Microtubules

What are microtubules?

hollow cylinders of a/B-tubulin polymers

What do microtubules function in?

providing mechanical support, tracks for transport, positioning of ER golgi, cell locomotion

What is the subunit of microtubules?

a/B-tubulin dimer

How much homology is there between alpha and beta tubulin?

40%

What do alpha and beta tubulin both bind?

GTP

What tubulin is a GTPase?

beta

Where is beta tubulin found?

at the very plus end

Where is alpha tubulin found?

the minus end

What are microtubules made of?

13 protofilaments

How do a/b-tubulin heterodimers bind?

head-to-tail protofilaments

What happens as the concentration of alpha/beta-tubulin heterodimers increases?

will eventually reach the Cc for polymerization

What is the Cc for pure tubulin in vitro?

7uM for the plus end

What is T-form?

the GTP bound beta-tubulin

What is D-form?

hydrolyzed GDP bound beta-tubulin

What do microtubule plus ends display?

alternating phases of growth and shrinkage

What should you think with tubulin polymerization properties?

dynamic instability

How do individual microtubules behave?

independently from each other

What happens when the rate of polymerization > rate of GTP hydrolysis in the polymer?

GTP cap maintained, MT will grow

What happens when the rate of polymerization < rate of GTP hydrolysis in the polymer?

GTP cap is lost, MT will shrink

What happens in a fast growing microtubule?

GTP is hydrolyzed with a delay, resulting in a stabilizing GTP cap

When does the microtubule depolymerize?

when the rate of GTP hydrolysis exceed the rate of new tubulin addition

How fast is GDP-tubulin at the plus end lost?

at a rate of 50x that of GTP-tubulin

What do GTP-tubulin islands allow?

rescue

Do microtubules use sequestration?

yes

What does expression of stathmin protein do?

bind free tubulin subunits and inhibit assembly and elongation

What does each stathmin protein bind?

two dimers

What does phosphorylation of stathmin do?

inactivate it

What can be used to precisely control microtubule polymerization?

a tug of war between kinases and phosphotases

What is the microtubule organizing center (MTOC)?

a structure found in eukaryotic cells from which microtubules emerge

What does the MTOC act as a major site of?

microtubule nucleation, where microtubules begin to form within the cell

What are the functions of the MTOC?

microtubule nucleation, spindle formation during mitosis, organelle positioning, cilia and flagella formation

What is the microtubule nucleation function of MTOC?

centrosome seeds and anchors microtubules, helping define the shape and polarity of the cell

What is the spindle formation during mitosis function of MTOC?

helps organize the mitotic spindle that separates chromosomes during cell division

What is the organelle positioning function of MTOC?

by organizing the microtubule network, the centrosome helps positions organelles like the Golgi and nucleus

What is the cilia and flagella formation function of MTOC?

the mother centriole can become a basal body, anchoring a cilium or flagellum

What do centrosomes contain?

peri-centrosomal nucleation (PCM) complexes surrounding pair of centrioles

What do centrioles within centrosomes become?

basal bodies, which are nucleation centers for cilia and flagella

What do duplicated centrosomes become?

the spindle poles of dividing cells

What do microtubules originate from?

gamma-tubulin ring complexes

What does gamma-tubulin do?

decreases the Cc for tubulin assembly

What is XMAP215?

a plus end polymerase

What does the ratio of XMAP215 and kinesin-13 do?

determines dynamics of microtubules

What is kinesin-13?

destabilizes the tip by favoring the formation of curved protofilaments

What does XMAP215 do?

both stabilizes and enhances the rate of polymerization of microtubules

How does XMAP215 enhance the rate of polymerization?

binds free tubulin dimers via TOG domains, increasing tubulin concentration at growing end, accelerates the addition of tubulin dimers

How does kinesin-13 depolymerize microtubules?

separating protofilaments from their lateral neighbors in the lattice and peeling them away from the central axis

What are microtubule plus-end tracking proteins (+TIPs)?

diverse group of protein that accumulate at + ends leading to stabilization, preventing depolymerization, and promoting microtubule elongation

What protein is important for sequestration?

stathmin

What protein is important for minus end capping and nucleating?

y-tubulin

What protein is important for plus end polymerase?

XMAP215

What protein is important for plus end destabilizing proteins?

kinesin-13

What protein is important for plus end stabilization?

EB1

What are depolymerizing anti-tubulin drugs from plants?

colchicine, vinblastine

What are stabilizing anti-tubulin drugs from plants?

taxol

What are anti-tubulin drugs from planets used in?

anti-cancer chemotherapy

What cells are more sensitive to perturbation of MTs?

mitotic cells

What gave the first evidence of microtubule motors?

extruded axoplasm assays

What is step 1 of an extruded axoplasm assay?

cytosol is squeezed from the axon with a roller onto a glass coverslip

What is step 2 of extruded axoplasm asay?

addition of ATP shows movement by video microscopy

What ends can kinesins go to?

plus and minus

What end can dyneins go to?

minus end

What is the first step of kinesin mechanochemical cycle?

ADP state, no MT binding

What is the second step of kinesin mechanochemical cycle?

ADP release, MT binding

What is the third step of kinesin mechanochemical cycle?

ATP binding, linker zips onto head, trailing head thrown forward

What is the fourth step of kinesin mechanochemical cycle?

ATP hydrolysis, linker released

What is the fifth step of kinesin mechanochemical cycle?

Pi release, unbinding trailing head from MT

What are the important kinesins?

kinesin-1, kinesin-5, kinesin-13

What does kinesin-5 do?

bipolar and slides microtubules past each other

What does kinesin-13 do?

depolymerizes MTs

What do microtubules and their motors generate?

the intracellular membrane network

What are dyneins a result of?

a fusion of 6 hexameric rings into a single protein

What is the main ATPase of dyneins?

AAA1

What are the largest and fastest of the molecular motos?

dyneins

What is the first step of dynein movement?

binding, dynein binds to the microtubule with its motor domain

What is the second step of dynein movement?

ATP binding, causes a conformational change that releases dynein from the microtubule

What is the third step of dynein movement?

power stroke set up, linker region repositions, preparing dynein for the next step forward

What is the fourth step of dynein movement?

ATP hydrolysis, dynein rebinds to the microtubule one step closer to the minus end

What is the fifth step of dynein movement?

power stroke, release of phosphate triggers a power stroke, linker shifts position, pulling the cargo forward

What is the sixth step of dynein movement?

repeat

What are cilia/flagella?

specialized eukaryotic cell structures/organelles that use microtubules and dynein

What is motile cilia important for?

cell movement or movement of fluids

Where are cilia rooted at?

basal bodies, which are derived from centrioles

What is found at the core of a cilium?

specialized microtubule doublets in a 9 + 2 arrangemnt

What is the core of the cilium called?

axoneme

What do cilia use for movement?

ciliary dyneins

What does dynein do with linkers present?

causes bending of the microtubules

What do dyneins normally allow?

MTs to slide past each other

What are ciliopathies?

mutations in many factors important in cilia

What occurs with recessive primary ciliary dyskinesia?

situs inversus