seam forms

Question 6

B tubulin is different from a tubulin because

Answer 6

B tubulin can be hydrolyzed from GTP to GDP

Question 7

What are the types of micritubule structures?

Answer 7

1. singlet: most common, found in cytoplasm made of 13 protofilaments
2. doublet: found in cilia and flagella, A MT (13 PF) + B MT (10 PF)
3. triplet: basal bodies & centrioles: A MT (13 PF) + B MT (10 PF) + C MT (10 PF) - stable tend to not polymerize and depolymerize

Question 8

cytoplasmic MT are found in

axonemal MT are found in

Answer 8

cytoplasmic: singlet nerve axon cytoplasm MT

axonemal: doublet cilia/flagella MT

Question 9

assembly of microtubule begina at

Answer 9

MT organizing centre (MTOC) which is associated with (-) end and (+) end grows away

Question 10

What is the centrosome?

Answer 10

main cellular MTOC that contains centrioles and pericentriolar matrix

Question 11

What are other MTOC examples?

Answer 11

• spindle poles in mitotic apparatus
• basal body: MTOC for cilia/flagella
• nerves: MT are not continuous (axon + dendrites: orientation varies)

Question 12

centrioles are found in

Answer 12

animals (not plants)

Question 13

centrioles are

Answer 13

2 barrel-like triplet MT that are perpendicular to each other

Question 14

the pericentriolar matrix is:

Answer 14

space around centrioles where proteins gamma tubulin and augmin are found

Question 15

gamma tubulin and augmin are

Answer 15

proteins to make singlet cytoplasmic MTs
gamma tubulin: starts polymerization of singlet MT
augmin: increases polymerization
both form gamma tubulin ring complex

Question 16

when centrioles replicate…

Answer 16

pericentriolar matrix also replicates and they make a mother and daughter MT which are different

Question 17

gamma tubulin ring complex provides …

Answer 17

nucleating sites for MTs = this is the minus end

Question 18

a nucleating site is…

Answer 18

where polymerization of MT can occur on

Question 19

augmin can (rarely) facilitate

Answer 19

MT branching

Question 20

to study MT polymerization…

Answer 20

Take flagellar nucleus (piece of flagella) which acts as a nucleating site, which is a doublet MT = stable and won’t depolymerize

Add tubulin diners to the nucleus until critical concentration for polymerization is reached

Question 21

Below critical concentration =

Answer 21

depolymerization

Question 22

MT – side polymerization and depolymerizes faster because …

Answer 22

plus

bc
minus end has gamma tubulin ring complex = capped = blocking polymerization and depolymerization

Question 23

Time of elongation of protofilament depends on

Answer 23

nucleus presence

Question 24

What is dynamic instability?

Answer 24

MTs are always growing and shrinking independently of each other **Catastrophe:** MT stops polymerization and starts depolymerizing (for ex. Below critical conc.) **Rescue: ** MT stops depolymerizing and starts polymerizing (for example: above critical conc.)

Question 25

dynamic instability depends on

Answer 25

presence or absence of GTP-beta-tublin cap

Question 26

GTP-beta-tubulin cap provides

Answer 26

lateral cohesion: protofilaments are attached to each other, which increases when alpha and beta tubulin are GTP bound

Question 27

As the protofilament grows, GTP is

Answer 27

hydrolyzed to GDP in beta tubulin

Question 28

Most beta tubulin in protofilament that is not on the plus end is

Answer 28

in GDP form by hydrolysis = less lateral cohesion

Question 29

how does MT prevent fraying?

Answer 29

minus end has gamma-Tu-RC and + end has GTP-B-tubulin cap which prevents fraying --> fraying = depolymerization

Question 30

how does depolymerization occur?

Answer 30

hydrolysis catches up to + end = all B-subunits are in GDP form * Exact mechanism of assembly is unclear as is role of seam

Question 31

2 requirements for polymerization are

Answer 31

1. above critical concentration 2. all B-tubulin GTP form

Question 32

Colchicine is

Answer 32

MT depolymerizing drug -> remove drug -> see how polymerization occurs

Question 33

Taxol is

Answer 33

stabilizes MT -> MT don’t depolymerize -> remove drug -> see how depolymerization occurs * Anti-cancer drugs: need depoly. of MT in mitosis so taxol can be used to stop depolymerization in cancer cells

Question 34

What are MAPs

Answer 34

Microtubule Associated Proteins * coats MT and stabilizes or destabilizes * can be regulated or bundle MT

Question 35

Example of MAP regulation

Answer 35

Phosphorylation can promote disassembly/destabilization e.g. CDK in cell cycle regulate MAPs

Question 36

MAPs that prevent depolymerization are

Answer 36

MAP2 and Tau - binds to MTs

Question 37

Structure of MAPs MAP2 and Tau?

Answer 37

MT binding domain and projection domain (sticks out of MT to attach to next MT)

Question 38

difference between Tau and MAP2

Answer 38

Large projection domain = MTs are far apart from each other (ex MAP2) * Small projection domain = MTs are close together (ex. Tau) * Size of projection domain can regulate MT spacing

Question 39

What are +TIPs?

Answer 39

special MAPs associated with the (+) end of MTs that when present helps/stabilizes polymerization and when absent depolymerization occurs

Question 40

example of a +TIP?

Answer 40

EB1 * binds to unique structures found on growing (+) end and potentially stabilizes and reducescatastrophe * EB1 moves along the seam of MT towards (+) end possibly transporting something

Question 41

What are XMAP215 and CLASP?

Answer 41

* MAPs (not +TIPs) found in (+) end and have TOG domains * TOG domains bind to growing protofilament at (+) end to help stabilize (+) end to prevent catastrophe by keeping dimers together

Question 42

What does kinesin-13 do?

Answer 42

- with ATP destabilizes MTs by removing terminal dimers

Question 43

What does stathmin do?

Answer 43

* protein that promotes **protofilament bending** by **decreasing** lateral cohesion * bind tubulin dimers in the curve may promote **fraying** and **GTP hydrolysis** * stathmin can be **inactivated** by phosphorylated

Question 44

depolymerization/destabilizing promoting proteins?

Answer 44

* kinesin-13 * stathmin

Question 45

stabilizing proteins?

Answer 45

* MAP2 and Tau * +TIPs (EB1) * XMAP215 and CLASP