Midterm 2 Flashcards
What is the subunit for MTs?
What are its dimensions?
What’s the polarity?
Stable or unstable?
alpha beta-Tubulin dimers
one dimer is 8nm long
- end=at alpha side, + end=at beta side
Stable.
What are the specific bindings of each dimer of the dimeric tubulin subunit?
alpha permanently binds to GTP
beta binds to both GTP and GDP because it can hydrolyze GTP– which participates in the formation of the protofilament, and is also associated with the polymerization of MTs
what are the dimensions of the protofilament hollow tube structure?
What is it?
What are the other structures that more hollow tubes form? + properties
Where can they each be found?
Singlet Microtubule:
- 25nm in diameter
- 100s of um long
- slanted at seam so protofilament 1 interacts with protofilament 13.
- 13 protofilaments
- unstable, can be dis/assembled quickly (dynamic)
- found in cytoplasm
Doublet and Triplet Microtubule: (aka, Axonemal MT)
- 13 protofilament as first tube, then 10 as next
- stable
- found in cilia
True or False: Cytoplasmic MTs are stable.
False. They are unstable and dynamic.
What do MTs need in order to be assembled?
Give me some examples of those.
What’s significant about their organization with MTs?
Exception?
MTOCs: Microtubule Organizing Centres
- centrosomes, basal bodies (in cilia), mitotic spindle poles
MTs - end is associated with MTOCs, therefore MTs extend to periphery from their + end.
—-Exception! Dendrites (mixed orientation of MTs)
How do you build MTs?
Where was one example where they were built?
What happens if you go back to under Critical Concentration?
On a graph, what will the change in MT mass to dimer mass look like relative to Cc?
By adding alphabeta-tubulin dimers until you hit the Critical Concentration of tubulin dimers bound to GTP, at which point the polymerization (small molecules become bigger ones) of the subunits happens to spontaneously form MTs!
The subunits are added to the flagellar ‘nucleus’ on either side, making the + and - end
If subunits are less than Cc, then you lose tubulin dimers (faster at + end).
On a graph, you’ll see that once you hit Cc, you won’t see increase in dimers anymore but instead increase in Mts. The dimer mass stays same as Cc.
Explain the structure/composition of centrosomes
What is the pericentriolar matrix made of?
Centrosomes, a MTOC, contains 2 centrioles (90 degrees from each other) which are surrounded in pericentriolar material/matrix, and has MTs embedded within this matrix (but not touching the centrioles)
Pericentriolar matrix composed of gamma-tubulin and augmin complex proteins.
Explain Centrioles (structure) Where are they found? What organisms?
- not in plants
- basically MT barrels. Made up of 9 triplets bound together to make a barrel/centriole
What is the role of Gamma Tubulin Ring Complexes?
Where are they situated?
What else accompanies it?
Gamma-Tubulin Ring Complexes (gamma-TuRC) are on the - end of MTs (the part that’s in the matrix), and provides a nucleating site for MTs.
They induce polymerization of alphabeta-tubulins.
Augmin proteins also support the production of MTs via the polymerization of GTP-bound subunits
Expand on the polarity of tubulin polymerization
(how polarity impacts MT formation)
What conditions are needed for the depolymerization of MTs?
MTs dis/assemble faster at the + end (aka, subunits are added faster there)
can be disassembled in lab at 4C (our body doesn’t get that cold)
What is responsible for Dynamic Instability of MTs?
What does the cap do?
The presence/absence of GTP-beta-tubulin “cap” determines the dynamics of the MT length because it keeps the + end from fraying.
- when GTP-beta-tubulin is present, this cap signals polymerization – MT assembly/growing. The cap provides for the lateral cohesion of protofilaments == smooth blunt + end for dimers to be added onto.
- when GDP-beta-tubulin is present, you have weak cohesion btwn the protofilaments, and so they flop out creating the Ram’s Horn structure– which has frayed + end, and thus shrinking MT.
Describe the function of Colchicine
Colchicine is a MT-disrupting drug that depolymerizes MTs. Only the centrosomes remain stable because they have different MTs.
The rest is depolymerized, and so you won’t see filament networks in the cells treated with this drug.
Describe the function of Taxol
Taxol is a MT-disrupting drug that stabilizes MTs, making them not dynamic anymore which is actually bad bc this impairs the proper function of the MTs during mitosis.
Used as an anti-cancer drug because it inhibits replication/mitosis.
What is the role of Microtubule Associated Proteins (MAPs)?
Effect of their action?
Two examples of MAPs
What can regulate MAPs?
- they alter the stability and interactions/distance btwn the MTs in a cell
- they have 2 domains: MT Binding Domain (binds the length of the MT to stabilize it) & Projection Domain (projects out 90deg from other domain and affects the interaction btwn MTs)
- MTs that are more spread out are able to transport larger loads
- MAP2 protein makes the distance larger btwn the MTs
- Tau protein bundles MTs closer to each other (it’s physically shorter)
- phosphorylation of these proteins (by Cyclin-dependent kinase CDK in cell cycle) inactivates them by promoting their disassembly
What are +TIPs
What do they do?
