11.6-11.11 Flashcards
true or false: dynamic instability is a non-equilibrium process
true
what drives dynamic instability
GTP hydrolysis by tubulin
during assembly, B-tubulin is stimulated to do what?
hydrolyze bound GTP to GDP
is GTP hydrolysis or polymerization faster
polymerization
does a growing microtubule have mostly GDP or GTP B tubulins
GDP
true or false: a tubulin always has GTP
true
which dissociates faster: GDP tubulin or GTP tubulin
GDP tubulin
what does the exposure of GDP at the end of a microtubule result in
rapid depolymerization
what is catastrophe the result of
a growing microtubule losing its GTP cap
what does rescue require
GTP tubules to re-cap the end of a shortening microtubule
does the GTP cap hold the protofilaments straight or curved
straight
do GDP tubules favor curve or straight conformation
curved
what triggers hydrolysis of GTP
incoming heterodimers stimulate hydrolysis to previously added heterodimer
size of GTP-tubulin cap
unknown, but could be single layer
nucleation
forming base from which microtubules eminate
are heterodimers first attached on the - or + end
-
MTOCs (microtubule organizing centers)
almost always used by cells to nucleate microtubules
are MTOCs associated with the minus or plus end of microtubules
minus
most common animal cell MTOC
centrosome
centrosome composition
pair of centrioles at right angles to each other and some pericentriolar material
centriole composition
9 sets of triplet microtubules arranged in a circle to form walls of barrel-like structure
what are the centriole triplet microtubules made from
a and B tubulin, and S- and E- tubulin
pericentriolar matrix
composed of about 100 different proteins, including y-tubulin as part of yTuRC
yTuRC (y-tubulin ring complex)
the MTOC that does the actual nucleation of the microtubules
structure of yTuRC
arranged as one turn of a very shallow helix, shape of lock washer
- resembles one turn of helix made from microtubule protofilaments as they form the hollow tube in a spiral fashion
about how many spindle fibers per sister chromatid
40-50
where does the yTuRC nucleate microtubules from
their minus end
if the MTOC is near the nucleus, where will the plus ends bw
toward cell periphery
when does the centrosome get larger
interphase
when does each centriole duplicate
S phase (at right angles toward themselves)
additional MTOC in motile animal cells
basal bodies
basal bodies
serve as templates for assembly of axoneme
axoneme
bundle of microtubules that will form core of cilia and flagella
basal body structure
nine triplet microtubules
difference between basal body and yTuRC/centrioles
basal body: nucleates axonemal microtubules
yTuRC/centrioles: nucleate astral ray microtubules
how do fungi nucleate and organize microtubules
spindle pole body embedded in nuclear envelope
how do plant cells nucleate and organize microtubules
nucleating sites distributed throughout cell cortex
how do epithelial cells nucleate and organize microtubules
nucleation sites near apical end of cell
what do various MTOCs all contain
y-tubulin - use similar mechanism to nucleate microtubules
at what phase do centrosomes reach the poles of cells?
prophase
how can microtubule dynamics be visualized
fluorescent tubulin:
- expressing tubulin fused to fluorescent protein
- injected cells with purified tubulin tagged with fluorescent dye
what is FRAP used to obtain
images of microtubule turnover
do interphase or mitotic microtubules have a longer half-time/rate of turnover
interphase
how does dynamic instability differ between in vivo and in vitro
- plus ends grow faster in vivo
- switch between growth and shortening more frequently in vivo
- pauses common in vivo
where are dynamic instability switches more frequent
toward edges of cell near plasma membrane
do all cells have dynamic microtubule populations?
no, some cells have more stable populations
modifications of more stable microtubules
- capped at plus ends
- contain more modified tubulin
where are stable microtubules more abundant
nonmitotic, differentiated cells
how do free microtubules exist
- broken off from anchored microtubules
- have been released from centrosome
where can free microtubules be stabilized
at minus end
treadmilling
dynamic instability of microtubule plus end is biased toward net growth while the minus end shortens
why do cells have dynamic microtubules
need to adapt to new situations or new stages of CDC
ex. of how microtubules search interior of cell
during prophase they need to find and connect their plus ends to kinetochores
what happens to microtubules that don’t hit a kinetochore
rapidly fall apart
what happens to microtubules that hit kinetochore
stabilized
where can dynamic microtubules detect a signal
plasma membrane - can induce polarization and begin to change shape
how are some proteins and organelles transported
have ability to hold on to tip of growing or shrinking microtubule
how is most movement in cells generated
molecular motors
do proteins usually bind to plus or minus end
more plus
MAPs (microtubule associated proteins)
family of proteins that bind to microtubules and:
- speed/slow growth/shortening
- function as linkers between the microtubule tip/sides and membrane vesicles or other structures
+TIPs
MAPs that bind to microtubules only at plus ends
how long are +TIPS bound
short time - continually falling off and being added
ex. of +TIP
CLIP-170
CLIP-170
stabilizes microtubules and promotes rescue by linking endosomes to microtubules
how to some MAPs make microtubules less stable
- disrupt GTP cap
- cut microtubules into pieces to make more ends to shorten
- bind free tubulin subunits
katanin
cuts microtubules by binding to their walls and disrupting contacts between tubulin subunits
what energy does katanin uses
ATP
MCAK (mitotic centromere associated kinesin)
molecular motor that disrupts GTP cap by binding at microtubule ends and destabilizing the tip structure by favoring formation of protofilaments that curve away from microtubule wall
is MCAK recycled?
yes
what is MAP activity often regulated by
phosphorylation and dephosphorylation
molecular motors
microtubule-binding proteins that use repeated cycles of ATP hydrolysis to power continuous movement along the side of a microtubule
2 families of molecular motors
- kinesins
- dyneins
kinesins
usually move toward plus ends of microtubules
dyneins
only move toward microtubules’ minus ends
for typical fibroblast cell, where do dyneins and kinesis usually transport:
dynein: toward center of cell
kinesin: toward plasma membrane
what contributes to direction of movement and navigation by the motor?
polarity of polymer
why have no motors been identified on intermediate filaments
because it doesn’t have polarity
two main methods of motor protein function
- motor proteins walk along anchored microtubule
- motor proteins are anchored and move microtubule
characteristic shape of molecular motors
pair of large identical globular domains at end of long rod-shaped domain
- sometimes second pair of smaller globular domains at other end
what binding sites are on the large globular domain (head/motor domain)
polymer and ATP
what are footsteps always taken by
heads
what does the tail domain bind to
cargoes
composition of molecular motors
several polypeptides, bulk is homodimer held together by coiled-coil interactions along rod-shaped region
stathmin
MAP that binds to free tubulin
kinesin with N-terminal motor domain
move vesicles toward plus end
kinesin with C-terminal motor domain
move vesicles toward minus end
kinesin with middle motor domain
regulated dynamics by using ATP hydrolysis to weaken microtubule cap
what is kinesin with middle motor domain similar to
MCAK
kinesin with bipolar motor domain
allows binding to 2 microtubules to slide them past each other
- mitosis and cytoskeletal rearrangement
cytoplasmic dynein
homodimer with 2 motor domains
axonemal dyneins
cilia/flagella - heterodimers or heterotrimers, 2-3 motor domains per molecule
