6: Microtubule Functions Flashcards
What is the function of a microtubule? What does it need to carry out this function?
- vescile transport in both directions and at different speeds
- requires motor proteins which need ATP energy
model orgamism for MT transport?
squid axon
how is axonal transport studied?
- radioactive AAs injected into large axon body
- this makes radioactive proteins
- some of these proteins get transported towards + end of MT
- isolate different fragments of the axon and run on SDS page gel
- each fragment is a different distance from initial injection site
- proteins get separated on the gel and you will see bands of different sizes
- repeat process at several time intervals and run on separate gel to see movement of proteins by the different band sizes
- notice that some proteins move faster than others and some proteins move in groups
- isolate protein bands to determine what the protein is and which proteins work together
axonal transport does not occur through
diffusion
What is kinesin?
Microtubule’s plus end directed motor protein
moves towards plus end
What is the structure of kinesin? What function do each component have?
ATP is used for
2 heavy chains: head (ATPase activity and MT binding ability), flexible neck/linker and stalk regions
2 light chains: variable (depending on cargo recognizing and binding)
movement to (+) end by linker region bending
What would a kinesin protein look like on an SDS page?
2 heavy chains are the same = 1 high MW band
2 variable light chains = 2 low MW bands
kinesin has similar movement to
myosin (actin motor protein)
What is kinesin 1?
structure
- conventional - found in cytoplasm
- 2 same heavy chains and 2 sometimes variable light chains
What is kinesin 2?
structure
- heterotrimeric
- 2 different heavy chains + 1 light chain-like
3 bands on SDS page
kinesin 1 and 2 are involved in
organelle transport
What is kinesin 5?
structure + function + location
- bipolar (has 4 heavy chains - one on each side) ie 2 head domains and 2 linker regions on each side
- involved in mitosis
- does not bind to cargo - binds to stalk domain of another 2 heavy chains
- sits between parallel MTs for MT sliding
What is kinesin 13?
structure + function
- uses ATP hydrolysis to remove dimers off MT ends (depolymerization)
- heavy chain (2 head domains + linker/neck)
- not moving towards (+) end
- can remove from both (+) and (-) end, but usually (-) end has proteins bound to in
In kinesin, ATP hydrolysis causes
conformation change of kinesin = head moves 16nm (dimer is 8nm) = antereograde (towards + end) movement
how do we stop kinesin from movement when there is no cargo?
- heavy chains folded so that head domain can’t bind to MT
- no ATPase activity
What is cytoplasmic dyenein?
- MT minus end directed motor protein for retrograde transport
What is the structure of cytoplasmic dyenein
- 2 heavy chains have 2 head domains (with ATPase and MT binding, linker/neck region and stalk)
- linker bends for movement
- stem = tail
- linker + stem (tail) interact with dynactin hetero complex to recognize and bind cargo
What is dynamitin?
protein in dynactin heterocomplex that helps release cargo when it reaches location
What is P150glued?
- binds to MT
- no motor force
- helps hold dyenein + cargo in place
kinesin and dynein can move
proteins (including kinesin moving dynein to plus end so it can function again) and organelles (ex. golgi, lysosome, mito)
How is tubulin modified? Why do we use this method instead of MAPs?
posttranslational acetylation of lysin residue of alpha tubulin
this stabilizes MT and promotes kinesin-1 movement
MAPs can get in the way of transportation
differences between cilia and flagella?
cilia: 2-10um and sweeps material acriss tissue (many)
flagella: 10-2000um and propels cells (few)
what is an axoneme?
underlying structure of cilia and flagella
what does an axoneme look like?
-
9+2 array: surrounded by 9 doublet MTs which are connected by nexin with 2 singlet MTs in the middle (function unknown)
-** radial spoke heads **are attached to A tubule of outer doublets facing centre - holds structure in place - **axonemal dyein: **permanently bound to A tubule and head domains projects towards B tubule of next doublet
- all MTs are stable
- > 250 proteins
How is basal body related to axoneme?
basal body is MTOC for cilia and flagella: triplet MT in basal corresponds to doublet MT in axoneme (same number) and the transitional zone is the area between triplet and doublet
basal body may or may not have central singlet MTs
cilia/flagella usually come in
pairs
similarity & differences btwn basal bodies and centrioles
2 basal bodies/centrioles 90 degrees to eo
both MTOC
basal bodies directly polymerized into the axonemes BUT centrioles do not generate singlet MT, these singlets arise from PCM
axonemal dyenein is attached to the
A tubule of the triplet in the axoneme (cilia and flagella)
how does cilia/flagella move?
