Gamma TuRC (MTOC) Flashcards
What does the gamma tubulin ring complex (TuRC) do?
Nucleates microtubules - highly conserved function.
Why is cryo-EM replacing x-ray crystallography to determine structures?
It can achieve a greater resolution.
Which proteins regulate TuRC?
Pericentrin, CM1- domain proteins.
What is the minimal unit of a MT?
Heterodimer of alpha and beta tubulin.
How closely related are alpha and beta tubulin?
30-35% identity.
Which nucleotide is involved in MT assembly?
GTP - it is bound and hydrolysed.
Which end of a MT is fast-growing?
The plus end.
Which end of a MT is slow-growing?
The minus end.
Where is the minus end of a MT usually embedded?
The microtubule organising centre (MTOC).
How can alpha and beta tubulin families interact to form a MT?
Laterally and longitudinally.
What causes MT depolymerisation / catastrophe?
GTP hydrolysis by beta tubulin, ie the loss of the GTP cap. The GDP tubulin (rest of MT is intrinsically unstable and breaks apart.
Why are MTs dynamic (can be polymerised or depolymerised)?
It allows them to explore and be reorganised quickly.
What property allows motor proteins to move along MTs?
They are polar, and the proteins can read their directionality.
Which MT end do kinesin like proteins move towards?
Plus.
Which MT end do dynein like proteins move towards?
Minus.
How does MT polarity help with cellular organisation?
Minus ends all at the centrosome, plus ends all at the periphery.
What are MTs used for within the cell?
- Polarity
- Spindle assembly
- Intracellular trafficking
- Chromosome segregation
How do MTs aid differentiation?
They are organised differently in different cell types.
How are MTs organised in neurons?
Minus ends are towards cell body and plus ends are towards axon terminal.
How are MTs organised in epithelial cells?
Minus ends are towards apical surface and plus ends are towards basolateral surface.
What does the centrosome divide to become during mitosis?
The spindle poles.
Why must the microtubules be organised?
Because otherwise trafficking due to their polarity would have no significant effect.
What are microtubule organising centres (MTOCs)?
Microtubule nucleation sites (where the microtubule initially forms).
What is critical concentration?
The subunit concentration necessary for polymer net assembly ie tubulin. About 15µM.
What do centrosomes do?
Nucleate microtubules at concentrations below their critical concentration, allowing MT assembly at low tubulin concentrations. They also localise the growth of microtubules to one location in the cell.
Why do microtubules not spontaneously assemble?
The concentration of tubulin is low (below critical concentration).
What is gamma tubulin?
Another low abundance (1%) form of tubulin that is conserved in eukaryotic cells with 60-70% identity. Only 30% identity to alpha and beta tubulin.
Where is gamma tubulin enriched?
The electron dense cloud surrounding the centrosome(s). ‘Pericentriolar material’. Indicates involvement of microtubule nucleation activity.
How was the localisation of gamma tubulin determined?
Immuno-electron microscopy (antibodies coupled with gold bound to it, showing electron dense areas surrounding centrioles).
Which technique was used to discover gamma tubulin forms a much larger complex than alpha and beta tubulin dimers?
Density gradient sedimentation. Larger complexes sediment more quickly than smaller ones - not actually to do with density!
What did purification / staining of gamma tubulin complexes with antibodies reveal?
- Their ‘lock-washer’ ring structure
- That this complex was able to promote MT nucleation at lower concentrations than purified tubulin.
How did the gamma tubulins build MTs?
By interacting with alpha / beta tubulin dimers; template for the MT formation. Brings them together in a more stable way than spontaneous assembly.