+TIPs are a type of Microtubule Associated Protein (MAP) but bind only to the + tips/ends of the MT
- they stabilize MTs by adding additional proteins to help with polymerization and possibly reduce catastrophe
What 2 proteins mediate the MT disassembly?
Kinesin-13: removes terminal tubulin dimers using ATP
Stathmin: induces GTP hydrolysis to get rid of cap; binds to curved part of Ram’s Horn. (inactivated by phosphorylation)
Describe the structure of Kinesin-1
2 Heavy chains (head, flexible neck (linker), stalk)
- ATPase activity
- binds to MT
2 Variable Light chains
- are specific to the cargo they bindDescribe the Movement of Kinesin-1 (Anterograde transport)
What happens when there’s no cargo?
Anterograde
- light chain binds to specific cargo, ATP hydrolyzes at head, linker changes conformation swinging the trailing head to become leading domain, each head moving 16nm
- if no cargo, Kinesin-1 is in folded conformation where the light and heavy domains are closely interacted
Know Kinesin-2, Kinesin-5, Kinesin-13
Kinesin-2: heterotrimeric (2 different heavy chains, 1 light chain)– for organelle transport
Kinesin-5: dipolar (4 heavy chains) – for MT sliding
Kinesin-13: just 2 short heavy chains — for MT end disassembly — not a motor protein
Explain posttranslational tubulin modification
acetylation of the lysine residue of the alpha tubulin of the MTs.
This stabilizes the MT + thus, promotes kinesin-1 movement along the MTs by cleaning up the surface of the MTs so that motor proteins can bind.
Describe Cytoplasmic Dynein (structure)
What process is it responsible for?
Cytoplasmic Dynein
- is a motor protein that is minus-end directed (+ —> -)
- just heavy chain with a head (ATPase + stalk(MT-binding domain)) and linker and stem/tail
- the linker and stem interact with Dynactin hetero complex so that it can recognize and bind cargo for Retrograde Transport of them - involved in retrograde transport
Explain the interaction of Dynein to Dynactin hetero complex
The linker + stem region of Dynein interact with Dynactin Hetero(many diff components) Complex via Dynamitin (a protein in Dynactin) so that it can recognize and bind cargo for Retrograde Transport of them.
The adaptor on Dynactin binds the cargo.
p150glued is a protein (not motor) that anchors the system onto the MT
What happens with abnormal levels of Dynamitin in the complex of Dynein-Dynactin?
irregular levels of Dynamitin causes the complex to explode apart – no more interaction.
Explain Retrograde Transport of cargo
minus-directed.
ATP binds to head of Dynein—> ATP hydrolysis —> linker domain changes conformation –> induces a stroke —> post-stroke interphase which moves head towards minus end.
How do Anterograde and Retrograde cooperate?
Often the cargo for these transports are the motor proteins, so Kinesin-1 and Dynein work to ensure that motor proteins are being passed to the right spot along MTs for transport
Compare Cilia and Flagella
Cilia and Flagella are made of same structure(axonemes),and are an extension of the plasma membrane, just different size + function.
Cilia: 2-10um, Flagella: 10-200um
Cilia: sweep material from surface of tissue,
Flagella: propel cells
What is the structure of Axonemes?
Axonemes (structure of cilia and flagella) are extensions of the plasma membrane, coming out of the basal body.
Its MTs are organized in a 9+2 array (9 doublets, and 2 singlets).
With Nexin binding the doublets in a circle.
The A tubule in the doublet has 2 Axonemal Dynein coming off it
Relate the Axoneme to the Basal Body.
Expand on the basal body
The flagella (The axoneme part) continues and attaches to the basal body in the cell via a transitional zone. The transition zone is made up only of doublets.
The basal body is a MTOC that is similar to centriole in that it is made up of triplets.
- but: it's different because the axoneme (MTs in doublet) extends all the way to basal body, while the MTs don't extend all the way to centrioles from centrosome.)Axonemal Bending
- axonemal dynein binds its tail permanently to tubule A, and binds its head to neighbouring tubule B to move it.
- protease removes Nexin, and the dynein is activated so sliding occurs
- if Nexin still there, dynein is activated and moves head but sliding is prevented, so bending occurs
- bending can be local –> wavy looking
Intraflagellar Transport
What motor proteins are used?
Purpose?
Transport of materials up (tip-directed - to +) and down (base-directed + to -) the flagella.
Uses Cytoplasmic Dynein and Kinesin-2 to move up and down the flagella using MTs as tracks.
For stability and signalling events
What end of the MT is the tip? Minus or plus?
Plus end is the tip. minus end is in centrosome.
Primary Cilium
Where is it found?
A single sensory organelle that emerges from the centrosome of Interphase cells (not mitotic). Arranged in 9+0 array.
Stabilized by the acetylation of the tubulin.
Negative embryonic consequences if mutated.
