Flashcards in exam 3b missing lecture Deck (89):
What are the three filament systems?
Actin filaments, microtubules, intermediate filaments
Two kinds of microtubules
Size of microtubules? What do they bind to?
50 kDa globular GTP-binding proteins
What kind of enzyme activity does beta-tubulin have? What does it form?
GTPase activity. It forms heterodimers.
How do microtubule heterodimers assemble? What do they form?
Head-to-tail. They form a protofilament.
How is a microtubule hollow tube formed? What is its diameter?
13 protofilaments assemble side-by-side to form a protofilament. Its diameter is 25 nm.
Where is there structural polarity in microtubule structures? What does that mean?
Within heterodimer, protofilament and microtubule itself. One positive end and one negative end.
What is the length of a microtubule?
Describe microtubule and its GTP activity
It includes one alpha tubulin and one beta tubulin stacked on top of each other. The GTP of the beta tubulin is exchangeable and hydrolyzable. The GTP of the alpha tubulin is nonexchangeable and nonhydrolyzable.
Let’s assemble microtubules in vitro. What do we need?
We need purified tubulin plus MG2+ plus GTP
Microtubule in vitro assembly conditions (temp) for animal tubulin.
37 degrees Celsius
Rate of assembly equation? Rate is dependent on?
konC. Dependent on concentration of monomer.
Rate of disassembly equation? Disassembly rate is dependent on?
Koff. Dependent on nothing. Disassembly rate is independent of monomer concentration.
What is equilibrium equation?
konC = koff, so Cc = koff/kon
Is GTP hydrolysis required for assembly?
How does Conc of monomer compare to Cc when assembly occurs? What is the free energy change?
Conc of monomer is above Cc, and the free energy change is zero. (which is what it has to be in order for assembly to be done)
How does Conc of monomer compare to Cc when disassembly occurs? What is the free energy rate?
Conc of monomer is below Cc, and the free energy change is above zero.
When a tubulin is assembled onto a bundle of parallel microtubules, when end of polarity does it prefer?
The plus end
What does GTP tubulin have a high affinity for?
In microtubule, at what rate does beta-tubulin hydrolyze GTP?
At stochastic (random) rate
When might GTP hydrolysis catch up with microtubule assembly? What happens to GDP tubulin at that point?
When the rate of assembly slows. At that point, GDP tubulin at the end will be exposed.
Does GDP tubulin have high affinity for microtubule?
No. it has low affinity. So when it is uncapped, there is rapid disassembly.
What does GTP tubulin cap assembly do?
It stabilizes GDP and allows for regrowth.
How does GTP hydrolysis work to disassemble microtubules?
GTP hydrolysis changes the dimer subunit (alpha and beta subunit) conformation. It weakens the bond in the polymer and changes a straight protofilament into a curved protofilament. Changing GTP to GDP weakens the bond between the polymers and causes depolymerization, leaving a single GDP-tubulin dimer that can only bind to another polymer once its GDP has been exchanged for GTP. This is called dynamic instability.
Does dynamic instability of microtubules occur only in vitro?
No. it occurs both in vitro and in vivo.
At what ends of the microtubule does dynamic instability occur?
Both plus and minus ends, but Cc differs at the two ends due to GTP hydrolysis.
When are minus ends often capped in microtubules?
For microtubules what is catrastrophe? Rescue?
Disassembly is catastrophe. Rescue is reassembly.
What regulates dynamic disassembly?
Half life of microtubules in interphase cells? In mitotic cells?
In interphase cells, 10 minutes. In mitotic cells, less than 2 min.
What happens to cytoskeleton during cell division?
Extensive remodeling of the microtubule cytoskeleton. Polymerization and depolymerization.
How dos drug Taxol (paclitaxel) affect microtubules?
It binds and stabilizes microtubules, inhibiting dynamics.
How do colchicine and colcemid affect microtubules?
Bind microtubule subunits and prevent their polymerization
How do Vinblastine, vincristine affect microtubules?
Bind microtubule subunits and prevent their polymerization
How does Nocodzole affect microtubules?
It binds subunits and prevents their polymerization
What do microtubule organizing centers (MTOCs) do?
They organize microtubule growth in vivo.
What nucleates microtubule growth? From what ends?
Protein complexes containing gamma-tubulin ring complexes (gammaTuRCs). From the minus ends.
How is gamma-TuSC formed?
Two copies of gamma-tubulin associate with a pair of accessory proteins to form the gamma-tubulin small complex.
How does gamma-TuSC form gamma-TuRC?
Seven copies of the gamma-TuSC associate to form a spiral structure in which the gamma-tubulin lies beneath the first, resulting in 13 exposed gamma-tubulin subunits in a circular orientation that matches the orientation of the 13 protofilaments in a microtubule.
What is microtubule seam?
Longitudinal discontinuity between two protofilaments. Results from the spiral orientation of the gamma tubulin subunits.
What is the centrosome in animal cells?
How are gamma-TuRCs arranged in centrosome?
Embedded in protein-rich matrix.
What is inside centrosome?
A pair of centrioles that organizes the matrix.
Where is centrosome located? How are microtubules attached to it?
