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
What is the stoichiometry of paclitaxel to MT dimer?
1:1
however typical microtubule (10,000 tubulin units) requires 5,000 paclitaxel molecules
How does laulimadlide/peluroside ligands work?
- binds to site
- MT stabilised by strengthening lateral contacts between protofilaments
How does vinca alkaloids work?
- Vinca site is located at the + end surface of β-tubulin
- Binding alters the surface of + end forming a ‘wedge’
which interferes with incorporation of new heterodimers - The + ends remain curved and microtubule wall cannot form
- cause tubulin oligomers, decreasing the free tubulin pool
- depolymerise the MT
How does vinca effect the dynamics of MT?
- One or two ligands at the end of a microtubule will reduce dynamics by 50 %
How does Maytansine work?
- Maytansine site is close to the vinca site
- In growing MT, maytansine binding pocket of the (+)-end accommodates the (-)-end of the next α-tubulin unit
- forming a wedge
- impedes elongation
Problem with Colchicine
- Very toxic
- but looking in to combrestatin instead
How does Colchicine work?
- Colchicine binding site on ALPHA tubulin (between the α and β units)
- Slow binding, practically irreversible
- Binding stabilises in curved conformation
- SLOWs but doesn’t prevent polymerisation
What cancers does paclitaxel work in?
Ovarian, mammary and lung tumours
What cancers does vinca work in?
Haematological cancers
What are the possible modes of resistance against MTAs?
- Over expression of ABC-transporters –> P-glycoprotein
- Overexpression or binding of MT associated/regulatory proteins
- Mutation in tubulin
- Up and/or down regulation of different tubulin isotypes which change dynamics
- Overexpression of multidrug resistance (MDR-1) gene
- Changes in lipid composition
- Over-expression of IL-6
- Post translational modifications
Most important thing to remember for MTAs and how they work
changes in dynamics of MT
What are microtubules crucial for?
- Cell shape
- Transport of vesicles
- Cell signalling
- Mitosis
What are the side effects of Vinca ligands and Taxane ligands?
- Reversible Myelosuppression
- Peripheral Neuropathy
Overview of study of paclitaxel and resistance…
- A549 lung cancer cells became dependent on paclitaxel
- Withdrawal lead to faster dynamics
- Cell adapted to slow effects of drug by making everything else go faster
- Cells resistant and dependent on drug
In paclitaxel what else might favour resistance?
- Expression of endogenous microtubule-depolymerizing factors
Where do Vincas originate from?
Periwinkle leaves
Where do maytensine ligands originate from?
Maytenus ovatus
Where do colchicine ligands originate from?
Autumn crocus
Where do Taxane ligands (paclitaxel) orginate from?
Taxus Brevifolia
Where do Laulimalide/Peluroside ligands originate from?
Sea sponges
What other hallmark of tumour have antimitotic drugs been investigated in as a treatment and an example of this?
- Angiogenesis –> acting as vascular-targeting agents
- rapidly depolymerize microtubules of newly formed vasculature to shut down blood supply to tumours
Example: Combrestatin drugs in clinical trail
Name of APC transporter which causes resistance
- P-glycoprotein
(ATP-dependent drug efflux pumps)
Where is the taxane pocket located?
Near ML surface on inside of tubulin
Where is the Laulimalide/Perluroside binding pocket located?
Opposite side of ML surface with respect to taxane outside the MT wall
Where is the Vinca Ligand binding site located?
At + end of BETA tubulin
Where is the Maytensine site located?
Close to the Vinca site
How does Colchicine bind to the MT?
- likely it doesn’t bind to the microtubule tip
- binds to the soluble pool first and is then incorporated
