5: Cytoskeleton Overview Flashcards
what is the cytoskeleton?
intricate network of protein filaments that extend throughout the cytoplasm
How do MTs have polarity?
one side is alpha (-) end
one side is beta (+) end
MT subunits are
alpha and beta tubulin which are 55kDa each. these form a stable dimer of 8nm
diameter of MT?
length of MT?
25nm
up to 100s of µm long (project out of cell - flagella)
alpha-beta dimers form
protofilaments when B subunit is in GTP subunit
are MTs perfectly circular?
no
seam forms
B tubulin is different from a tubulin because
B tubulin can be hydrolyzed from GTP to GDP
What are the types of micritubule structures?
- singlet: most common, found in cytoplasm made of 13 protofilaments
- doublet: found in cilia and flagella, A MT (13 PF) + B MT (10 PF)
- triplet: basal bodies & centrioles: A MT (13 PF) + B MT (10 PF) + C MT (10 PF) - stable tend to not polymerize and depolymerize
cytoplasmic MT are found in
axonemal MT are found in
cytoplasmic: singlet nerve axon cytoplasm MT
axonemal: doublet cilia/flagella MT
assembly of microtubule begina at
MT organizing centre (MTOC) which is associated with (-) end and (+) end grows away
What is the centrosome?
main cellular MTOC that contains centrioles and pericentriolar matrix
What are other MTOC examples?
- spindle poles in mitotic apparatus
- basal body: MTOC for cilia/flagella
- nerves: MT are not continuous (axon + dendrites: orientation varies)
centrioles are found in
animals (not plants)
centrioles are
2 barrel-like triplet MT that are perpendicular to each other
the pericentriolar matrix is:
space around centrioles where proteins gamma tubulin and augmin are found
gamma tubulin and augmin are
proteins to make singlet cytoplasmic MTs
gamma tubulin: starts polymerization of singlet MT
augmin: increases polymerization
both form gamma tubulin ring complex
when centrioles replicate…
pericentriolar matrix also replicates and they make a mother and daughter MT which are different
gamma tubulin ring complex provides …
nucleating sites for MTs = this is the minus end
a nucleating site is…
where polymerization of MT can occur on
augmin can (rarely) facilitate
MT branching
to study MT polymerization…
Take flagellar nucleus (piece of flagella) which acts as a nucleating site, which is a doublet MT = stable and won’t depolymerize
Add tubulin diners to the nucleus until critical concentration for polymerization is reached
Below critical concentration =
depolymerization
MT – side polymerization and depolymerizes faster because …
plus
bc
minus end has gamma tubulin ring complex = capped = blocking polymerization and depolymerization
Time of elongation of protofilament depends on
nucleus presence
What is dynamic instability?
MTs are always growing and shrinking independently of each other
Catastrophe: MT stops polymerization and starts depolymerizing (for ex. Below critical conc.)
**Rescue: ** MT stops depolymerizing and starts polymerizing (for example: above critical conc.)
dynamic instability depends on
presence or absence of GTP-beta-tublin cap
GTP-beta-tubulin cap provides
lateral cohesion: protofilaments are attached to each other, which increases when alpha and beta tubulin are GTP bound
As the protofilament grows, GTP is
hydrolyzed to GDP in beta tubulin
Most beta tubulin in protofilament that is not on the plus end is
in GDP form by hydrolysis = less
lateral cohesion
how does MT prevent fraying?
minus end has gamma-Tu-RC and + end has GTP-B-tubulin cap which prevents fraying –> fraying = depolymerization
how does depolymerization occur?
hydrolysis catches up to + end = all B-subunits are in GDP form
* Exact mechanism of assembly is unclear as is role of seam
2 requirements for polymerization are
- above critical concentration
- all B-tubulin GTP form
Colchicine is
MT depolymerizing drug -> remove drug -> see how polymerization occurs
Taxol is
stabilizes MT -> MT don’t depolymerize -> remove drug -> see how depolymerization occurs
* Anti-cancer drugs: need depoly. of MT in mitosis so taxol can be used to stop depolymerization in cancer cells
What are MAPs
Microtubule Associated Proteins
* coats MT and stabilizes or destabilizes
* can be regulated or bundle MT
Example of MAP regulation
Phosphorylation can promote disassembly/destabilization e.g. CDK in cell cycle regulate MAPs
MAPs that prevent depolymerization are
MAP2 and Tau - binds to MTs
Structure of MAPs MAP2 and Tau?
MT binding domain and projection domain (sticks out of MT to attach to next MT)
difference between Tau and MAP2
Large projection domain = MTs are far apart from each other (ex MAP2)
* Small projection domain = MTs are close together (ex. Tau)
* Size of projection domain can regulate MT spacing
What are +TIPs?
special MAPs associated with the (+) end of MTs that when present helps/stabilizes polymerization and when absent depolymerization occurs
example of a +TIP?
EB1
* binds to unique structures found on growing (+) end and potentially stabilizes and reducescatastrophe
* EB1 moves along the seam of MT towards (+) end possibly transporting something
What are XMAP215 and CLASP?
- MAPs (not +TIPs) found in (+) end and have TOG domains
- TOG domains bind to growing protofilament at (+) end to help stabilize (+) end to prevent catastrophe by keeping dimers together
What does kinesin-13 do?
- with ATP destabilizes MTs by removing terminal dimers
What does stathmin do?
- protein that promotes protofilament bending by decreasing lateral cohesion
- bind tubulin dimers in the curve may promote fraying and GTP hydrolysis
- stathmin can be inactivated by phosphorylated
depolymerization/destabilizing promoting proteins?
- kinesin-13
- stathmin
stabilizing proteins?
- MAP2 and Tau
- +TIPs (EB1)
- XMAP215 and CLASP
