Lecture 10 - Spindle Mitosis Poisons Flashcards
What are microtubules?
- components of the cytoskeleton
Microtubule structure overview
- Alpha and Beta Tubulin heterodimers connected by a non-covalent bond
- Have a + end associated with GTP cap
- Have - end surface
- ML surface
- H3 surface
- 13 filaments in spiral structure
Overview of dynamics of microtubules
- Dynamics at positive end faster than dynamics at negative end
- Rate of addition/loss faster at + end
How do microtubules expand?
- Heterodimers free floating in proximity can be added on to either end
What is treadmilling and what effect does this give?
- add heterodimers at one end whilst the are being lost from the other end
- gives effect of microtubule moving in space
4 factors which characterise dynamism of microtubules
- Rate of MT growth
- Rate of MT shrinkage
- Catastrophe
- Rescue
What is catastrophe?
Frequency of transition from growth (or pause) to shrinking
What is rescue?
Frequency of transition from shrinking to growth (or pause)
What is the role of GTP/GDP in microtubule stability?
- Both α and β units have a GTP/GDP binding site at the (+) end
- Within heterodimers the α+ retains GTP tightly bound (due to interaction to the β (-)-end), and is non-exchangeable
- At β (+)-end, Tubulin-bound GTP hydrolysed to tubulin–GDP and inorganic phosphate (Pi ) at the time or after tubulin adds to the microtubule ends
- The Pi dissociates from the microtubule, leaving a microtubule core consisting of tubulin with stoichiometrically bound GDP.
- MT end containing tubulin-bound GTP or GDP–Pi is stable, or ‘capped’, against depolymerization.
- Hydrolysis of tubulin-bound GTP and release of Pi induces conformational changes in the tubulin molecules –> destabilize the microtubule polymer –> CATASTROPHE and shortening of MT
What is difference between MT dynamics in vivo and in vitro?
Slower in vitro
How is functional diversity achieved in microtubules?
- Microtubule associated proteins (MAPs)
- Soluble tubulin
- Microtubule surfaces and ends
- Isotypes
- Post-translational modification
Role of MT during onset of mitosis?
- onset of mitosis –> network of MT replaced by new population which are more dynamic
- result of an increase in catastrophe and decrease in rescue
What is the process of hunting in prometaphase by MT?
microtubules ‘hunt’ by rapidly elongating and shrinking, probing the cytoplasm until they find the chromosome kinetochore
Why would we target microtubules?
- play role in mitosis
- …by organising the chromosomes, and cleaving daughter cells
- cancer characterised by rapid replication
- naturally occurring toxic molecules by plants which target them
What are the 2 groups of agents which bind to microtubules?
Surfaces of the globular part of tubulin has several binding sites that allow binding of:
- Microtubule Stabilizing Agent (MSA’s)
- Microtubule Destabilizing Agents (MDA’s)
What do the microtubule targeting agents affect?
MT dynamics, leading to mitotic arrest and cell death
How many MTA binding sites are known?
- 6
4 on β-tubulin and two on α tubulin
What are the MTAs which bind to beta tubulin?
- taxane
- laulimalide/peloruside
- vinca
- maytensine
What are the MTAs which bind to alpha tubulin?
colchicine and pironetin
What are the microtubule stabilizing agents?
- Taxane ligands
- Laulimalide/peluroside ligands
What are the microtubule destabilising agents?
- Vinca ligands
- Maytensine ligands
- Colchicine ligands
- Pironetin ligands
What is the name of the drug containing taxane ligands?
Paclitaxel
What is the mode of action of paclitaxel?
- Acts to strengthen lateral contacts between adjacent protofilaments – leads to MT stabilisation
- Taxane pocket in MT is near ML surface on inside of tubulin
- Bind poorly to soluble pool of tubulin
- Bind high affinity to β subunit
What binds well/not well to taxanes?
Heterodimers in solution don’t bind well to taxanes (poorly to soluble tubulin)
Bind high affinity to β subunit
