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Kinetochores Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

What is a centromere?

A

The chromosomal region where the kinetochore is formed. DNA!

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2
Q

What is a kinetochore (KT)?

A

The proteinaceous complex that connects the centromere to the mitotic spindle. Protein!

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3
Q

Why are centromeres / kinetochores essential to eukaryotic life?

A

They are essential for eukaryotic division, which allows DNA to be passed into future generations.

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4
Q

What can problems with KTs cause?

A

Problems with chromosome segregation which lead to cell death, severe disease e.g. cancer, or birth defects e.g. downs syndrome.

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4
Q

What is the function of KTs?

A
  • Ensure duplicated chromosomes to be separated in a 1:1 ratio for chromosome segregation in daughter cells by bi-oriented attachment to spindles.
  • Regulate cell cycle progression; unattached KTs activate SAC which inhibits progression from metaphase to anaphase by APC.
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5
Q

Who was the first person to detect chromosome segregation?

A

Flemming in 1879. He didn’t know about genetic material, but described chromosomes and spindles through drawings.

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6
Q

Why are newt lung cells good for chromosome study?

A
  • The chromosomes are large (easy to visualise with DIC)
  • The cells are transparent so can monitor chromosome behaviour
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7
Q

When does anaphase occur?

A

When every chromosome in the cell is aligned at the metaphase plate.

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7
Q

What are spindles?

A

Dynamic MT polymers made of tubulin.

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8
Q

What is bi-oriented attachment?

A

Sister kinetochores always bind spindles from opposite poles.

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9
Q

What are the states of dynamic MTs?

A
  • Catastrophe (disassembly)
  • Rescue (assembly)
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10
Q

Where do KTs initially interact with MTs?

A

The lateral side of the plus end (not right at the tip). Much larger surface area for KTs to interact with.

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11
Q

Where are KTs ultimately localised on the MT?

A

The plus tip.

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12
Q

How are the chromosomes moved on the KT MTs?

A

The MT (net) disassembles (still some reassembly) but the kinetochore remains attached, so the chromosome moves towards the MT minus end / spindle pole.

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13
Q

What happens when cells go into anaphase before bi-oriented attachment is established?

A
  • Chromosomes may get stuck in the middle; not pulled to either end by the spindles (merotelic).
  • Both sister chromatids may go to one pole / daughter cell (syntelic or monotelic).
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14
Q

What are MTs called before binding and during lateral binding of KTs?

A

Astral / centrosomal MTs.

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15
Q

What are MTs called after the switch to end on binding by KTs?

A

KT MTs.

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16
Q

How are the chromosomes moved on the lateral MTs?

A

They are moved towards the minus end by motor proteins.

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17
Q

How do KTs shift from lateral association to end on association at the MTs?

A

We don’t know - ongoing research.

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18
Q

What does the sister KT that is not associated with the MT do?

A

Captures MTs from opposite spindle pole once KT has converted to end on association. Bipolar attachment. Also don’t know how this happens.

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19
Q

Why don’t sister KTs bind to MTs from the same pole?

A

The have a ‘back to back’ orientation. Face opposite ways.

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20
Q

What is mono-oriented (syntelic) attachment?

A

Both chromosomes / KTs attached to MTs from the same spindle.

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21
Q

What is mono-oriented (monotelic) attachment?

A

Only one KT binds to MTs.

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22
Q

What is merotelic attachment?

A

One sister KT binds to MTs from both spindle poles.

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23
Q

How do cells detect bioriented attachment?

A

Detecting tension between kinetochores due to MT pulling forces.

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24
Q

How do we test the importance of tension?

A
  • Apply tension to the mitotic chromosomes with mono-oriented attachment using a glass microneedle - very difficult experiment. Monitor KT/MT attachment using Abs.
  • Experiments in yeast minichromosomes.
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25
Q

What does applying slight tension to mono-oriented chromosomes do?

A

Detach 1 chromosome from MTs and allow it to reorient and form bioriented attachment. Anaphase can occur.

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26
Q

How can experiments monitor tension?

A

Yeast are easy to study. They have a minichromosome (≈2kb) which contains a centromere. When replicated, tension can be measured between 2 minichromsomes’ KTs. Or, an artificial centromere can be introduced into one minichromosome and tension can be observed between these 2 KTs attached to spindles.

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27
Q

How can the position of mini chromosomes be monitored in yeast?

A

Using TetRepressor-GFP, which binds to the Tet operator in the minichromosome.

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28
Q

What must be included in the minichromosomes in order to stimulate its replication.

A

ARS - autonomous replication sequence.

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28
Q

How was it discovered that 2 centromeres in a minichromosome induced biorientation?

A

When replication was inhibited (ARS excised) and the second centromere was activated in the minichromosome it became stuck between the 2 spindle poles. But when only 1 centromere was active the minichromosome was pulled to 1 pole.

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29
Q

How is the absence of tension during chromosome segregation corrected?

A

The kinase Aurora B phosphorylates the kinetochores which destabilises their attachment to the MTs. This allows them to reattach in the correct orientation. The phosphatase PP1 can then remove the phosphates to stabilise the attachment in the right orientation.

