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Gamma TuRC (MTOC) Flashcards

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1
Q

What does the gamma tubulin ring complex (TuRC) do?

A

Nucleates microtubules - highly conserved function.

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2
Q

Why is cryo-EM replacing x-ray crystallography to determine structures?

A

It can achieve a greater resolution.

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3
Q

Which proteins regulate TuRC?

A

Pericentrin, CM1- domain proteins.

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4
Q

What is the minimal unit of a MT?

A

Heterodimer of alpha and beta tubulin.

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5
Q

How closely related are alpha and beta tubulin?

A

30-35% identity.

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6
Q

Which nucleotide is involved in MT assembly?

A

GTP - it is bound and hydrolysed.

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7
Q

Which end of a MT is fast-growing?

A

The plus end.

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8
Q

Which end of a MT is slow-growing?

A

The minus end.

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9
Q

Where is the minus end of a MT usually embedded?

A

The microtubule organising centre (MTOC).

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10
Q

How can alpha and beta tubulin families interact to form a MT?

A

Laterally and longitudinally.

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11
Q

What causes MT depolymerisation / catastrophe?

A

GTP hydrolysis by beta tubulin, ie the loss of the GTP cap. The GDP tubulin (rest of MT is intrinsically unstable and breaks apart.

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12
Q

Why are MTs dynamic (can be polymerised or depolymerised)?

A

It allows them to explore and be reorganised quickly.

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13
Q

What property allows motor proteins to move along MTs?

A

They are polar, and the proteins can read their directionality.

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14
Q

Which MT end do kinesin like proteins move towards?

A

Plus.

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15
Q

Which MT end do dynein like proteins move towards?

A

Minus.

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16
Q

How does MT polarity help with cellular organisation?

A

Minus ends all at the centrosome, plus ends all at the periphery.

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17
Q

What are MTs used for within the cell?

A
  • Polarity
  • Spindle assembly
  • Intracellular trafficking
  • Chromosome segregation
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18
Q

How do MTs aid differentiation?

A

They are organised differently in different cell types.

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19
Q

How are MTs organised in neurons?

A

Minus ends are towards cell body and plus ends are towards axon terminal.

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20
Q

How are MTs organised in epithelial cells?

A

Minus ends are towards apical surface and plus ends are towards basolateral surface.

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21
Q

What does the centrosome divide to become during mitosis?

A

The spindle poles.

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22
Q

Why must the microtubules be organised?

A

Because otherwise trafficking due to their polarity would have no significant effect.

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23
Q

What are microtubule organising centres (MTOCs)?

A

Microtubule nucleation sites (where the microtubule initially forms).

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24
Q

What is critical concentration?

A

The subunit concentration necessary for polymer net assembly ie tubulin. About 15µM.

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25
Q

What do centrosomes do?

A

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.

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26
Q

Why do microtubules not spontaneously assemble?

A

The concentration of tubulin is low (below critical concentration).

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27
Q

What is gamma tubulin?

A

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.

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28
Q

Where is gamma tubulin enriched?

A

The electron dense cloud surrounding the centrosome(s). ‘Pericentriolar material’. Indicates involvement of microtubule nucleation activity.

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29
Q

How was the localisation of gamma tubulin determined?

A

Immuno-electron microscopy (antibodies coupled with gold bound to it, showing electron dense areas surrounding centrioles).

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30
Q

Which technique was used to discover gamma tubulin forms a much larger complex than alpha and beta tubulin dimers?

A

Density gradient sedimentation. Larger complexes sediment more quickly than smaller ones - not actually to do with density!

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31
Q

What did purification / staining of gamma tubulin complexes with antibodies reveal?

A
  • Their ‘lock-washer’ ring structure
  • That this complex was able to promote MT nucleation at lower concentrations than purified tubulin.
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32
Q

How did the gamma tubulins build MTs?

A

By interacting with alpha / beta tubulin dimers; template for the MT formation. Brings them together in a more stable way than spontaneous assembly.

