6: Microtubule Functions
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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