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30
Q

What does inactivation of aurora B result in?

A

Lots of chromosomes being stuck without bioriented attachment.

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31
Q

How were kinetochores first detected?

A

Bound by Abs in antisera (blood serum containing Abs) of people with autoimmune disease.

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32
Q

Which KT proteins did Earnshaw et al pull down using antisera?

A

CENP-A, CENP-B and CENP-C.

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33
Q

What is CENP-A?

A

A centromere-specific histone H3 variant; copurifies with core histones (DNA binding). KT protein. Found at KT base.

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34
Q

Which budding yeast protein is human CENP-A orthologous to?

A

Cse4

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35
Q

How many human KT proteins do we now know of?

A

≈100

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36
Q

How many structural proteins are in the budding yeast / human KT (conserved)?

A

40, with 8 sub complexes. (Also many regulatory proteins).

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37
Q

Inner KT proteins have what function?

A

Centromere / DNA binding.

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38
Q

Outer KT proteins have what function?

A

MT binding.

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39
Q

Inner KT protein copy number?

A

2-4.

40
Q

Outer KT protein copy number?

A

≤8.

41
Q

Which outer KT proteins does aurora B phosphorylate to destabilise MT binding?

A

Ndc80 and Dam1DASH.

42
Q

How does lack of tension stimulate Aurora B pathway?

A

We still don’t know.

43
Q

What is spindle assembly checkpoint (SAC)?

A

A mechanism involving 5-8 spindle checkpoint proteins (organism dependent) that is activated proteins localise to unattached KT). It inhibits anaphase promoting complex (APC) and metaphase to anaphase progression.

44
Q

What happens to SAC when the KT binds to MTs?

A

It is silenced, and APC is activated so metaphase can progress to anaphase.

45
Q

What does APC activate?

A

Cleavage of cohesin that holds sister chromatids together.

46
Q

What happens in a dicentric (2 KTs) chromosome?

A

One of the KTs gets inactivated (don’t know how).

47
Q

What could happen if there were 2 active KTs on a chromosome?

A

The chromosome could bind MTs from both poles causing missegregation.

48
Q

How are cleaved regions of chromosome (no centrosome) segregated?

A

KTs are assembled on them de novo.

49
Q

How is the 1 KT per chromosome thought to be regulated?

A

Epigenetics.

50
Q

What is monocentric?

A

One centromere location on the chromosome. Most eukaryotes e.g. humans, yeast, trypanosomes.

51
Q

What is holocentric?

A

There is centromere activity all along the chromosome; KTs can assemble anywhere.

52
Q

What might be the advantage of being holocentric?

A

Ensures complete chromosome segregation even if the chromosome is cleaved (MTs attached all the way along). Prevents cell death / cancer.

53
Q

Which organism’s DNA doesn’t condense much during mitosis?

A

Budding yeast.

54
Q

What are telocentric chromosomes?

A

Chromosomes with centromeres very close to the telomeres.

55
Q

Which organisms have telocentric chromosomes?

A

Mice.

56
Q

Which organisms have holocentric chromosomes?

A

C. elegans, silkworms, butterflies.

57
Q

What are metapolycentric chromosomes?

A

Chromosomes with multiple clustered centromeres.

58
Q

Which organisms have metapolycentric chromosomes?

A

Pisum (pea).

59
Q

What is a point centromere?

A

A 125bp centromere where only one MT can attach via a KT e.g. in budding yeast.

60
Q

What is a regional centromere?

A

A centromere of several kb - several Mb where multiple MTs can attach at once, e.g. in fission yeast, humans, trypanosomes.

61
Q

Why is it easier to study MT behaviour in cells with point centromeres?

A

There are much fewer MTs! So the imaging is clearer. E.g. in budding yeast there are 40 MTs but in kangaroo rats there are hundreds.

62
Q

Why is O. tauri hypothesised to have fewer MTs than chromosomes?

A

They are so small they don’t synthesise MT protein.

63
Q

Which organisms do not have KTs?

A

Prokaryotes (bacteria and archaea).

64
Q

When did KTs evolve?

A

During the evolution of archaea to eukaryotes.

65
Q

How can we hypothesise what a KT was like in the earliest eukaryotic common ancestor?

A

By studying early-branching eukaryotes (don’t know which branched off earliest).

66
Q

Which eukaryotic group is more popular for KT research (95%)?

A

Opisthokonts (yeast, worms, flies, humans).

67
Q

Why might mostly studying KTs in one eukaryotic group be problematic?

A

The KTs / chromosome segregation in other groups may be different. There are lots of pathogens in the other groups so we should characterise them.

68
Q

Why do kinetoplastids need studying?

A

Their genome does not contain any sequences indicating canonical (normal) KT proteins, despite other mitotic components being conserved.

69
Q

What are kinetoplastids?

A

> 30 species of unicellular flagellated eukaryotes in the Discobids group. Have a unique organelle called a kinetoplast. Parasites that cause a range of understudied tropical diseases.

70
Q

What is a kinetoplast?

A

An organelle containing a mtDNA cluster found in kinetoplastids.