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33
Q

Gamma complex peptides (GCPs) properties:

A
  • Found in complex with gamma tubulin.
  • Conserved in mammalian cells.
  • Similar sequences to each other - GRIP motifs 1 and 2 (gamma ring interacting proteins).
34
Q

How do we know gamma tubulin is in complex with GCPs?

A

They cosediment in density gradient sedimentation.

35
Q

What is the gamma TuRC made of?

A

Gamma tubulin and GCPs 2/3/4/5/6.

36
Q

What is the gamma TuSC made of?

A

Gamma tubulin and GCPs 2/3.

37
Q

Does gamma TuSC promote MT nucleation alone?

A

No.

38
Q

Does gamma TuRC promote MT nucleation alone?

A

Yes.

39
Q

What is the composition of gamma TuRC?

A

Helix of gamma TuSCs (and some TuSC-like complexes with other GCPs). Stalks slot into the centre of the ring, in line with gamma tubulin of turn below. Not radially symmetric. See slide 19, lecture 5

40
Q

Which eukaryotes are missing GCPs 4/5/6?

A

Budding yeast. But they still nucleate MTs fine!

41
Q

How was a 3D image of gamma TuSC built?

A

‘Class average’ images of gamma TuSC in various orientations were combined by a computer.

42
Q

What is the structure of gamma TuSC?

A

Y shape - stalk and arms are GCP 2 and 3 and on the tips of the arms are gamma tubulins.

43
Q

What is the structure of GCP4?

A

Almost identical to GCP 2 / 3 ‘bent finger’. Conferred by only the GRIP motifs.

44
Q

What do the GRIP motifs in GCPs confer?

A

‘Bent finger’ shape.

45
Q

Do all GCPs have the ‘bent finger’ shape?

A

Yes

46
Q

What is the contribution of GCP 4 / 5 / 6 in gamma TuRC?

A

They dimerise with any other GCPs to make gamma TuSC-like complexes with the same function as gamma TuSC.

47
Q

Is gamma TuRC structure variable?

A

Yes - there can be a range of combinations of gamma TuSCs and TuSC-like proteins contributing.

48
Q

Is the gamma TuSC order conserved in metazoans?

A

Yes. 4 x 2/3, 1 x 4/5, 1 x 4/6, 1 x 2/3 etc.

49
Q

How many subunits are in 1 turn of the gamma TuRC helix?

A

13 (6 and a half dimers).

50
Q

What does the lack of radial symmetry in gamma TuRC indicate?

A

It is not an active nucleator.

51
Q

Why are GCP 4 / 5 / 6 dimers on one side of the ring?

A

They form the base from which the gamma TuRC will assemble in metazoan cells.

52
Q

What is at the base of the ‘cone’?

A

A single actin molecule and 2 copies of Mzt1 microprotein. Form the luminal bridge with N termini of GCP 3 and 6.

53
Q

What are outstanding technical issues surrounding cryo EM structure of gamma TuRC?

A
  • Unassigned densities
  • Conflicting assignments
  • Missing/unresolved regions of proteins
  • Missing/unresolved interactors
54
Q

What are outstanding biological issues surrounding cryo EM structure of gamma TuRC?

A
  • MT nucleation by gamma TuRC is relatively inefficient, consistent with structure (too flat and too wide compared to a MT).
  • Is conformational change needed to “activate” g-TuRC?
    How would it work? Self-induced? Interactors?
  • Gamma TuRC composition and mutant phenotypes are different in different organisms; not one structure anyway and may behave slightly differently.
  • Normally, gamma TuRC that nucleates MTs is localized to MTOCs (nucleus).
  • Is cytosolic form (one being studied) missing key components?
  • How does gamma TuRC localize to specific sites?
55
Q

Where does pericentrin localise?

A

Pericentriolar material (PCM).

56
Q

How does gamma TuRC get targeted to specific cells?

A

Several partially redundant mechanisms including:
- Pericentrin
- CDK5RAP2/Cep215

57
Q

What is centrosomal maturation?

A

Gamma TuRC recruitment to centrosome / PCM is increased in mitosis by cell-cycle kinases, e.g. Plk1 (Polo-like kinase), CDK. MT nucleation capacity increases. May also suppress non-spindle nucleation.

