Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

1. BIOC 1301 S2 > Microtubule Motors > Flashcards

Microtubule Motors Flashcards

1
Q

What is another motor with a track like actin/myosin?

A

Kinesins and dynein walk on microtubules
Both are ‘linear’ motor proteins
They use ATP to drive movement
The microtubules have a ‘polarity’ allowing the motors to track this and move in the appropriate direction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are some key components of the cytoskeleton?

A

Microtubules are tracks for kinesin and dynein
Centrosome with a pair of centrioles
Actin filaments
Intermediate filaments - in between the size of actin and microfilaments, they have a structural role

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is the centrosome?

A

The microtubules are nucleated at the centrosome in cells
There are 2 centrioles within the centrosome - which will move to the poles during mitosis
It comprises of barrel shaped structures surrounded by ‘fuzzy material’
The fuzzy material = pericentriolar matrix, containing various proteins that helps anchor the microtubules
Gamma tubulin rings (proteins within the pericentriolar matrix) - this is involved in the start of microtubule polymerisation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are microtubules?

A

They are dynamic tubes growing from the centromere and then they collapse back
They are like highways along which motors carry membranes
They have moving vesicles along them, in both directions
Negative end is towards the centre and the positive end is towards the periphery of the cell (showing directionality)
○ Positive end = fast growing
○ Negative end = slow growing
They are the biggest type of filament in a cell - seen by immunostaining during interphase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the composition of microtubules?

A

They are made from tubulin
There are two types of tubulin: alpha and beta
There are around 6-7 genes for each alpha and beta tubulin (in mammals) = diverse types of tubulin
They are formed from the polymerisation of tubulin proteins - therefore the ‘building blocks’ are an alpha-beta tubulin complex (heterodimer)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is a protofilament?

A

Protofilament - a ‘stack’ of heterodimers forming a vertical line
Each protofilament is slightly offset creating a slightly helical structure
There are 13 protofilaments in a microtubule (diameter of 25 nm)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are the proposed properties that allow for microtubule purification?

A
  1. They were abundant in brain
  2. Microtubules don’t like to get cold - they depolymerise in the cold and repolymerise in the warm
  3. Glycerol stabilises them
  4. Calcium ions depolymerise them - Ca2+ can also activate proteases that destroy proteins, therefore EDTA or EGTA is added to chelate and bind the Ca2+ ions
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How are microtubules purified?

A
  1. Take a pig’s brain
  2. Homogenise in in EGTA + Mg2+ + GTP + buffer at 4 degrees
  3. Centrifuge at high speed
  4. Collect supernatant, add glycerol and warm to 37°C - solution goes cloudy, as MTs repolymerise and you can measure absorbance in a spectrophotometer
  5. Recentrifuged to sediment microtubules
  6. Repeat depolymerisation/repolymerisation = purer results
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What were the results of the purification of the microtubules?

A

SDS-PAGE was then used to visualise the results
There are 2 major protein bands at 55kDa and 50kDa
There are additional proteins stuck onto the sides of the microtubule - The tau proteins which stabilise the MTs
MAPs - microtubule associate proteins
Motor proteins - responsible for moving membranes along the MTs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the two types of motors moving along the microtubule tracks?

A

Dynein - moves to the minus end, bringing things in

Kinesin - moves to the positive end, moving things out

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Describe Kinesin?

A 
A dimer formed by two heavy chains
Motor domain - binds actin and nucleotide (hydrolyses Mg.ATP to generate movement)
Tail - Binds cargo
It has no lever
Mr - around 100 kDa
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the function of kinesin?

A

Most kinesins walk to the ‘plus’ (fast growing) ends of microtubules at the cell periphery and they are involved in:
• Cargo transport - can travel a long way (e.g. Along a 1 m neurone)
• Mitosis - help stabalise chromosomes along the equator
• Cilia and Flagella (kinesin-2) - 9+2 organisation of their microtubules

They have a high duty ratio - stays attached to microtubules for the majority of the ATPase cycle

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How was kinesin discovered/purified?

A

They were observed in squid axons (due to large diameter of the axon)

  1. Prepare purified microtubules (MT) and stabilise with Taxol
  2. Prepare a high speed supernatant from squid axons
  3. Incubate supernatant with MT in the presence or absence of AMP.PNP and pellet
  4. Then release bound protein with 1mM ATP
  5. Mix the pellets with MT in presence or absence of AMP.PNP – no 100 kDa band

AMP.PNP - inhibits movement

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How can we determine kinesin’s movement?

A

Using an in vitro assay purified kinesin is bound to the glass slide in a flow cell (This uses grease to stick the cover slip)
Microtubules are added (fluorescently labelled) and they stick to the kinesin
A solution containing ATP is added and this allows kinesin to go through its ATPase cycle and move the microtubules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What conclusions are drawn about kinesin’s movement?

