Lecture 3 - Intermediate Filaments and Microtubules.

Question 1

Out of the three types of filaments, which two show the most overlap?

Answer 1

Microfilaments and intermediate filaments.

Question 2

5 types of IF proteins?

Answer 2

Keratins (I are acidic & II are basic), Vimentin (III), Neuronal IF proteins (IV) and Lamins (V).

Question 3

Keratins (types I and II).

Answer 3

• They are found in the outer epithelia. Prominent in skin, hair and nails.
• Heterodimers - always found in pairs, one acidic and one basic.
• Red keratin staining used for their identification.

Question 4

Vimentin (type III).

Answer 4

• Widely distributed in many different cells.

- Supports cell membranes + keeps organelles correctly positioned.

Question 5

Neuronal IF proteins (type IV).

Answer 5

• Role in neurotransmission –> they determine the diameter of the axon, which in turn determines the speed of conduction.

Question 6

Lamins (type V).

Answer 6

• Support the inner nuclear membrane –> determine the structure of the nucleus.
• Play a role in the organisation of different types of chromatin.

Question 7

IF proteins share a general structure. Describe this.

Answer 7

They have a globular head and a globular tail (N- and C- termini) with a long alpha helical region inbetween.

Question 8

How do IF filaments assemble?

Answer 8

1. 2 IF monomers wrap around each other to form parallel dimers.
2. 2 parallel dimers then go head to tail = antiparallel tetramer.
3. 2 antiparallel tetramers stack end-on-end = protofilaments.
4. 2 protofilaments come together to form a protofibril.
5. 4 protofibrils wrap around each other to form the 10nm IF fibre.

Question 9

Where is the majority of sequence diversity found between IF proteins?

Answer 9

N- and C- termini globular domains. The alpha helical region is highly conserved.

Question 10

Give an example of a heteropolymer.

Answer 10

Keratins - will always be Type I and Type II.

Question 11

What determines which proteins become homopolymers and which become heteropolymers?

Answer 11

Spacer sequences in the alpha helical domain.

Question 12

There is one key difference in the formation of IF fibres in comparison to the formation of MTs and MFs. What is this?

Answer 12

Does not require ATP/GTP.

Question 13

Keratins and the epidermis.

Answer 13

The skin has several layers of cells. First layer = skin stem cells in the basal epidermal layer. As they differentiate they change keratin expression.

Question 14

Name two diseases associated with IFs.

Answer 14

Bilstering disease - epidermolysis bullosa simplex (EBS).

Hutchinson Gilford Progeria

Question 15

Blistering disease - Epidermolysis Bullosa Simplex.

Answer 15

Caused by mutations in the N- and C- termini. Protofilaments cannot form. The basal epidermis cannot adhere properly; epidermis and dermis separate easily.

Question 16

Hutchinson’s Gilford Progeria.

Answer 16

Caused by mutations in the LMNA gene. It is a premature ageing disease that produces an abnormal form of Lamin A.

Question 17

Which type of tubulin binds GTP reversibly?

Answer 17

Beta tubulin. Can hydrolyse GTP for GDP. Alpha tubulin binds GTP irreversibly.

Question 18

Assembly of MTs?

Answer 18

1. Tubulin dimers come together in a head-to-tail manner; protofilament formation.
2. Protofilaments come together laterally. Form a sheet that curves to form the 25nm tube.
3. Have a (+) end and a (-) end. Beta tubulin is found at the (+) end.
4. If the rate of addition is greater than the rate of hydrolysation then a GTP cap will form.
5. Assembly is concentration dependent - must be above Cc for addition at both ends (greater at the (+) end).
6. Once pool is depleted = depolymerisation = greater at the (-) end due to GTP cap.

Question 19

What does MTOC stand for? Where are they located?

Answer 19

Microtubule Organising Centre. Just outside the nucleus.

Question 20

Main functions of the MTOC?

Answer 20

They direct the organisation of MTs, the movement of vesicles and the orientation of organelles.

Question 21

In animal cells, what is the MTOC called?

Answer 21

The centrioles.

Question 22

Cells with flagellar have an additional MTOC. Name this structure and explain its function.

Answer 22

Basal body. Organises MTs in the flagellar so they can generate the force for movement.

Question 23

Name the two motor proteins that associate with the MTs for vesicular transport.

Answer 23

Kinesins and dyneins (for MFs it is Type I and V myosins).

Question 24

Kinesins and dyneins perform movement in opposite directions. Explain this.

Answer 24

Kinesins –> movement AWAY from the MTOC –> Anterograde transport.
Dyneins –> movement towards the MTOC –> Retrograde transport.

Question 25

MTs and MFs are located in different areas of a cell. How can a single cargo access all areas?

Answer 25

Carry both kinesin and myosin motors, which can be switched.