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Microtubules Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

what is the subunit component of microtubules?

A

tubulin heterdimers of alpha and beta

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2
Q

when alpha beta tubular heterodimers assemble, what do they form?

A

protocilaments

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3
Q

how many protofilaments are there normally in a microtubule?

A

13

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4
Q

what is at the growing end of a microtubule?

A

a GTP-rich cap. This cap becomes hydrolysed to GDP over time. These molecules are bound to the tubular heterodimers. The GTP cap s more stable that the GDP tubular areas. The microtubules form by the formation of an initial sheet which tapers

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5
Q

what happens to the conformation of the tubulin heterodimers when GTP is hydrolysed?

A

they acquire a higher curvature, this puts strain on the lattice and fragilises it. this results in a peel back of the microtubule

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6
Q

explain why microtubules are highly dynamic even in normal conditions

A

they undergo rapid cycles of growth and shrinkage. Growth is associated with a GTPcap, there is normally rapid growth with GTP-capped end of microutubules. this hydrolisation of the GTP can happen so quickly that the cap is lost. this is the catastrophe phase and there is rapid shrinkage which will enable a new cap to form. this is the rescue stage. By measuring the velocity of growth and shrinkage and frequency of rescue and catastrophe, you can study the dynamics of different microtubules

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7
Q

what are nucleators?

A

they are protein complexes which act as a template from which microtubules can polymerise. Once heterodimers bind to the, polymerisation is thermodynamically favourable.

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8
Q

what is the main microtubule nucleator?

A

gamma tubulin ring complexes such as gamma TUSC

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9
Q

how does gamma TUSC work?

A
  • they form gamma tubular ring complexes when it initially binds to the alpha beta complexes in a ring, they are slightly out of place. Once in an activated conformation, they form a microtubule. The specific dynamics of this activation are unclear, but the proteins MTo1 and MTO2 have been implicated in activation at microtubule organising centres.
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10
Q

where are gamma tubular ring complexes enriched?

A

at the centrosomes, the complexes associate at the - end of the microtubules and then they have the _ ends radiating out

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11
Q

what is a centrosome?

A

the main place in the cell where microtubules are organised

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12
Q

what can gamma TUSCs use to bind them to the side of existing microtubules to form branches?

A

augmin

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13
Q

at the centrosome, where are nucleating sites found?

A

in the centrosome matrix which forms a sphere around a pair of contrioles.

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14
Q

what are 5 types of proteins that can interact with microtubules?

A
  • cappers and +tips which bind to either end of the microtubules
  • sequesterers which sequester heterodimers
  • bundling proteins and stabilisers with will break up microtubules faster than normal
    motors: dynein and kinesin
  • nucleators: gamma TUSC
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15
Q

in what direction to dyneins move?

A

all are minus end directed

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16
Q

in what direction do kinesins moves?

A

different families have different directionality’s

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17
Q

what do motors need to bind to function?

A

they are Atlases so they need to bind and hydrolyse ATP to work

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18
Q

what are two roles of kinesin?

A

they can act to transport things but also to slide antiparallel microtubules apart

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19
Q

what are the 4 phases of mitosis?

A

prophase, metaphase, anaphase and telophase

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20
Q

what happens in prophase, pro metaphase, metaphase, anaphase (a&b), and telophase?

A

prophase: chromosomes are already condensed and replicated and the centrosomes have been duplicated
prometaphse: chromosomes become captured by microtubules that extend from the spindle poles and then become aligned along the metaphase plate. Each chromosome being connected at its kinetochore.
metaphase: spindles become bipolar
anaphase A: chromosomes move towards each pole
anaphase B: the poles move apart
telophase: two daughter cells separate from each other.

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21
Q

how was it found that there are differences in the dynamics of microtubules in metaphase compared to interphase?

A

they transfected cells with tubular GFP, one part of the cell is exposed to a high laser power to bleach the chloroform of the GFP, then they look how long it takes for fluo recovery to occur. You find that recovery takes only 2 minutes in metaphase- a lot longer in interphase.

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22
Q

what is the main way that mitosis microtubule studies occurred?

A

using ovum extract from xenopus. If you use the cytoplasmic extract, with ATP and nucleic acids from frog sperm and add tubulin, microtubules forms spontaneously and the spindle forms as if cell division was occurring.

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23
Q

what is the general structure of the mitotic spindle?

A

two centrosomes with microtubules radiating out of them. The minus ends of the microtubules are found in the periphery of the centrosomes and the plus ends are radiating outwards.

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24
Q

what are the 3 main microtubule functions within the spindle?