Centrosome is in cytoplasm next to the nucleus. The minus end of each microtubule is embedded in the centrosome. The plus end grows out.
Do cells of land plants have centrosomes and centrioles?
Where are gamma-tubulin and gammaTuRCs in plants?
They are dispersed and nucleate the growth of microtubules.
Where do interphase microtubules lie in plants? What do they do?
In parallel arrays beneath plasma membrane to guide deposition of cellulose microfibrils in the cell wall.
What is augmin?
A protein that attaches to the sides of microtubules.
How does augmin attach to microtubules? What does it do?
It binds to gammaTuRC to initiate growth of a new microtubule “branch”.
How do microtubule associate proteins associate with microtubules? What is their role?
They bind to the sides of microtubules. They mediate interactions with other cellular structures and stabilize microtubules.
What is direction of microtubule polarity in axon?
Minus end at some and plus end at terminal branches.
What are two microtubule associate proteins?
MAP2, which is 25 nm long, and microtubule tau
How are microtubules spaced with bound MAP2 or tau?
MAP2 makes them farther apart than tau because MAP2 is stretched out away from microtubule whereas tau winds back to microtubule.
What are +TIPS?
Proteins binding microtubule plus ends
What do +TIPS do?
Regulate dynamic instability.
Name three +TIPS proteins?
XMAP215, kinesin 13, and EB1 protein
What does XMAP215 do? Results in?
It is a plustTIP protein that stabilizes plus end. Results in longer, less dynamic microtubules.
What does kinesin 13 do? Results in?
It is a catastrophe factor that induces catastrophe. Results in shorter, more dynamic microtubule.
What does EB1 protein do?
It binds to GTP-tubulin at plus end of microtubule. It directs growing microtubule to “target protein” on plasma membrane to initiate/maintain cell polarity. It leaves the microtubule when GTP cap is lost (disassembly).
What is katanin? How does it work?
Katanin is a microtubule severing protein. It promotes rapid microtubule depolymerization by severing microtubules.
How to microtubule motor proteins work?
They couple ATP hydrolysis to movement.
How do kinesin motors move?
They walk along microtubule toward “plus” end.
What do motor domains of motor proteins do?
They bind microtubule and bind and hydrolyze ATP
What domains are conserved in kinesin superfamily?
Only motor domain.
Describe domains of kinesin-1?
Motor domain at N end. Membrane vesicle cargo at C terminal.
Describe domains of kinesisn-5?
Motor domain at N end. Slides two microtubules past each other. Forms tetramers where two dimmers associate by their tails (C ends)
Motor domain is in middle of heavy chain. Has lost typical motor activity. Instead binds to microtubule plus ends to promote depolymerization.
C-terminal kinesin. Travels in opposite direction from majority of kinesins, towards the minus end instead of plus end of microtubule. Functions in mitotic spindle.
What is processive movement of kinesin along a microtubule?
Processive motor proteins carry out many cycles of nucleotide hydrolysis and movement before leaving the track. (so, how much and long they move before leaving track)
What are dynein proteins? How large are they and what kind of domain do they have?
They are motor proteins. Very large (>500 kDa) with an “AAA” domain.
How is cytoplasmic dynein structured?
It is a homodimer.
How do dynein subunits make enzyme processive?
They alternate movement.
What do dynein proteins do?
They transport cargo along microtubules towards the “minus” ends
What does dynein require to associate with membrane-enclosed organelles?
A large number of accessory proteins.
What is dynactin?
A large complex that includes components that bind weakly to microtubules, components that bind to dynein itself, and components that form a small, actin-like filament made of the actin-related protein Arp1.
Where is dynein located (2)?
Cytoplasm and cilia.
Describe cytoplasmic dynein.
Two heavy chains that contain domains for microtubule binding and ATP hydrolysis (each), connected by a long stalk. Bound to heavy chain are multiple intermediate chains and light chains that help mediate many of dynein’s functions.
Two types of axonal transport
Anterograde transport and retrograde transport
What is anterograde transport?
Movement from soma to terminus.
What is moved via anterograde transport? At what rate?
Vesicles, mitochondria, proteins, microtubules and intermediate filements, Herpes Simplex. Move at 2 micrometers per second.
What is retrograde transport?
Movement from terminus to cell body.
What is moved via retrograde transport?
Mainly materials to be degraded in lysosomes but also tetanus toxin, viruses including Herpes Simplex, Rabies
How do vesicles in axonal transport? What is axon’s polarity?
Along microtubule “tracks”, so polarity is important. Minus end at soma. Plus end at terminus.
Let’s take the pigment granule movement in melanophore pigment cells. When is color least concentrated? Most? What causes changes in concentration?
Color is least concentrated when pigment cells are dispersed from center. Color is most concentrated when cells are aggregated at center. When cAMP is decreased, cells aggregate at center. When cAMP is increased, cells disperse.
What does Golgi localization in animal cells depend on?
What is FtsZ?
A tubulin homolog in bacteria
How is FtsZ amino acid sequence conserved? What kind of structure does it have?
About 17% conserved with alpha, beta-tubulin. It has a crystal structure similar to tubulin.
How does FtsZ work? What is its job?
It binds and hydrolyzes GTP to form protofilaments.