lecture 8

Question 1

Microtubule motor proteins

Answer 1

Dynein and Kinesin

Question 2

What do microtubule motor protein do?

Answer 2

Transport membrane bound vesicles, proteins and organelles at expense of ATP

Question 3

What direction do kinesins move cargo?

Answer 3

(+) end of microtubule - anterograde

Question 4

What direction do dyneins move cargo?

Answer 4

(-) end of microtubule - Retrograde

Question 5

What does kinesin family domain contain?

Answer 5

• ATP binding site
• Microtubule binding site of motor
• Neck domain takes steps - connects motor domain to stalk domain
• Cargo binding domain

Question 6

Kinesin 1

Answer 6

Globular, bulbous region at one end of molecule - region enlarged when antibody that binds to motor domain is present

• Head or feet hydrolyse ATP to generate energy for neck movement
• Has a stalk, tail and binding site in foot

Question 7

Kinesin 1 cargo transport

Answer 7

• Each requires 1 ATP movement in neck
• Cargo binding site
• Anterograde

Question 8

Describe the different kinesins types

Answer 8

Kinesin 1 (conventional) - 2 heads, stalks the same

Kinesin 2 (heterotrimeric) - head and stalks differ

Kinesin 5 (bipolar) - 4 head and stalks - sliding - mitosis to separate chromosomes

Kinesin 13 (KinI) - End disassembly

Question 9

Describe the process of kinesin movement along microtubules

Answer 9

1. Forward motor binds beta-tubulin, releasing ADP
2. Forward head binds ATP
3. Conformational change in neck linker causes rear head to swing forward
4. New forward head releases ADP trailing head hydrolyses ATP and releases Pi

Question 10

Catalytic core of kinesin is similar to catalytic core of what other molecule?

Answer 10

Myosin

Question 11

Cytosolic dyneins

Answer 11

• Retrograde (transport cargo towards - end of microtubule)
• Very different structure to kinesins
• Do have ATPase and microtubule binding domains
• Linked to cargos by large complexes of microtubule binding proteins

Question 12

Dynein structural components

Answer 12

• ATPase domain (head)
• Microtubule binding domain (stalk)
• Dynactin binding domain (Stem)

Question 13

Dynactin

Answer 13

• Near domain hydrolyses ATP which generates a force
• Results in far mT binding domain detaching and moving forward one step
• Cargo binding domain - huge complex which includes dynamitin, CapZ, Arp1
• Required for cargo attachment
• P150glued - helps dynein attach to microtubule at start of transport process

Question 14

What happens at the end of a microtubule?

Answer 14

• Motor proteins hitch a ride back with cargo of other motor proteins
• 600nm per second - centre to periphery of cell in 2-3 seconds
• Sciatic nerve over 1m long in some people - days rather than hours

Question 15

Tau tangles

Answer 15

• Cause Alzheimer’s disease
• Microtubule disintegrates with subunits falling apart
• Causes tangled clumps of tau proteins

Question 16

Cilia and flagella

Answer 16

• Contain microtubules
• 9+2 arrangement with 9 outer doublet microtubules and 2 central pair microtubules
• 13-protofilament microtubule and 10-protofilament microtubule with each outer doublet
• Central pair contains 13 protofilaments each

Question 17

What does cell movement depend on?

Answer 17

Axoneme bending

Movement requires bending – force generated by axonemal dyneins (ATP)

Crosslinking prevents sliding and results in bending

Question 18

Axonemal dynein

Answer 18

1. Cargo binding domain permanently fixed to A tubule
2. Force generated by ATP hydrolysis in heads
3. Force attempts to slide
4. Nexin crosslinks prevent sliding
5. Wave like motion

Question 19

Nexin links

Answer 19

Microtubules slide past each other if:
- Nexin links removed by protease
- ATP provided

Question 20

3 types of microtubules in spindle

Answer 20

Kinetochore - Connect chromosomes and spindle poles via kinetochore attachment site

Polar - Overlap, Hold poles together, regulate pole-to-pole distance

Astral - Radiate from each spindle pole towards cortex of cell, position spindle and determine plane of cytokinesis

Question 21

Size of intermediate filaments

Answer 21

10-12nm

Question 22

Do intermediate filaments have motor proteins?

Answer 22

No

Question 23

What are intermediate filaments

Answer 23

• Formed from family related proteins e.g. keratin or Lamin
• Strong, ropelike structure
• Provide nuclear membrane support or for cell adhesion

Question 24

5 classes of intermediate filaments

Answer 24

Class I - Acidic keratins - epithelial cells - tissue strength and integrity

Class II - Basic keratins - epithelial cells - tissue strength and integrity

Class III - Desmin (muscle) - Sarcomere
GFAP (glial cells) - Organisation
Vimentin (mesenchymal cells) - Integrity

Class IV - Neurofilaments - neurons - axon organisation

Class V - Lamins - nucleus - nuclear structure and organisation

Question 25

What do intermediate filaments do?

Answer 25

- Mechanical support to plasma + nuclear membrane

Question 26

Why are intermediate filaments more stable than microfilaments and microtubules?

Answer 26

alpha helical rod structure of subunits which overlap along filament

Question 27

Basic structure of intermediate filaments

Answer 27

- 10nm diameter filaments - Globular N-terminal and C-terminal domains connected by central helical core domain - Responsible for filament organization and interaction - Core domain conserved among all IFs - mediates dimer and tetramer formation - N and C terminals differ and therefore distinguish IFs - Parallel dimer then antiparallel tetramer - C terminals at both ends. N terminals in middle

Question 28

Protofibrils

Answer 28

Tetramer forms protofilament Protofilaments form protofibrils Protofibrils twist into rope like microfilament

Question 29

What di IFAPs do in IFs

Answer 29

Connect different intermediate filaments

Question 30

Medical importance of intermediate filaments

Answer 30

- Markers for specific cell types e.g. stem cells - Mutation to IF genes can also result in diseases like Epidermolysis bullosa simplex - Mutation in keratin 14 gene