Spatial control of cell division Flashcards
1
Q
Cell division is needed for…
A
- growth
- healing
- replacing old cells
2
Q
G1 phase checkpoint
A
- restriction point
- checks for: cell size, nutrients, growth factors, DNA damage
- can arrest cell if environmental conditions are not met
3
Q
G2 phase checkpoint
A
- checks for: DNA damage, DNA replication completeness
- if damage is detected cell will pause at G2 checkpoint to allow for repairs
- if damage is irreparable, the cell can undergo apoptosis
4
Q
Spindle checkpoint (M checkpoint)
A
- checks for: chromosome attachment to spindle at metaphase plate
- if chromosome is misplaced, cell will pause mitosis allowing time for spindle to capture stray chromosome
5
Q
Function of microtubule motor proteins
A
- move across microtubule, transporting cellular cargo within the cell
- different classes provide motion through interaction with tubulin subunit
- either (+) end (kinesin) or (-) end (dynein) motors depending on direction of movement
- two classes; both display ATPase activity (energy derived from hydrolysis of ATP)
6
Q
Role of securin
A
- stops protein separase (which promotes separation of sister chromatids)
7
Q
Mitotic checkpoint
A
- MPS1 kinase localised to a kinetochore
- recruits other proteins -> assembles inhibitory complex (MCC)
- MCC inhibits large ubiquitin ligase needed for mitotic exit (anaphase promoting complex)
- MT attach and the checkpoint signal gets shut down
8
Q
How are MT interactions removed
A
- kinase (AurB) phosphorylates protein on kinetochore to remove MT interactions by adding phosphate (-) which repels a negatively charged microtubule
9
Q
How does the checkpoint signal get shut down following MT attachment
A
- MPS1 is removed from kinetochores by
- phosphatases are activated to dephosphorylate key checkpoint proteins
- microtubule motors (dynein) strip the checkpoint proteins from the kinetochore down microtubules towards spindle poles
10
Q
How can we test the hypothesis that MPS1 and MT binds the same binding site on NDC80/Hec1
A
- immunofluorescence - can quantify the location and/or activation state of proteins (e.g. phospho-antibodies)
- in vitro biochemistry - an isolated system to distinguish direct from indirect effects
11
Q
How could you test whether MPS1 is directly responsible for MPS1 localisation in cells?
A
- knockdown/replacement - to characterise how proteins are localised in cells, move NDC80 (or mutants of) to other locations in the cell and examine whether MPS1 appears as well
- synthetic biology - to build complexes at artificial locations; shows that competition between NDC80 and MPS1 for MTs enables rapid checkpoint silencing
12
Q
How can error correction be shown experimentally?
A
- Live cell imaging: fluorophores measure localisation and/or activity status (FRET assays)
- Aurora B kinase localises to centromere
- phosphorylates the kinetochore to remove MTs (electrostatic charge)
- kinetochores under tension are removed away from Aurora B activity, therefore they are stabilised
13
Q
How can FRET reporters be used to measure kinase activity
A
- if proteins are close enough for wavelengths to excite each other, we can see emission from one protein to another
- used to detect activity status of a protein
- level of emission dependent on relation orientation (i.e. level of phosphorylation)
14
Q
Sensing chromosome bi-orientation by spatial separation of AurB kinase from kinetochore substrates
A
- using FRET-based biosensors, we can measure localised phosphorylation dynamics in living cells
- repositioning AurB closer to kinetochore prevented stabilisation of bi-oriented attachments and activated the spindle checkpoint
- centromere phosphorylation is the same irrespective of MT attachment
- kinetochore phosphorylation is reduced upon MT attachment
15
Q
Role of Aurora B kinase
A
- regulates chromosome-spindle attachments by phosphorylating kinetochore substrate that bind MTs
