microtubules

Question 1

what are the three types of cytoskeletal filaments?

Answer 1

• actin microfilaments
• aB tubular microtubules
• intermediate filaments

Question 2

what are the properties of the cytoskeleton?

Answer 2

• dynamic but organised structure
• formed from the polymerisation of small subunit proteins
• provides cellular structure and organisation
• each type of cytoskeletal filament has unique properties that allow it to perform specific functions for the cell

Question 3

what are the functions of intermediate filaments?

Answer 3

cell strength and mechanical support

Question 4

what are the functions of actin microfilaments?

Answer 4

• cell structure and shape

- cell migration

Question 5

what are the functions of microtubules?

Answer 5

• organisation of intracellular structures and organelles
• intracellular vesicle trafficking
• chromosome segregation during mitosis

Question 6

what is the structure of microtubules?

Answer 6

• dimer of alpha and beta tubulin in a 13 protofilament hollow tube
• a tubulin has a GTP binding site which is constantly GTP bound
• B tubulin has an exposed GTP binding site
• microtubules have a polarity

Question 7

what is the ‘minus end’ of a microtubule?

Answer 7

the end with a-tubulin at the end

Question 8

what is the ‘plus end’ of the microtubule?

Answer 8

the end with B-tubulin at the end

Question 9

which end of the microtubule are new subunits added and removed from?

Answer 9

added- plus end

removed - minus end

Question 10

what is the significance of the B-tubulin at the ‘plus end’ of a microtubule?

Answer 10

the exposed GTP/GDP binding site permits hydrolysis, allowing it to regulate dynamics

Question 11

what is the role of the centrosome?

Answer 11

it is the major microtubule organising centre of the cell

Question 12

what is the structure of the centrosome?

Answer 12

consists of two pairs of centrioles and is surrounded by a protein network that is capable of nucleating microtubules

Question 13

what end of the microtubule is nucleated at the centrosome?

Answer 13

the minus end

Question 14

how is microtubule dynamics regulated by GTP hydrolysis?

Answer 14

• dimer with GTP bound is added to the plus end, over time, this can be hydrolysed
• this causes a bend in the protofilament, de-stabilising the microtubule
• this causes GDP bound subunits to dissociate
• if GTP is bound to the plus end, this is a stabilised microtubule

Question 15

what is meant by dynamic instability of the microtubule?

Answer 15

there is a cap of GTP bound a/B tubulin, this stabilises the plus end

• in this state, the microtubules are able to grow
• however, it can switch to an unstable state rapidly when GTP is hydrolysed, causing curvature of the filament, de-stabilisation and rapid de-polymerisation of the microtubule

Question 16

what is the function of dynamic instability?

Answer 16

it allows rapid re-organisation of microtubules in response to appropriate cell stimuli

Question 17

what are the functions of microtubule associated proteins?

Answer 17

regulate microtubule dynamics by:

• stabilising filaments
• destabilising filaments
• linking filaments to other molecules or structures

Question 18

how do MAPs control microtubule dynamics?

Answer 18

controlling when microtubules:
remain stable
depolymerise
grow

Question 19

how does Tau regulate microtubules?

Answer 19

binds to the side of the microtubule, keeping it in rigid conformation
this allows them to form parallel bundles

Question 20

what is kinesis-13 and how does it regulate microtubule dynamics?

Answer 20

• end binding protein that promotes catastrophe during mitosis
• causes curvature of the protofilaments, destabilising them
• they are motor proteins that use ATP

Question 21

what feature of the microtubule allows motor proteins to travel in opposing directions down the microtubule?

Answer 21

polarity

Question 22

which direction do kinesins move cargo?

Answer 22

towards the plus end, at the periphery of the cell

Question 23

which direction do dyneins move cargo?

Answer 23

towards the minus end, at the centrosome

Question 24

how do kinesins move?

Answer 24

a conformational change upon ATP hydrolysis allows one subunit to be moved in front of the other

Question 25

what are the major functions of microtubules?

Answer 25

• determinant of intracellular organisation and movement
• positioning of organelles
• intracellular trafficking
• cell polarity
• formation of the mitotic spindle
• formation of cilia and flagella

Question 26

give an example of when microtubules might contribute to cell polarity

Answer 26

in epithelial cells

the minus end of the microtubule is aligned to the apical domain and the plus end is aligned to the basolateral domain

Question 27

which techniques might you use to investigate microtubule function?

Answer 27

• use of an inhibitor of the protein
• expression of a mutant that impairs or abolishes protein function
• knocking down protein expression by RNAi

Question 28

what is nocodazole?

Answer 28

a drug used to depolymerise microtubules. binds to free a/B tubulin, preventing it from incorporation into growing microtubules

Question 29

how can we demonstrate which motor protein is responsible for a function?

Answer 29

• dynein inhibition

- kinesin inhibition

Question 30

how can we inhibit dynein?

Answer 30

• use inhibitor such as EHNA

- use RNA interferences to knock down expression of one of the dynein subunits

Question 31

what happens when you overexpress a kinesin dominant headless mutant?

Answer 31

• binds to cargo by the tail domain
• outcompetes endogenous protein but cannot bind to the microtubule
• prevents cargo from being transported to its destination