microtubules 2 Flashcards
what do tubulin heterodimers associate to form?
protofilaments
what does the loss of the GTP cap result in?
results in a switch from growth to rapid shrinkage
what does a chymograph do?
converts movie into 2 dimensional image
what is being shown in this image
dynamic instability of microtubules
give examples of things the cell uses to regulate the cytoskeleton (microtubule associated proteins) . what do each of them do?
-polymerases: accelerate growth
-depolymerases: destroy GTP cap and trigger collapse
-+TIP proteins: recognizes growing end
-severing enzymes: sever in the middle
-nucleation factors: initiate birth of new microtubules
true or false, once microtubules “die”, they arent rebuilt
false, they are born and reborn continuously
true or false, micortubules only born in cells undergoing specific processes (ex: mitosis)
false, nucleated in all cells always, even in those not doing anything specific like mitosis. cells are constantly nucleating microtubules
what experiment allowed us to see that microtubules are predominantly nucleated by the centrosome?
experience where we take cells, shift from 37 degrees to 4 degrees, then put them in cold. all microtubules will collapse, but if we put back to 37 degrees, regrowth from centrosome only
true or false, microtubules are temperature dependent
true. they hate the cold. like warm temp (ex: 37 degrees)
what does the centrosome consist of?
2 centrioles, pericentriolar material and gamma-TURCs
what angle is between the mother and the daughter centriole
90
what is a centriole composed of?
microtubule triplets (3 microtubules smacked together)
what is the pericentriol material? what is its major component?
surrounds centrioles. electron dense amphormous proteins (appear dense in electron microscopy)
major component: gamma tubulin
what does the gamma tubulin ring complex allow for?
provides a template for microtubule nucleation
why is gamma tubulin important, even though it isnt part of the tubulin heterodimer like alpha and beta?
cause accessory proteins can organize it into the gamma tubulin ring complex
true or false, all micortubules are polymerized by the gamma tubulin ring complex
false. initially we thought this, but then it was discovered that some microtubules come from a place that doesnt have the ring complex
true or false, microtubules elongate from gamma tubulin ring complex
true
what is the analogy between the gamma tubulin ring complex for microtubules and the ARP 2-3 complex for actin?
-both provide a template
-both adopt a shape that looks similar to the polymer they’re trying to produce
which end of the micortubule is capped by the gamma tubulin ring complex?
the negative end
give an example of a place where there are microtubules, but no gamma tubulin ring complex
-in neurons (axons specifically)
-in the growth cone (tip of axon where it grows from), there are NO gamma tubulin rings, yet for the axon to elongate, lots of new microtubules need to be born in that region
what is responsible for nucleation in the neurons (just a hypothesis, not proven yet)?
doublecortin protein (related to Gary’s research)
what are the two domains of doublecortin?
-N-DC
-C-DC
what is the hypothesis about doublecortin and its mutation?
hypothesis that doublecortin is responsible for the nucleation of microtubules in axons. when there is a single amino acid mutation, neurons are unable to migrate to form cerebral cortex, which leads to neurodevelopmental diseases
basically, neurons arent able to migrate out properly to form the cortex, because there is an issue with microtubule formation, which is a core component of the neuron
true or false, double cortex syndrome is genetic when a female has it (she can pass it to her girls)
true
what is type 1 lissencephaly?
-single amino acid substitution in doublecortin in men.
-presents itself as smooth cerebral cortex.
-symptoms include speech issues and development issues
what is double cortex syndrome?
-single amino acid subsitution in doublecortin in females
-secondary layer of gray matter
-presents itself as epilespy
what are the 2 ways in which cells can nucleate polymers? which way is doublecortin thought to work?
-template by base-plate: ARP 2-3, gamma tubulin complex
-arrange monomers: bring subunits together ex: spire
-doublecortin hypothesis is that it acts like a spire
how does a spire work?
-for actin
-4 domains that bind to actin subunits to make nucleus (brings them together)
what are MAPS? what do they do? give some examples
-microtubule associated proteins regulate the cytoskeleton in many diff. ways
- some can help control nucleation process by interacting with the template (such as gamma tubulin complex)
-some inhibit and some promote nucleation
what are severing enzymes
ATPases that cut microtubules in the middle. another way for the cell to control/remodel their microtubules
true or false, some MAPs interact directly with the nucleation template (ex: gamma tubulin complex)
true. some also dont though. in the end, whether they interact with them directly or not, they all lead to same thing (either promoting or inhibiting polymerization)
how does the severing enzyme work?
form is a hexameric ring with a pore on the inside. the pore catches the c terminal of the tubulin protein’s tail, and yanks it though, which creates holes and defects (cutting it)
what is the purpose of the severing enzyme?
not actually to destroy microtubules. their role is rather to remodel the microtubules, by multiplying them. for example, severing enzymes would make 100 long microtubules and turn them into 500 short ones
how do severing enzymes not detroy the microtubule, since severring them in their middle exposes their GDP lattice and gets rid of the cap?
saving factors come in after to stabilize the cut microtubule. if it wasnt for them, the microtubule would fall apart
what type of ATPase are severing enzymes?
AAA ATPases
what do +TIP proteins do?
track growing of microtubule ends. always beehive of activiity happening as microtubule is growing
where do the +TIP proteins bind?
GTP cap
which +TIP recognizes the GTP cap? what does this allow for?
-EB family proteins (specifically EB1)
-allows other +TIP proteins to hitch a ride
what do depolymerases do?
remove tubulin from end and trigger catastrophe (removes the GTP cap)
give an example of a depolymerase and explain what it does
kinesin 13, which is an ATPase. it hydrolyzes ATP and uses that energy to get on the + end of the microtubule and tear it apart
specific example of kinesin 13 that prof did experiment on? what was the experiment?
MCAK. put microtubules on glass cover, and then added MCAK. then the polymer shrinked, and protofilament forced into a curved structure (unfolding=catastrophe)
what happens during a catastrophe?
-The microtubule starts peeling apart
-Tubulin dimers rapidly fall off, leading to fast shrinkage of the microtubule.
give an example of a polymerase microtubule associated protein
XMAP215
what does XMAP215 do?
-accelerate microtubule growth
true or false, XMAP215 only lowers activation energy for the Kon
false, also for Koff, and the reaction favoured depends on the concentration of reactants
-this is because it acts as a catalyst for microtubule polymerization, therefore it lowers delta G for both the forward and reverse reaction
what happens to microtubules based on the concentration of tubulin in the presence of XMAP215?
no tubulin in solution: inverse reaction favoured, cause its reactants (microtubules) have a greater concentration than its products (tubulin). leads to shrinkage
a little bit of tubulin: equilibrium. no net growth or shrinkage
lots of tubulin: forward reaction accelerated. net growth
reaction with highest reactant concentration is favoured
what exactly does XMAP215 do to be a catalyst? what were the theories on the way it does this?
-stabilizes weakly attached tubulin dimers at the ends
two hypothesis:
-at first, was thought that tubulin binding domains (TOG domains) wrapped around a single tubulin domain to form a complex
-that theory was then disproven, as scientists realized that only 1 domain fit onto the heterodimer. what actually happens is that one domain reaches out, grabs a heterodimer and brings it to the microtubule end (like a formin)
what does XMAP215 act like?
like a forming
compare proteins involved in actin with proteins involved in microtubules
give an example of a specific severing enzyme
katanin