71
Q

What diseases do kinetoplastids cause?

A
  • Sleeping sickness (African trypanosomiasis)
  • Chagas disease
  • Leishmaniasis
72
Q

Which kinetoplastid is easiest to study?

A

Trypanosoma brucei

73
Q

What is evidence for spindles / MTs in trypanosomes (despite no KTs)?

A

Inhibition of MT polymerisation inhibits DNA segregation.

74
Q

How is the KT-like structure in trypanosomes different from canonical KTs?

A

There is no inner centromere (1 µm space between sister KTs). This is usually where Aurora B and cohesin accumulate.

75
Q

How was the first trypanosome KT protein identified?

A

YFP-tagging 30 uncharacterised genes that were upregulated in S-phase or mitosis. Found one that looked like a KT protein based on localisation pattern.

76
Q

What does KKT1 stand for?

A

Kinetoplastid kinetochore 1

77
Q

What is KKT1 localisation during the cell cycle?

A
  • Synthesised in S phase.
  • Remains present in G2.
  • Localises to metaphase plate in metaphase.
  • Localises at the leading edge of the separating chromosomes in anaphase.
78
Q

How were 24 other trypanosome KT proteins identified?

A

Copurification with KKT1 using immunoprecipitation and mass spec. Repeat copurification with 12 first discovered proteins. Named KKT 2-25.

79
Q

Where are trypanosome KT proteins conserved?

A

Only the kinetoplastid group.

80
Q

Is the kinetoplastid KT structure similar to the canonical KT structure?

A

No - implies this system evolved separately.

81
Q

Why might KKT proteins be useful in study?

A
  • Characterisation of their function may improve understanding of the canonical KT function.
  • They are specific to kinetoplastids so may provide a good drug target (stop parasite division).
82
Q

How were the KKTs binding MTs and DNA identified?

A
  • Protein sequence analysed
  • Localisation timing analysed
  • RNAi depletions and gene deletions to see which KKTs are essential for proliferation
83
Q

Which KKT is longest?

A

KKT1

84
Q

Which KKTs have DNA binding motifs (AT hook and SPKK)?

A

KKT2 and KKT3

85
Q

Which KKT has MT binding motif?

A

KKT4 - but not a similar motif to other eukaryotes!

86
Q

How does KKT4 associated with MTs?

A

Binds to the lateral side at tip. Remains bound during MT depolymerisation.

87
Q

Proteins involved in which processes have we identified various KKT proteins to have similarity to?

A
  • Mitosis
  • Meiosis
  • DNA damage response pathway.
88
Q

What is the synaptonemal complex (SC)?

A

A meiosis-specific structure that forms between homologous chromosomes and promotes genetic exchange by recombination.

89
Q

What are the components of the chromosomal axis of the SC?

A

SYCP2/3 (KKT16/17/18), cohesins, HORMADs

90
Q

What is the hypothesis for where KKT proteins evolved from?

A

Evolved from meiotic synaptonemal complexes (that are found in most eukaryotes) that became limited to 1 region and gained MT binding activity. Supported by some KKTs having similarity to proteins involved in homologous recombination by SC.

91
Q

Why might kinetoplastids have unique KTs?

A
  • Derived condition: kinetoplastids ancestrally had canonical kinetochores, which got lost and replaced by unique KKT proteins
  • Ancestral condition: kinetoplastids never had canonical kinetochores
  • Kinetoplastids are one of the earliest branching eukaryotes and the last common ancestor could have had either type of KT protein, or no KT.
92
Q

How are prokarytic chromosomes segregated with no KTs?

A

Don’t know but idea is the 2 long DNA polymers spontaneously segregate to increase the entropy of the system (works in simulation).

93
Q

What is the most likely explanation for the different KT systems in kinetoplastids and other eukaryotes?

A

Kinetoplastids repurposed the homologous recombination machinery in a common ancestor that did not have a KT. Other eukaryotes invented a new KT system.

94
Q

What would the existence of homologous recombination machinery but not KTs in the last eukaryotic common ancestor suggest?

A

That meiosis evolved before mitosis.

95
Q

Where is the canonical KT system thought to have come from?

A

The repurposing and combining of various processes (Lecture 7, slide 75).

96
Q

What is unique about dinoflagellate and parabasalid KTs?

A

They are embedded in the nuclear envelope as the spindle MTs assemble outside of the nucleus.

97
Q

Do we know how KTs insert into the nuclear membrane and establish bi-oriented attachment?

A

No - it is still being researched. It appears to have evolved twice independently though from looking at phylogenetic trees.

98
Q

Which group has no canonical or kinetoplastid-like KT proteins?

A

Diplonemids; a diverse and abundant plankton with unknown roles in the ecosystem.

99
Q

Why have diplonemids not been studied much yet?

A
  • They were not recognised in water samples due to their rRNA not being recognised by common sequencing techniques.
  • Only one model we can study easily (Paradiplonema papillatum).
100
Q

Why is Paradiplonema papillatum easy to study?

A
  • Culturable in sea water
  • Genome / transcriptome sequenced
  • Transformation possible

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