Section 7: Cytoskeleton and microtubules Flashcards
1
Q
What is the cytoskeleton?
What is it composed of?
A
- Cytoskeleton: an intricate network of protein filaments that extend throughout the cytoplasm of almost all cells
- Composed of 3 types of structures:
- Actin - makes microfilaments (smallest)
- Various proteins - make intermediate filaments (intermediate)
- αβ-tubulin dimer - makes microtubules (largest)
2
Q
Describe some roles of the cytoskeleton
A
- Organelle/protein trafficking
- Cilia/flagella
- Karokinesis/cytokinesis
- Muscle contraction
- Cell adhesion
- Cell migration
- Extravasation
3
Q
Structure of microtubules?
A
- Made up of 13 αβ-tubulin dimers
- 1 monomer of tubulin is 55 kDa (non-dimer form)
- Polarity
- The dimers polymerise into a long chain (protofilament)
- They polymerize at the β (+) end
- The tubes do not roll perfectly; a seam is present
- Results in a jagged end
4
Q
Describe the α and β subunits and their GTP state
A
- α is permanently bound to GTP
- β can hydrolyse GTP, so it may be bound to GTP or GDP
- As the polymer grows, the GTP on the β (+) end is hydrolysed
5
Q
State the 3 arrangements of microtubule protofilaments
A
6
Q
What are the two types of microtubules?
A
- Cytoplasmic (everywhere, except…)
- Axonemal (cilia and flagella)
7
Q
How are microtubules organised?
State the main types of organization
A
- Microtubules originate from a MTOC (microtubule organising centre)
- The (-) end is associated with the MTOC, but an exception is dendrites (no MTOC)
8
Q
What are centrosomes?
What do they contain?
A
- Centrosomes are the major MTOC in non-mitotic cells
- Contain 2 centrioles inside (perpendicular barrel shaped structures)
- Centrioles divide, create a mother and daughter centriole (unknown why they differ)
- Centrioles are triplet microtubules not found in plants (likely due to large vacuoles that take up space)
-
Pericentriolar matrix/material surrounds the centrioles
- These are singlet molecules
- γ-tubulin and augmin complex trigger the singlet polymerisation
9
Q
What is the function of γ-tubulin?
A
- The γ-tubulin ring provides nucleating sites for microtubules
- Nucleation sites accelerate initial polymerization
- Without a nucleus/nucleation sites, microtubule growth is slow and lagged (lag phase)
- It works with many other proteins (ex., augmin)
- It is located at the (-) end
10
Q
How does the microtubule nucleus work?
A
- αβ dimers are rapidly added to the nuclei created by the γ-tubulin
- If the concentration of αβ dimers is above the Cc (critical concentration), polymerisation occurs
- If the concentration of αβ dimers is below the Cc (critical concentration), depolymerisation occurs
- Temperature is also a factor that determines polymerisation/depolymerisation (ex., microtubules disassemble at 4oC
11
Q
Describe the dynamics of microtubules
A
- The (+) ends are constantly growing and shrinking (dynamic instability)
- They “provide new roads constantly” for the material they are transporting/moving
- Experiences catastrophe and rescue
- This is regulated by the concentration of αβ dimers and the critical concentration
- How it moves is specific to the microtubule environment
12
Q
What does dynamic instability depend on?
A
- It depends on the presence of a GTP β-tubulin “cap”
- Recall that polymerisation requires GTP β-tubulin
- GTP β-tubulin prevents the ends of the microtubules from ‘fraying’ at the (+) end via lateral cohesion
- Smooth ends are GDP β-tubulin ends; smooth ends are what fray
- The hydrolysis and revitalisation of GTP β-tubulin is what causes the dynamic instability
13
Q
State the 2 microtubule disrupting drugs
A
- Colchicine: depolymerises microtubules (can get rid of them)
- Taxol: stabilises microtubules (they won’t shrink or grow)
14
Q
What are MAPs?
A
- Microtubule associated proteins
- They:
- Alter microtubule stability
- Bundle microtubules (via projection domains)
- Ex., MAP2 and Tau
- Tau has a smaller projection domain (for tighter bundling
- They have microtubule binding domains
- They are regulated (by phosphorylation)
15
Q
What happens when a MAP is phosphorylated?
A
When MAPs are phosphorylated, they are released and decrease microtubule stability, leading to depolymerisation
17
Q
Describe the 2 microtubule end proteins
A
- Kinesin-13: removes terminal dimers, destabilises the (+) end
- Requires ATP
- Stathmin: binds tubulin dimers at the “curve”
- Promotes GTP hydrolysis (removes β cap)
- Inactivated by phosphorylation
Thus, both can cause depolymerisation even above Cc
18
Q
What is the main function of microtubules?
A
- The main function of microtubules is for vesicle transport (bidirectional)
- Utilises motor proteins (which use ATP)
19
Q
How were motor proteins identified?
A
Squid giant axon is the model system
- Inject radiolabeled amino acids into the squid axon
- The proteins will be transported along the axon
- After a time point, the nerve fragments are isolated at given distances from the injection site
- Protein is isolated and loaded onto an SDS-PAGE gel
- The movement of the proteins can be observed across the time points
- Note that they move at varying speeds
- Note that they often move together in complexes
- Kinesin was identified
21
Q
How does kinesin move cargo?
A
- Light chains recognise the cargo, which has an appropriate receptor
- Heavy chains have ATPase (ATP hydrolysis) and microtubule binding activity
- It uses ATP hydrolysis to walk to the (+) end with its cargo
22
Q
State the 4 classes of kinesin and describe their structure
A
- Kinesin 1 (main)
- Kinesin 2 (main)
- Kinesin 5
- Does not bind cargo
- Involved in microtubule sliding when they move to the (+) end
- Kinesin 13
- Does not bind cargo
- Uses ATP hydrolysis to rip off dimers and depolymerise (+) and (-) end
23
Q
How is cargo transported anterograde?
A
- Kinesin 1 and kinesin 2 move cargo anterograde (+) end using ATP hydrolysis
- 1 ATP = 16 nm of head movement (essentially, β to β dimer)
- They are inactive and folded when cargo is not present
24
Q
How is cargo transported retrograde?
A
- Cytoplasmic dynein moves cargo retrograde (-) end using ATP hydrolysis
- Dynein has a large head domain with ATPase activity
- Uses the dynactin hetero complex to recognise and bind cargo
25
Q
How does the dynactin complex work?
A
- The dynactin complex links dynein to cargo
- This interaction is regulated by dynamitin
- Inappropriate dynamitin levels result in dynactin and dynein exploding apart
- p150Glued helps the cargo move but it is not a motor protein (provides no force)