58
Q

What happens in mitosis when pericentrin is knocked down?

A

MTs are not well organised at spindle poles because gamma tubulin is not being localised to a single site.

59
Q

What do mutations in the pericentrin gene cause?

A

MOPD II/Seckel syndrome - very similar dwarfisms.

60
Q

What is MOPD II/Seckel syndrome?

A

A primordial dwarfism (from start):
- Not associated with growth hormone deficiency
- Small stature
- Small heads (proportional to body size though)
- Range of intellect due to decreased brain development

61
Q

Why does the pericentrin mutation cause dwarfism?

A

There is a lack of increase in cell number during development.

62
Q

What are the competing interpretations of how pericentrin mutation leads to disease?

A
  • MT nucleation model (defects in chromosome segregation therefore lots of daughter cells are apoptosed).
  • DNA damage model (ATR protein in the centrosome senses DNA damage and arrests cell cycle. ATR localised to centrosome by pericentrin. Mutation in it means cells progress to mitosis with damaged DNA which also means lots of daughter cells are apoptosed).
63
Q

Where do fission yeast nucleate MTs from in interphase?

A

Multiple sites on the surface of the nucleus and in the cytoplasm. No single centrosome.

64
Q

Where do fission yeast nucleate MTs from in mitosis?

A

Spindle pole bodies (SPBs).

65
Q

Where do fission yeast nucleate MTs from in cytokinesis?

A

MTOCs at the acto-myosin ring.

66
Q

What phenotype do mutations in mto1 and mto2 cause?

A

Curved cell shape (fission yeast). Due to microtubule defects.

67
Q

What is unusual about MT formation in fission yeast in interphase?

A

There is GCP2 associated along the MT, not just at the MTOC.

68
Q

What do the MTO1 and MTO2 proteins do?

A
  • Complex with each other and recruit the gamma TuRC to the MTOCs.
  • Activates the gamma TuRC.
69
Q

How big is the MTO1/2 complex?

A

Similar size to gamma TuRC.

70
Q

How was it discovered the MTO1/2 complex has an activation function as well as localisation?

A

Localisation domains were removed in vivo - protein truncated, so the complex was free floating in cells. Sufficient to activate MT nucleation. Replicated results in vitro.

71
Q

What is Mto1/2bonsai?

A

The truncated version of Mto1/2 without the localisation domain.

72
Q

What is the activation domain in Mto1?

A

The CM1 domain - ≈60aas, highly conserved between many organisms.

73
Q

What happens when CM1 is mutated?

A

Can’t bind to gamma TuRC, MT nucleation inhibited.

74
Q

Which human protein contains the CM1 domain?

A

CDK5RAP2

75
Q

What do mutations in CDK5RAP2 cause?

A

Primary autosomal recessive microcephaly (small brain size (particularly cortex), but not dwarfism).

76
Q

When does primary autosomal recessive microcephaly show up?

A

Consanguineous populations (marriages within families).

77
Q

Why are primary autosomal recessive microcephaly / CDK5RAP2 mutations hard to study?

A

We have lots more complex brain development than other model organisms like mice.

78
Q

What are hypotheses for why CDK5RAP2 mutations cause fewer cells?

A
  • Not enough stem cells (radioglial cells (RDGs)) to make the neurons (SCs have defects in division).
  • Stem cells prematurely switch to differentiating; not enough stem cells produced before neurons start forming from them.
  • Defects in neurogenic divisions; neuronal cells not produced correctly when SC divides.
79
Q

How can defects in spindle orientation (MT formation) affect neuron production from stem cells (RGC)?

A
  • Division with a vertical cleavage plane produces 2 new RGCs.
  • Division with an off-vertical cleavage plane produces 1 RGC and 1 neuron (neurogenic division).
80
Q

What have we discovered recently about gamma TuRC?

A
  • It changes conformation to become symmetric once it begins nucleating. Does first nucleation confer change?
  • Also looks like CDK5RAP2 is able to confer the conformational change for gamma TuRC to become radially symmetric.
  • Actin molecule kicked out when gamma TuRC becomes active. Does it keep it inactive?

Cell Biology (7 decks)

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