Lecture 7 - Microtubules

Question 1

What are microtubules composed of?

Answer 1

alpha-beta tubulin heterodimers

Question 2

Shape of microtubules

Answer 2

Hollow, tube like cylinder

Question 3

What do microtubules take part in

Answer 3

Cell polarity, cell motility, structural support, and intracellular transport

Question 4

Diameter of tube like cylinder

Answer 4

25nm

Question 5

GTP binding sites in ab tubulin heterodimer

Answer 5

Both have GTP binding sites

GTP never hydrolysed by alpha tubulin

GTP hydrolysed by beta tubulin when heterodimer incorporated in microtubule

Question 6

How do microtubule-binding drugs work?

Answer 6

• Prevent microtubule assembly (colchicine) or disassembly (cancer drug taxol)
• Inhibits cellular processes that depend on microtubules and polymer rearrangement

Question 7

GTP on alpha-tubulin

Answer 7

• Never hydrolysed or exchanged with free nucleotide

Question 8

GTP on beta-tubulin

Answer 8

• Hydrolyses bound GTP to GDP
• Exchanges GDP for GTP after subunit disassembles

Question 9

Size of ab-tubulin dimer

Answer 9

8nm

Question 10

Microtubule singlets, doublets, triplets

Answer 10

• Singlet
• Doublet e.g. cilia, flagella (9+2 arrangement)
• Triplet e.g. basal bodies, centrioles

Question 11

Microtubule organzing centres (MTOCs)

Answer 11

• Nucleate and organise microtubules
• Almost all microtubules originate from MTOCs
• (-) ends associate with MTOC

Question 12

Main MTOC in animal cells?

Answer 12

Centrosomes

Question 13

Are spindle poles a MTOC?

Answer 13

Yes

Question 14

Where is MTOC located in nerve cells?

Answer 14

Base of axon

Question 15

What are basal bodies?

Answer 15

• MTOC that incorporates into ‘mother centriole’ - the centriole inherited from previous division

Question 16

Microtubule polarity

Answer 16

• head-to-tail assembly
• A crown of alpha-tubulin at (-) end
• A crown of beta-tubulin at (+) end
• Nonpolarized animal cells, (-) ends associated with MTOCs
• (+) ends extend towards cell periphery
• Motor proteins dynein towards -
• Kinesin (towards+) move in aspecific direction along the microtubule

Question 17

Centriole structure

Answer 17

EM of a centriole – heart of MTOC

Barrel like structures at right angles to each other

9 triplet microtubules arranged in a ring like structure

Surrounded by pericentriolar material

Question 18

What is the third type of tubulin?

Answer 18

Third type of tubulin - gamma

Found in the pericentriolar region

Binds alpha/beta dimers

Template for nucleation

Question 19

Tubulin ring complex

Answer 19

Gamma-TuRC

Gamma tubulin ring complex

Gamma is in red - nucleates the microtubule – starting with a –End at the MTOC

Question 20

Kinetics of microtubule assembly

Answer 20

Initially have dimers - at a certain concentration microtubules can form

At that point the concentration of dimers plateaus as they are being constantly incorporated

Question 21

Microtubules critical concentrations requirements

Answer 21

Microtubule growth dependent on dimer concentration

High growth

Low disassembly

Critical concentration higher at –End then +End

Question 22

Cc Values

Answer 22

• Tubulin dimers assemble into microtubules above the Cc concentration
• Cc different for the + and - ends
• Steady-stae Cc tubulin dimers add to + end at same rate as leave - end - Stays same length, microtubules move down

Question 23

Treadmilling

Answer 23

It can happen, but unsure of concentrations required

Question 24

Dynamic instability

Answer 24

Model is one of dynamic instability

Because they can alternate quickly between growth and disassembly

Centred on the GDP bound to the beta-tubulin

If there’s lots of free dimers – added rapidly – GTP not hydrolysed quickly – exposed beta tubulin is bound to GTP

If supply dries up the exposed beta tubulin will hydrolyse its GTP to GDP – microtubule become unstable - disassembly

Question 25

Microtubule associated proteins (MAPs)

Answer 25

Stabilising MAPs - Promote assembly, increase microtubule assemly, crosslink microtubules

MAP2

Tau tangles on Alzheimers kills brain cells

Cognitive decline

Question 26

Examples of MAPs

Answer 26

MAP2 - Long projection

Tau - Similar binding - Shorter projection

MAP2 KO nerve cell – spacing is disrupted – nerve function changed

Question 27

Tau tangles

Answer 27

Alzheimers disease

Tau tangles

Progression of disease results in an increase in Tau tangles and we know they kill nerve cells

There is another protein involved – beta-amyloid

Beta-amyloid outside the nerve cells

Tau tangles inside the nerve cells

For many years the pharmaceutical companies have tried to target Beta-amyloid (easier target) - billions RD spent – Abs that clear plaques developed

But no cognitive improvement observed
Beta-amyloid not a good indicator of cognitive function
But Tau tangles well correlated
Tau much more important than beta-amyloid

Question 28

Drugs targeting Tau tangles

Answer 28

Salbutamol – asthmatics – relaxes smooth muscle

Being repurposed for Alzeimers – low ethical barriers as well tolerated

Inhibits Tau agreggation

Question 29

End binding microtubule proteins

Answer 29

+TIPs (plus end tracking proteins influence assembly and disassembly) eg EB1 (end binding 1)
Kinesin 13 helps disassembly
Stathmin binds to curved protofilaments and enhances their disassembly

Question 30

Ending binding 1 (EB1)

Answer 30

EB1 – attaches to end of microtubules

Importantly also associates with other end tracking proteins (TIPS)

Question 31

Role of EB!

Answer 31

• Stimulates spontaneous nucleation and growth of microtubules
• Promotes both catastrophes and rescues

Question 32

Kinesin 13 and Stathmin

Answer 32

Roles of Kinesin 13 and Stathmin much clearer

Both involved in disassembly of microtubules from the plus end

Even though they are structurally very different