A
  • the spindle microtubules which like the poles
  • the kinetochore microtubule bundles (K-fibres) that link chromosomes to the poles
  • the astral spindles that tether the centrosome to the actin cortex and this plays a role in anaphase.
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25
Q

how does kinesin and the microtubules connect the two poles of the spindle?

A
  • the kinesin-5 is a plus ended motor which bind different microtubules at each end. This keeps the microtubules form either end under tension, keeping the poles together
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26
Q

how do spindles assemble and how was this shown?

A

if you put dyneins together in a tube with lots of microtubules, they will move towards the minus end, with each ‘foot’ on a different microtubule. This causes a radial structure to form. If you put kinesin 5 with microtubules (+ end directed) you get each head associated with a microutule each that will want to go towards the + end and you end up with the linking of microtubules at the linked at the + ends of microtubules. This gave rise to the theory that in all of these things are put together then a spindle will spontaneously assemble. if you do this in purified egg extract with chromosomes then this happens.
- if you inhibit either kinesin 5 or dynein you lose the structure

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27
Q

what is the kinetochore?

A

a complex which binds K-fibres to chromosomes. the outer kinetochore binds microtubules and transducers microtubule motor activity signals. The inner kinetochore is the chomartin interface. There is also the inner centromere which regulates chromatid cohesion

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28
Q

briefly, what is the aim of metaphase?

A

to ensure that each sister chromatid is attached to only one opposite pole (biorientation) to its pair and that every one is attached to a k-fibre.

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29
Q

what are the 5 main stages of metaphase?

A
  • the capture of chromosomes by microtubules
    conversion of chromosomes to the metaphase phase
    verification of kinetochore attachment: anaphase wait signal
  • verification of spindle biorientaton
  • satisfaction of spindle assembly check point
30
Q

how are chromosomes captured by microtubules and become orientated at the end?

A
  • microtubules grow out of the centrosome
  • by chance they come in to contact with a chromosome via the kinetochore and it bound laterally
  • chromosomes slide towards the minus end of the microtubule
  • the kinetochore established an end on interaction with a microtubule
  • sister kinetochore attaches microtubules from the opposite pole.
31
Q

how does conversion occur?

A

it occurs via micro tubule polymerisation and via the kinesin CENP-E. The microtubule polymerises at the kinetochore and then the chromosome is moved towards the + end.

32
Q

wat is the origin of the anaphase wait signal

A

the kinetochore

33
Q

how was it shown that the kinetochore is the source of the wait signal?

A
  • if you take a spindle which has a chromosomes with an unattached kinetochore, you find that upon destruction of the kinetochore, the cell enters anaphase SO this suggests that the wait signal was from the kinetochore
34
Q

how does Mad2 orchestrate the anaphase wait signal generally and the initial evidence for it?

A

an unbound kineotchore binds mad2. when the kinetochore binds the microtubule, a dynein-dependent process releases Mad2 from the kinetochore. If you inhibit a dynein during this process, anaphase is blocked. if you add dynein inhibitor then a mad-2 antibody, anaphase ensues. This show that it is dynein disinhibiting anaphase via mad2.

35
Q

describe in detail the molecule mechanisms behind how mad protein orchestrates the anaphase wait signal

A

KNL1 and NDC80 are core kinetochore proteins that form a scaffold for checkpoint protein recruitment. MPS1 is recruited to NDC80 and phosphorylates KNL1. This allows BUB1 and BUB3 to bind. these form another complex with bub3 and bubr1. Bub is then phos by MPs1 and recruits mad2. Mad1 converts mad2 into a closed conformation when bound. this closed mad2 is then released and forms a complex with BUB3 and BUBR1 to bind to CDC20. This prevents cdc20from binding to APC/C. thereby inhibiting APC/C role in degrading cyclinB and Securing to trigger anaphase.

36
Q

how is the wait anaphase signal silenced by microtubule binding?

A

loss of mad1/2 from the kinetochore halts Mad2 conversion and Mitotic checkpoint formation.
- dynein localises to the kinetochore through RZZ-spindly, and microtubule binding to kinetochores allows RZZ-spindly to be transported by dynein to the poles. Mad1 and mad2 and bubr1 are also removed by dynein during this process, coupling microtubule binding to stripping of kinetochore checkpoint proteins. This allows cdc20 to bind apc/c and degrade proteins

37
Q

what is the evidence that not all anaphase wait signal deactivation is dynein dependent?

A

depletion in spinldy, the dynein receptor, does not completelyy prevent mad2-mad2 removal, suggesting that spindly might normally work with another paralell removal pathway.

