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What are the monomers of the microtubules? Explain their relation with GTP.
alpha tubulin: GTP bound to this; tight; never comes off; never hydrolyzed
beta tubulin: GTP is loosely bound; can exchange it with cytosolic GTP and can be hydrolyzed
Structure of the microtubule in terms of its direction of monomeric arrangement.
Alpha and beta tubulins put together are __.
- appears helical
- - there's pitch to them
- - not horizontal (have angle to them)
- heterodimers because they are not exactly alike
Explain the positive and negative ends of the microtubule.
+ end: most of the action seen here whether added or removed
- end: less action
They can be added or removed from either end, but the + end is the more active end
Each vertical row in a microtubule is a __.
Molecular weight of a microtubule: __
Sedimentation coefficient: __
How many binding sites for GTP? For Mg++?
- 110-120000 (each tubulin may weigh 55k-60k)
- 2 (one on each molecule of tubulin)
- 2 (one on each molecule of tubulin)
Each dimer binds several different __. > __(numerical value)
- What are alkaloids?
- Where are they mostly produced?
- all organic with N and rings
Biochemistry of microtubules
- major protein: __
--studied from lots of types of cells (common in any kind of __ because they are __)
- similar in __ and __
- remarkable __
--- Example: isolated from sperm and chick brain cells and then sequenced. What was found?
What does this demonstrate? Why?
- euk cell
- the principle structure to move chromosomes in mitosis
- physical and chemical properties
- the first 24 amino acids are exactly the same
- shows that tubulin has been conserved evoluntionarily
- - you can't change it much and expect it to function
When dealing with assembly and disassembly, what is the process called and explain it.
- it is the equilibrium process
- it depends on promoting factors whether formation will occur or not
- If you have a pool of dimer subunits, assembly is favored.
If you isolate the __, they will form __.
What do you see in a cross section of the microtubule? Measurement?
- the cortex: ring of subunits= 1-13 (outside)
--the core: hollow inside
25 nm in diameter
What are all alkaloids? What do they bind? What are they used in?
What is taxol used in? vincristine?
- all are antimitotic
- bind to microtubules
- used in treatment of cancer
ovarian and breast cancer
childhood leukemia, brain tumors, breast cancer, Hodgkins, etc.
What do microtubules have that can bind MT to each other, to other components of the cytoskeleton and can cause cilia and flagella to move?
Kinesins were found in __
Dyneins were found in __
Cytoplasmic dynein was found in __.
cilia and flagella
__ of microtubules by __ occurs. There are separate regions for the binding of the __ to the tubule and to each other.
What are the steps of formation to the elongating microtubule?
- 1) tubulin dimers
- 2) associate to create oligomers
- 3) associate to create protofilaments
- 4) sideways association to create sheets of protofilaments
- 5) fold around to close and form a closing microtubule
- 5) now able to elongate into an elongating microtubule
Microtubules are assembled from subunits composed of one molecule of __ and one molecule of __ bound together tightly as a __, called an __, or simply a __.
- αβ-tubulin dimer
- tubulin dimer
At the start of the __, several __ can aggregate into clusters called __, some of which go on to form linear chains of tubulin dimers called __. The __ can then associate with each other side-by-side to form __.
- nucleation process
- tubulin dimers
Sheets containing __ or more __ can close into a tube, forming a __. __ continues by the addition of __ at one or both ends.
- elongation of the microtubule
- tubulin subunits
The function of the microtubule determines __.
What determines whether formation or disassembly occurs?
What are the three major factors that determine assembly and disassembly?
- the equilibrium process
- concentration of dimers
Microtubule growth in vitro depends on the concentration of aB-tubulin dimers.
The concentration of dimers when polymerization and depolymerization are balanced is called __.
Microtubules grow when dimer concentrations are high and depolymerize when dimer concentrations are low.
the critical concentration
Criticial concentration is lower at the __, meaning dimers are __. What does this explain?
You can't make microtubules unless you have __. It also determines what?
What kind of concentration is needed to get action.
- plus end
- more likely to assemble
- why more association at the plus end occurs
a higher concentration than the critical concentration
Explaiin GTP in terms assembly and disassembly.
GTP binding produces what so what happens?
one molecule of GTP and one Mg bind to each monomer
a conformational change in the tubulin so that dimers are more likely to bind to one another
GTP causes __ for one another. How many are in each molecule of tubulin. What happens to GTP?
- higher affinity
- hydrolyzed to GDP
What do MAPs do?
- shift the equilibrium to favor assembly and can stabilize a microtubule, making it more difficult for the microtubule to depolymerize; can lead to very stable microtubules
- - stability makes it harder for them to fall apart
Once dimers assemble, there may be changes in the microtubules, signlaing what?
MAPs to bind
Ca++ concentration affect on assembly/ disassembly.
True or False:
This is Ca++ primary role.
role unclear; concentrations that are greater than 1 mM cause depolymerizatioin, but increasing Ca++ concentration from 0 to 1 enhances polymerization
Role of calmodulin in assembly/ disassembly
During metaphase, where is it?
- role unclear; it's a Ca++ binding protein, so it regulates Ca++ concentration
- - distribution different in different areas
at the poles
Microtubule organizing centers (MTOCs)...assembly and disassembly role.
- polymerization of microtubules occurs in association with MTOCs
- the - end of hte microtubule is usually tightly bound to a MTOC. This may prevent depolymerization; they are firmly anchored, which may explain why very little occurs at the - end
Several types of MTOCs exist. Which ones?
1) MTOCs contain microtubules that do not connect to polymerizing microtubules (ex: centrosome)
2) MTOCs contain microtubules that do connect to polymerizing microtubules
3) CTOCs that do not contain microtubules
Temperatures effect on assembly and disassembly.
higher temperatures favor polymerization, lower temperatures favor depolymerization
Pressure's effects on assembly and disassembly
low pressure favors polymerization, high pressure favors depolymerization
All drugs are __.
Colchine may cause __.
Vincristine and vinblastine are very similar and may induce __.
Cortisol is __ and similar to __.
- clumping, causing less availability
What does colchicine do?
binds to free dimers and competes for binding sites that are normally required for dimer interaction during polymerization
Explain colchicine-dimer complexes.
they bind to the + end and block addition to dimers; colchicines alone can't bind to intact microtubules--> inhibit polymerization of microtubules
Where do colchines bind?
bind in a location that other dimers bind
Explain vincristine and vinblastine
link dimers into large, semi-crystalline aggregates, making the dimers unavailable and pushes the equilibrium toward disassembly
causes microtubules to depolymerize; similar to colchicine
binds tightly to microtubules and stabilizes them; pushing the equilibrium toward assembly, preventing depolymerization
What is the reason for the equilibrium shift?
because youhave MTs that can assemble and disassemble