Erika Holzbaur Flashcards
Microtubule remodeling – a fast or slow process?
Fast!
Tubulin – structure?
Heterodimer of alpha and beta tubulin, and there is a site for GTP binding on both; however, only beta tubulin hydrolyzes GTP; on alpha tubulin the GTP has a purely structural and non-exchangeable role.
How big are alpha and beta tubulin?
50 ka polypeptides
How many tubulin genes do mammalian cells have?
6 alpha tubulin and 7 beta tubulin genes – differences mostly map to the C terminal 15 aas of tubulin. Usually isotypes are exchangeable.
Are tubulins conserved?
Yes highly; only region of diversity is C terminal tail. Mutations in neuronal tubulins can cause neurodevelopmental diseases.
Do we have a crystal structure of tubulin?
No, but we have crystal structures with binding proteins attached. Some homology to Ras and GTP binding proteins.Structure of A and B are almost identical.
Microtubules
Hollow tube of 13 protofilaments, 25 nm in diameter. Microtubules may be assembled with tubes of 9-16 protofilaments, these are rare. Dimers arranged head to tail, so the polymer is polar. Beta is exposed at plus end, alpha at the minus end.
Will tubulin spontaneously assemble?
Yes, it will. If you incubate it at 37 degrees.
Are there covalent interactions between subunits?
Only noncovalent interactions
What kind of structure do microtubules form in the cell?
A B lattice. The symmetry of the helix is broken by a seam running down the length of the microtbule. Every tubulin dimer has the same contacts except the ones along the seam.
Similarities and differences between microtubules and actin filaments?
Similarities: both are formed from self associations of subunits, both are polar polymers, both have ends with significantly different kinetics of polymerization and depolymerization, polymerization does not require an exogenous energy source. Differences: actin filaments are thin and flexible, while microtubules are thicker and 300 times stiffer, therefore they can exert pushing forces.
What is the slow phase?
Nucleation… speeds up after that. There is a lag phase though.
Fast growing end? Slow growing end?
Plus end = fast. Minus end = slow.
What is the critical concentration?
C = koff/kon = kD. Above critical concentration net polymerization will be observed. Below this net depolymerization will be observed. ???
Dynamic instability
Microtubules stop growing suddenly and shrink back rapidly
Catastrophe
Sudden disassembly
Rescue
Turn their life around and start to expand again
How many microtubules are growing slowly and how many are rapidly shortening?
Many are growing, few are shortening
What explains dynamic instability?
GTP hydrolysis by beta tubulin could destabilize the end of the microtubule and act as the switc
What is the role of GTP hydrolysis?
It destabilizes the polymer lattice (GDP-bound tubulin binds less tightly to the filament end than GTP bound tubulin. The tubulin-GTP off rate is 1 s-1 vs 700s-1).
What do depolymerizing Mts look like?
Rams horns
How does the structure change with GTP hydrolysis?
It induces a compaction of the nucleotide binding site between adjacent dimers – this strains the structures.
How deep is the GTP cap?
> 20 subunits deep. May be 700-800. Shortening excursions in addition to catasstrophic depolymerization
Where is GTP tubulin?
May not just be at the very end but also throughout the polymer. Local islands of stability. Could these GTP remnants mediate rescue in the cell?
Where is tubulin in cells?
In general, plus ends are dynamic but minus ends are rooted at centrosome or capped by other structures. There is usually an array with plus ends radiating out.
Microtubule arrays within cell include
- Interphase (MTOC), 2. Cilia/flagella, 3. Mitotic, 4. Neuronal
How does polarity in dendrites work?
It’s mixed
MTOC
At the center you have a pair of centrioles; surrounding the centrioles is a cloud of pericentriolar material. Microtubules nucleate from sites in the pericentriolar material. The gamma tubulin ring complex is required for microtubule nucleation.
Gamma turc
25 S complex composed of 14 moles of gamma tubulin. This shares 35% of its sequence identity with alpha and beta tubulins; it is highly conserved; purified complex forms a ring, 28 nm in diameter. It blocks minus end growth but does not block plus end growth. Provides a template for growth. Has a plug shape at the bottom.
tempered dynamic instability
Refers to the fact that microtubules in the cell assemble and disassemble at much higher rates than seen in vitro at the same tubulin concentrations
Post translational modifications of tubulin
Detyrosination, acetylation, glutamylation, glycylation, phosphorylation, palmitoylation – most of these things occur on STABLIZED MICROTUBULES. They correlate with stabiity but they dont necessarily cause it.