38
Q

what is the PP1 method of silencing the anaphase wait signal? what about the direct effect of microtubule binding on mad1 and mad2?

A

Protein phosphatase 1 (PP1) associates with kinetochores and is important for checkpoint silencing
PP1 activity promotes removal of budding uninhibited by benzimidazole 1 (BUB1)–BUB3, although it is not known whether it promotes dissociation of BUB1–BUB3 from MAD1–MAD2. Other phosphatases might also be important for silencing.

Microtubules might silence the checkpoint by physically displacing MAD1 or inducing conformational change at the kinetochore that reduces MAD1 affinity. d | MPS1 activity promotes its own dissociation from the kinetochore

39
Q

what is monotely?

A

when only one kinetochore is connected to a pole and the other isn’t

40
Q

what is syntely?

A

when both chromatids are connected to the same pole

41
Q

what is merotely?

A

when one kinetochore is connected to two poles and one to one

42
Q

what is amphitely?

A

each kinetochore is connected to the opposite pole

43
Q

what does bi-orentiated mean?

A

amphitely

44
Q

what is the experimental evidence for a connection between tension on the kinetochore and phosphoylation?

A

chromosomes within a spindle were artificially grabbed and pulled. they were then fixed and an antibody against phosphorylated kinetochore revealed that phosphorylation decreases with tension

45
Q

how is tension applied to the kinetochor by the microtubules?

A

there is depolymerisation and polymerisation at the kinetochore ends of microtubules which apply force in each direction

46
Q

what was determined by looking at the movement dynamics of proteins of the kientocore and centromere during poleward and antipoleward movement?

A
  • that poleward movement is an activate process involving a motor and when the chromosome moves away from the pole, it moves in a massive-manner
47
Q

what does an increased tension on kinetochore-microtubule interaction cause?

A

when there is tension pulling the kinetochore away from the centromere: stabilises the correct attachments- this ensures that correct attachments are stabilised and wrong attachments are destabilised - allowing reorientation

48
Q

how was tension shown to stabilise attachments?

A

Nicklas found that applying tension to a unipolar attached chromosome (applying tension to the opposite side as if the other side were attached to a microtubule too), stabilised the opposite kinetochore microtubule binding, meaning that the presence of this tensions stabilised a binding that is normally unstable

49
Q

how was aurora first found?

A

Ipl1 mutants yeast showed increased ploidy and was shown to be required for correct chromosome segregation and to phosphorylate kinetochore substrates regulating microtubule binding. Ipl1- is the homologue of Aurora B in vertebrates.

50
Q

where is aurora B found?

A

the inner centromere as the enzymatic component of the chromosome passenger complex, which also includes INCENP, surviving and borealin

51
Q

what happens when Aurora B is inhibited?

A

incorrect attachments are stabilised when example when the same sister kinetochore is attache to a single spindle. Removing the inhibitor leads to the correction of these bindings.

52
Q

what type of enzyme is aurora B?

A

serine threonine kinase

53
Q

how did the use of FRET-based biosensors reveal the mechanisms of Aurora B function?

A
  • When a FRET based biosensor that reports Aurora B phosphorylation is constitutively phosphorylated when positioned close to Aurora B independent of the tension being applied. However, when the same sensor was positioned on the outerkinetochore, it was dephoshpylrated when tension was applied. Importantly, endogenous outer kinetochore substrates behave in a similar way to this FRE-based reporter: and are dephosphorylated when centromeres biorient
54
Q

how is aurora B thought to work to sense tension and therefore stabilise binding?

A

Aurora B is positioned on the inner kinetochore and can phosphoylate substrates that are positioned close to it. When there is no tension pulling the outer kinetochore away from the inner, Aurora can phos the outer kinetochore and destabilise attachments. When tension pulls the outer away, Aurora B can no longer phos and so the binding is stable.

55
Q

what happens when aurora B is targeted to the outer kinetochore?

A

the outer kinetochore is Constit phos, independent of tension and microtubule attachment cannot be stabilised.

56
Q

what is the general link between Aurora B phos and stabilisation?

