Microtubule functions
Axonal transport
can measure by using giant squid axon
when cytoplasm gets rolled out, can see many molecules moving around in the microtubules
the molecules that move around, move around at different speeds
conclusion that do move their structures around, ATP is required
how the technique is conducted
inject radioactive aa into the squid axon
radioactive aa is incorporated into protein machinery of the axons
the proteins produced are radioactive and move around the microtubules
because they are radioactive, they can be detected and collected at different sections of the axon
the proteins can be isolated with SDS page
the proteins are detected at different axon segments when waiting different amount of time
proves different proteins travel at different speeds in the microtubules
proves this is not diffusion
proves the proteins travel in groups
different proteins travel at a certain speed
injections must repeated while waiting longer to observed where a protein group has migrated to
kinesin
many different types exist
anatomy
2 heavy head chains
2 light chains that are variable to recognize different stuff
binds to the microtubule and have ATPase activity
to move to the + end
they can bind to cargo
composed of different domains
head
flexible neck (linker)
stalk (tail)
leads to the light chain
usually used for anterograde transport
some different types
Kinesin-1
Kinesin-2
Kinesin-5
Kinesin-13
the classic kinesin structure
2 different heavy chains and has a heterodomain for cargo binding
bipolar with 2 head domains that permit for microtubule sliding
they have a tail domain that overlap between the 2 kinesins
the head domains are always ATPase activity that bind to the microtubule
simply a head and neck domain that bind to the MT and remove dimers with ATP hydrolysis
works on both the + and - ends and favors MT disassembly
heterodomain is for organelle transport
ATP hydrolysis causes the kinesin head to undergo conformational change and step forward (16 nm each step)
regulated as it is inactive when folded
it is activated upon receptor binding
kinesin is positioned between and alpha and beta dimer
when not moving they are 8 nm apart
cytoplasmic dynein
involved in retrograde transport (goes to - end)
heavy chains
have ATPase activity
have a stalk that is part of the head
tail domain
along with the linker region,they interact with the dynactin hetero complex to recognize and bind cargo
the tail structure does not directly bind to the cargo
the dynactin complex
has many components such as actin, Arp1 filament and CapZ
one important protein, dynamitin helps regulate the binding to the cargo and releases when dynein reach the - end
too much dynamitin causes dynactin(protein complex) and dynein to explode apart
150glued binds the complex to the MT but is not a motor
cooperates with dynein in anterograde/retrograde transport where they themselves can be the cargo to go back and fourth
posttranslational modification of tubulin affect MT stability and transport
ex: acetylation of a lysin residue
axonemal bending
cilia and flagella
they are short/long versions of the same thing
cilila is 2-10 micro meters
flagella is 10- 2000 micro meters
sweeps material across tissues
propels cells
in both cases the structure is bending/sweeping to move stuff across the cell or moving the cells itself
more abundant than flagella
less abundant than cilia
axoneme
structure
9 doublet rings surrounding the a central pair of singlet microtubules (diff from cytoplasmic ones)
this is the 9+2 array
nexin and other proteins hold the doublets in place
the radial spoke heads does the same thing
they extend from a basal body, not a centrosome
has its own plasma membrane
axonemal dyenin can also be found such that it is stuck on the A-tubule and extends towards the B-tubule
from the basal body, it is a triplet microtubule and becomes a doublet at the transition zone
the center of the basal body or or may not have a singlet in the center
only A+B MT are extension from the basal body
this means the basal body contributes to MT polymerization while the centrioles do not
how bending occurs
it occurs through sliding with MT sliding past each other which is powered by axonemal dynein while being stopped by nexin
the basal body and nexin prevents sliding by holding the MT in place
can also be used for signalling
uses cytoplasmic dynein to transport retrograde and anterograde(this is to receive and give signals)
these are the same microtubules that are used for bending (the MT has multi-function)
example
interphase cells with non-motile primary cilium with no axonemal dynein that are important for signaling
the axonemal structure has no axonemal dynein and is only used for signaling
stabilized by acetylated tubulin
this cilium sends out important signals only in interphase
there is no MT present in the center
Karyokinesis
it is defined as the division of chromosomes
cytokinesis is the division of the cytoplasm
in interphase, the centrosome has not duplicated yet
when they duplicate, mitotic poles arise
MT stability and instability in interphase vs mitosis
in interphase XMAP215 stabilizes the MT
when XMAP215 is inactivated through phosphorylation, it increases the dynamic ability of MT during mitosis
nucleation of MT also increases a lot in mitosis
the mitotic apparatus
polar MT
Kinetochore MT
Astral MT
MT attached to kinetochore
MT going away from the poles
MT that is not capturing a kentitochore and can 2 can be found antiparallel to each other
how attachment occurs
(+) end goes to kinetochore but this part of the MT is still free to move
pulling is done by dynein
pushing is done by the motor protein, CNPE
metaphase
anaphase
to ensure kinetochore is attached on both ends
to ensure there is proper alignment
uses phorphorylation by aurora on protein Ndc 80
corticol anchor
one side is inactivated and other is activated to move the kinetochores to center
driven in 2 phases
anaphase A
anaphase B
chromosomes move to the ends
done through disassembly on both ends
poles is separating and cell starts elongating
driven by motor proteins
kinesin-5 is in the middle of polar MT moving to their + end to undo the overlap and pushes the poles apart
astral MT dynein is activated, depolymerization the MT and causes the pulling motion of the poles