- a pair of MT- nexin links them and dynein head is attached to one and head domain reaches to the other
- if there is nothing holding the MTs together = sliding (not in axoneme bc nexin + basal body + radial spoke heads holds in place)
- head of axonemal dynein is minus end directed motor – when head moves, causes localized bending bc sliding can’t occur
only some MTs will be bound to each other - bending is localized
cilia and flagella (as long projections) can be used as
antenna (to receive and give signals)
secrete ligands & have signal receptors
how does intraflagellar transport occur?
movement of material up and down unrelated to bending by using cytoplasmic dynein for retrograde to minus end and kinesin for antereograde to plus end
cilia and flagella signaling antenna is important in
embryos
many interphase cells contain
a non-motile primary cilium (no axonemal dynein)
antenna like for cell-cell signalling
also found in embryos
primary cilia are
stabilized by acetylated tubulin and not found in mitotic cells
2 components of mitosis are
karyokinesis: dividing chromosomes and MTs
cytokinesis: dividing cytoplasm and actin
What happens to the cytoskeleton in interphase?
antereograde and retrograde transport (long half-life) change to be responsible for chromosome separation rearrange (very short half-life0
centrosomes duplicate (2 -> 4)
interphase MT are – from mitotic MT
different
What happens to the cytoskeleton in prophase?
breakdown of interphase microtubule array and its replement by mitotic asters which separate
What happens to the cytoskeleton in prometaphase?
chromosomes captured and brought to spindle equator
What happens to the cytoskeleton in metaphase?
chromosomes aligned by spindle fibers
MTOC centrosomes = poles
pole MT are different from centriole MT
What happens to the cytoskeleton in anaphase?
spindle pole separation
how are pole different from centrioles?
centrioles:
* interphase
* antereograde and retrograde transport MTs
* gamma tubulin ring complex at minus end = no depoly
* fast half-life 5 mins
* more stable MT
poles:
* mitosis
* spindle apparatus
* different mechanism for polymerization and nucleating much faster
* short half-life 15sec
* less stable MT
* depoly at minus end possible
What causes mitotic MT instability?
XMAP215 activity decreases in mitosis = unstable
types of MT in mitotic apparatus
SPINDLE
polar MT: MT growing towards another pole, but doesn’t reach a chromosome
kinetochore MT: holds chromosome by kinetochore protein
–
astral MT: grows not towards other pole
kinetochore proteins are attached to
plus end of MT (such that it can still polymerize and depolymerize)
how do all chromosomes align at metaphase plate?
- movement by poly and depoly of MTs (poly + kinesin on one side and depoly + dynein on other)
- kinesin and dynein help MTs move to move everything to metaphase plate (ie plus and minus end directed proteins)
what must occur by the spindle apparatus?
all chromosomes must be captured on BOTH sides
what happens with the chromosome is not attached on both sides?
Ndc80 captures MT = conformational change of Ndc80
without tension, Ndc80 gets phosphorylated by Aurora B kinase
phosphorylated Ndc80 lets go of MT
what happens with the chromosome is attached on both sides?
Ndc80 captures MT = conformational change of Ndc80
with tension, Aurora B kinase is inhibited = no phosphorylation = movement of chromosome to metaphase plate
What is the function of astral MT?
bind to dynein (which are on ends of cells and bound to PM)
pole and active cortical anchor are minus end = dynein walks towards minus end to pull the pole by astral MT towards the side that is far from metaphase plate
other side is inactive cortical anchor
What are the two phases of anaphase?
A: chromosome moves closer to pole by kinetechore MT shortening by depoly of minus and plus end
B: poles push away from metaphase plate + cell elongating requiring motors
how does anaphase B occur?
polar MTs overlap and sliding between these by kinesin 5 (+ end directed motors so that heads move towards respective + ends) to undo overlap = pushing poles apart
can polymerize more on + ends so kinesin 5 can keep pushing
dynein pulling astral MT also pulls poles apart + astral MT depolymerizes at + end
What is RhoGTP used for in mitotic spindle apparatus?
gets localized by polar MTs, RhoGTP localizes actin which allows for contracile actin/myosin ring at metaphase plate
ring is formed as far away from 2 new forming nuclei