A

that its phos causes destabilisation

57
Q

give two explains of proteins in the outer kinetochore that are affected by Aurora B binding

A

ndc80/Hc1 and KNL1 strongly reduce their microtubule binding when they are phosphorylated- this likely happens by introducing negative charges that prevent interaction with neg charged microtubule

58
Q

explain how it was shown that conversion to the anaphase plate is not dependent on chromosomes being bi-orientated

A
  • monoorientated
59
Q

explain how it was shown that conversion to the anaphase plate is not dependent on chromosomes being bi-orientated

A
  • mono-orientated chromosomes are retreated back to the pole to which they attached
  • once at the pole they can bind CENP-E (plus end kinesin-7) on existing biorientated K-fibres. they are then transported towards the spindle equator- likely moving them towards free microtubules.
    within this mechanis, the probability that a monoorienttated chromosomes wll be transported towards the spindle equator progressivlet increases as more and more chromosomes become biorientated, which increases the density of K fibers at the in the spindle.
60
Q

how does congression occur normally in K-fibres?

A

the way that congression occurs on biorientated chromosomes is that there is polymerisation behind the kineotchore which pushes the chromosome towards the metaphase plate in addition to work with CENPE

61
Q

what is known about how chromosomes align in the plate?

A
  • in metazoans biorienatted kinetochores do not move in a direct movement to the middle of the spindle, they undergo oscillation. the oscillations are driven mostly by the action of the poleward-moving kinetochore and not the anti-poleward sister kinetochores. .
  • This indicates that kinetochores are mostly exerting a pulling force on chromosomes instead of a pushing force, implying that kinetochores use microtubule depolymerization as the main energy source for the alignment of bioriented kinetochores.
  • Nevertheless, it is important to note that kinetochores can also exert pushing forces on the spindle in mammalian cells, suggesting that microtubule polymerization can also contribute to force generation in certain circumstances
62
Q

what is the idea about how oscillation at the plate is organised?

A

Controlling chromosome movement on the metaphase plate. Bioriented sister kinetochores undergo regular oscillations on the metaphase plate. The direction of chromosome movement is primarily dictated by the pulling forces exerted by plus-end microtubule depolymerization. One proposed regulator of microtubule depoly- merization is the microtubule depolymerase MCAK, a kinesin-13. Kinetochore movement is also assisted by microtubule polymeriza- tion at the lagging sister kinetochore, which could be promoted by EB1 and requires CLASP. One proposed model for the control of the chromosome oscillations is that the microtubule depolymerase Kif18a accumulates in a length-dependent manner on microtubules, leading to a gradual increase of Kif18a on the lagging kinetochores. As a threshold of Kif18a is achieved, microtubule depolymerization is initiated at the lagging kinetochore, leading to a switch in direction

63
Q

what are the two proteins that need to be activated without APC to trigger anaphase?

A

cyclin B and securin

64
Q

what do cyclin B and securing do?

A

cyclin B activates CDK1 ativity and securing releases sister chromatids

65
Q

why is it thought that bi-oreinatted chromosomes oscilate?

A

This bi-stability appears to be part of the mechanism for aligning chromosomes at the spindle equator during prometaphase without the loss of mechanical linkage between sister and kinetochores and the spindle poles.
in mammalian cultured cells, low tension promotes the switch to kMT depol and high tension promotes switching to kMT polymerisation.
at high tension, kineotchores act like a slip clutch mechanism, switching to polymerisation to prevent detachment of depol ends.

66
Q

what is the pacman model of chromosome movement towards the pole?

A

in which chromosomes travel towards spindle poles along microtubules while chewing (depolymerising) the fibers at the plus-ends near kinetochores,

67
Q

how did the flux theory challenge the pacman model ?

A

experiments involving the UV-cutting of microtubules or photo-activating of fluorescent tubulin, which revealed that microtubules cannot be viewed as stationary tracks on which chromosomes move instead, even when the spindle maintains constant length, for example during metaphase, the entire microtubule lattice continuously moves towarda each spindle pole.

68
Q

what does microtubule spindle flux involve?

A

requires constant addition of tubulin subunits at microtubule plus ends and their corresponding removal from the microtubules minus ends at spindle poles.

69
Q

what does that paper claim the process of poleward movement is?

A

he direct observations of flux suggested that chromosome moveemnts may be powered by a traction fiber. In the current versions of this model, chromosomes are pulled polewards as K-fibers shorten as a result of reductions in rate of tubulin incorportation at the kinetochore, relative to that of microtubule depol at the pole. In this view, both pac-man and traction fibre mechanisms contribute to chromosome segregation.

70
Q

what was the initial evidence and thought behind poleward movement being due to the pacman model?

A

studies of the dynamic incorporation of labelled tubulin dimers revealed that, in mammalian cells, most tubulin subunit exchange takes place at the ends of microtubules distal to the spindle poles, the so-called plus ends.

Advanced molecular cell biology (6 decks)

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