Term 2 Lecture 5: Cytoskeleton, Microtubules Flashcards
Microtubules interphase role
Form ‘tracks’ allowing motor proteins to move vesicles and organelles around the cell. (Form mitotic spindle in mitosis)
Microtubule info
Hollow cylinders made of multiple protofilaments (tubulin) in a tubular arrangement - we don’t know why it’s hollow
25nm outer diameter
One end attached to a ‘microtubule organising centre’ (MTOC) or centrosome near golgi (where motor proteins are)
Grow/shrink rapidly by tubulin addition/loss.
Grow out of the centrosome towards cell periphery
Formed of 2 proteins, alpha and beta tubulin which form a dimer, a stable basic building block
Visible in TEM as relatively large and straight.
In mitosis cytoplasmic microtubules disassemble and reform as the mitotic spindle.
Alpha tubulin is always bound to GTP whereas beta tubulin can be bound to GTP or GDP form and nucleotide is exchangeable. The GTP of alpha tubulin is buried in its structure becoming more accessible in GDP hydrolysis of GTP →GDP which drives movement
Depending on microtubule function the MTOC can be named/function differently
Metaphase cell - centrosome
Mitosis - spindle poles
Cilia/flagella - basal body
Neuron - multiple types of MTOC
Tubulin can be tagged with GFP for dynamic observation of microtubules in living cells
In interphase only one centrosome replicated in mitosis and each migrates to opposite ends of the cell becoming 2 spindle poles
Cilia move by motor proteins moving along microtubules
Tubulin dynamics
At steady state tubulin dimers are preferentially added to + end and lost from - end
In cells MT (-) ends are usually anchored to a microtubule organisation centre (MTOC) therefore dynamics mostly occur at (+) end
The (-) end has a higher critical concentration (Cc) so needs a higher concentration of free tubulin to grow
The (+) end has a lower Cc so grows at lower free tubulin concentration
Structure of centrosomes
9 triplet microtubules
Assembled on a ‘cartwheel’ structure
Surrounded by pericentriolar material
Including gamma tubulin ring complex (gamma TuRC)
Gamma TuRC nucleates microtubules
Links them to nuclear envelope in plants
Links to gamma 2 complex in animal cells
→takes seconds to minutes for tubulin to lengthen/shorten
Dynamic instability of microtubules: sudden shortening followed by rescue
Unstable structures in vitro, stabilised in Vivo by proteins
Can be tagged to track them
Catastrophe: sudden disassembly of tubules
Rescue: sudden reassembly
(Random in vitro)
Why dynamic instability?
In-built instability due to GTP hydrolysis by beta tubulin
GTP bound beta tubulin binds more stably than GDP tubulin and ‘caps’ growing end
If GTP beta tubulin is added faster than it hydrolyses it’s GTP then the tubule grows
If addition is slower than hydrolysis the cap is lost and tubule shrinks rapidly (catastrophe) the protofilaments unzip curl back and break up
Molecular explanation for the instability of microtubules with GDP tubulin
Due to confirmational change in beta tubulin structure
-GTP beta tubulin forms straight protofilaments
-protofilaments become curved when beta tubulin converts GTP to GDP
This introduces tension into the microtubule, constrained only by the GTP cap.
Leads to dynamic instability (similar to a fraying rope)
Post translational (after synth) modifications of tubulin affect MT stability and function
Other factors affect tubulin stability such as tubulin binding proteins
MT achieve stability by post -translational modification e.g. acetylation, phosphorylation and methylation.
These modify the protein by changing the properties of the side chains.
E.g. alpha tubulin can be acetylated at Lysine K40, preventing growth/shrinkage
Acetylated alpha tubulin is found in transport tracks in interphase, centrioles and primary cilia.
MT binding proteins
MAP2 and Tau are examples of bundling proteins
Katanin severs microtubules (as in mitosis)
Stathmins bind subunits and prevent assembly
TIPs that remain bound to (+) and can link them e.g. to membranes
Microtubules and directional transport
Polarised microtubule system allows cargo movement along microtubule tracks
Nerve cells are highly polarised, very long cells, relying on diffusion alone transport could take years so instead transport relies on microtubule motors that drive transport up to 10cm per day (70 micrometres/min)
Two classes of motor proteins exist
Kinesin: drives movement towards (+) end, plentiful in most cells
Dynein: drives movement towards (-) end
- both can detach and reattach
- both contribute to separation of chromatids in mitosis
Kinesin dependent vesicle transport
The two kinesin heads (often referred to as feet) use ATP hydrolysis to coordinate ‘walking’ on microtubules. ATP binding to motor head domain causes confirmational change. This swings previously ‘trailing’ head forward to become the ‘leading’ head
Kinesins transport pigment granules, early endosomes, secretory vesicles, ER and mitochondria
Dynein dependent transport
Dynein is another motor protein it binds it’s cargo by adaptor proteins and transports towards (-) end.
Dynein power stroke is the result of ATP binding and hydrolysis causing the head domain to rotate, detach and move along the microtubule- like a ‘hop’
Dynein requires dynactin adaptor protein to link to cargo.
Cytoplasmic dyneins transport: lysosomes, late endosomes, pigment granules, ERGIC, golgi and mitochondria
Summary
Microtubules are tracks for vesicles, organelles and chromosomes.
Microproteins carry cargo along microtubules - dyneins towards (-) end and kinesins towards (+) end.
Kinesin “walk” is driven by ATP hydrolysis confirmational changes
Dynein movement by ATP binding and hydrolysis which induces head region rotation and hopping movement
