Kinetochores Flashcards
What is a centromere?
The chromosomal region where the kinetochore is formed. DNA!
What is a kinetochore (KT)?
The proteinaceous complex that connects the centromere to the mitotic spindle. Protein!
Why are centromeres / kinetochores essential to eukaryotic life?
They are essential for eukaryotic division, which allows DNA to be passed into future generations.
What can problems with KTs cause?
Problems with chromosome segregation which lead to cell death, severe disease e.g. cancer, or birth defects e.g. downs syndrome.
What is the function of KTs?
- 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.
Who was the first person to detect chromosome segregation?
Flemming in 1879. He didn’t know about genetic material, but described chromosomes and spindles through drawings.
Why are newt lung cells good for chromosome study?
- The chromosomes are large (easy to visualise with DIC)
- The cells are transparent so can monitor chromosome behaviour
When does anaphase occur?
When every chromosome in the cell is aligned at the metaphase plate.
What are spindles?
Dynamic MT polymers made of tubulin.
What is bi-oriented attachment?
Sister kinetochores always bind spindles from opposite poles.
What are the states of dynamic MTs?
- Catastrophe (disassembly)
- Rescue (assembly)
Where do KTs initially interact with MTs?
The lateral side of the plus end (not right at the tip). Much larger surface area for KTs to interact with.
Where are KTs ultimately localised on the MT?
The plus tip.
How are the chromosomes moved on the KT MTs?
The MT (net) disassembles (still some reassembly) but the kinetochore remains attached, so the chromosome moves towards the MT minus end / spindle pole.
What happens when cells go into anaphase before bi-oriented attachment is established?
- 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).
What are MTs called before binding and during lateral binding of KTs?
Astral / centrosomal MTs.
What are MTs called after the switch to end on binding by KTs?
KT MTs.
How are the chromosomes moved on the lateral MTs?
They are moved towards the minus end by motor proteins.
How do KTs shift from lateral association to end on association at the MTs?
We don’t know - ongoing research.
What does the sister KT that is not associated with the MT do?
Captures MTs from opposite spindle pole once KT has converted to end on association. Bipolar attachment. Also don’t know how this happens.
Why don’t sister KTs bind to MTs from the same pole?
The have a ‘back to back’ orientation. Face opposite ways.
What is mono-oriented (syntelic) attachment?
Both chromosomes / KTs attached to MTs from the same spindle.
What is mono-oriented (monotelic) attachment?
Only one KT binds to MTs.
What is merotelic attachment?
One sister KT binds to MTs from both spindle poles.
How do cells detect bioriented attachment?
Detecting tension between kinetochores due to MT pulling forces.
How do we test the importance of tension?
- 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.
What does applying slight tension to mono-oriented chromosomes do?
Detach 1 chromosome from MTs and allow it to reorient and form bioriented attachment. Anaphase can occur.
How can experiments monitor tension?
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.
How can the position of mini chromosomes be monitored in yeast?
Using TetRepressor-GFP, which binds to the Tet operator in the minichromosome.
What must be included in the minichromosomes in order to stimulate its replication.
ARS - autonomous replication sequence.
How was it discovered that 2 centromeres in a minichromosome induced biorientation?
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.
How is the absence of tension during chromosome segregation corrected?
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.
What does inactivation of aurora B result in?
Lots of chromosomes being stuck without bioriented attachment.
How were kinetochores first detected?
Bound by Abs in antisera (blood serum containing Abs) of people with autoimmune disease.
Which KT proteins did Earnshaw et al pull down using antisera?
CENP-A, CENP-B and CENP-C.
What is CENP-A?
A centromere-specific histone H3 variant; copurifies with core histones (DNA binding). KT protein. Found at KT base.
Which budding yeast protein is human CENP-A orthologous to?
Cse4
How many human KT proteins do we now know of?
≈100
How many structural proteins are in the budding yeast / human KT (conserved)?
40, with 8 sub complexes. (Also many regulatory proteins).
Inner KT proteins have what function?
Centromere / DNA binding.
Outer KT proteins have what function?
MT binding.