A

Kinesin-1 walks ‘straight’ along a single protofilament and takes 8 nm steps

To show the walking: an ‘optical trap’ assay is used
Small forces (pN in size) generated by a beam of light can hold onto a bead to which kinesin is attached
As kinesin steps along the microtubule, the bead is pulled along with it, so by detecting the position of the bead (about 1 micron in size) the position of kinesin can be determined
Kinesin moves 600nm before detaching (75 steps) in in vitro assays

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is the kinesin-1 ATPase cycle?

A
  1. Kinesin and ADP binds
    The microtubules accelerate ADP release x1000 fold
  2. ADP releases and the neck liner undocks
    There is a strong Apo binding state
  3. ATP binds to the kinesin and the neck linker docks
    During ATP state there is a strong microtubule binding
    Microtubules accelerate ATP hydrolysis x10 fold
  4. ATP hydrolysis results in an ADP.Pi state
    Here there is strong microtubule binding
  5. Pi is released - resulting in weak binding
  6. Kinesin detaches with the ADP
    ADP release is the rate limiting step
17
Q

What is significant about the two kinesin heads?

A

They are asynchronous (out of sync)
1. Rear head (ATP.Pi): neck linker docked, Front head (ADP) and neck linker undocked
2. ADP is lost by front head and Pi is lost by rear head
’Gating’ behaviour between the two heads for co-ordination
3. Rear head (ADP) detaches, neck linker will undock
4. Front head (was rear head) binds ATP, the Neck linker docks and helps to move rear (ADP) head forward

Powerstroke - this is the docking/undocking of the neck linker
It is the neck linker that is driven by the ATPase state of the motor

18
Q

What is the variety within kinesin?

A

Kinesin has 45 different genes
They can be monomers, homo/heterodimers or tetramers

All these kinesins have a motor domain on the N-terminal, but others have a central motor domain or even a C-terminal motor domain
C-terminus motor domains - they go the opposite direction along the microtubule
Middle/central motor domain - depolymerise the track of microtubule

19
Q

Describe Dynein motors?

A

Minus-end directed microtubule motor
ATPase (4 nucleotide pockets per heavy chain)
Work coupled to release of products of ATP hydrolysis
A member of the AAA+ superfamily of mechano-enzymes

Composition:
Heavy chain(s) (>500 kDa each)
Accessory chains (I, LI, L- mainly cargo binding)
Heavy chain contains the motor domain (ATPase and microtubule-binding domains)
20
Q

What is the AAA+ superfamily of mechano-enzymes?

A

AAA+ proteins assemble into ring shaped oligomers: typically homo-hexamers
AAA+ mechanoenzymes perform diverse functions: unfolding/destabilising

Dynein’s contain 6 AAA+ domains in a single polypeptide chain
They convert energy from ATP hydrolysis into force/stepping along microtubules
ATPase kinetic scheme is similar to that of the actin-based motor myosin

21
Q

What is significant of the iosforms of dynein?

A

The family tree = all the isoforms that can be in one cell
Only two isoforms of cytoplasmic dynein heavy chains
Only one of these (cytoplasmic dynein 1) is the main trafficker
Multiple functions achieved through light chain/intermediate variation and additional regulatory proteins

22
Q

What is dynein’s structure?

A

This is the 9+2 organisation
Each of the 9 outer doublets has rows of dynein arms that reach out to the next doublet and cause it to slide
Driven by ATP hydrolysis
Both stalk and tail flexible
This means distance varies between cargo and MT-binding site
Important to allow Powerstroke of motor without simultaneous movement of cargo

23
Q

What are the functions of dynein?

A

Cytoplasmic dynein 1:
Positioning/transport and functioning of: proteins, mRNA, receptors, organelles, vesicles etc…
Mitosis: Nuclear envelope breakdown, spindle organisation, chromosome movement and checkpoint release

Cytoplasmic dynein 2
Assembly of motile and immotile cilia

Axonemal dynein’s
Multiple (>14) different isoforms
Bending motions of cilia and flagella

24
Q

How does the powerstroke of dynein take place?

A

Tail connects to AAA1 ATPase domain in head via ‘linker’ domain
Linker changes its orientation relative to head and stalk
Linker movement could translate microtubule

We can see the linker’s change in conformation
When you hydrolyse ATP, The linker changes from straight to bent, causing it to come away form AAA4, this part of the molecule pulls on the stalk allowing the microtubule domain to bind = generation of the power stroke

25
Q

Describe the ATPase cycle for dynein?

A
  1. ATP hydrolysis and phosphate release, the linker is straight
  2. ADP is released, the linker docks and then ATP can bind
  3. ATP binding, causes the linker to bend
26
Q

What else does dynein require to work?

A

Dyactin activates the dynein
Dynactin is 1 mega Da
It binds to the tail of dynein
It makes sure the motor domains stick out correctly, in order to bind to the microtubule

27
Q

What do both kinesin and dynein have in common?

A

They have a common ancestral protein (G-protein) for both types of motors
They alternate between weak-strong binding and biased stepping in one direction - either a lever or docking mechanism

28
Q

What is tracking of kinesin and dynein important in?

A

Neurones:
Cell bodies of neurones are in the spinal cord and can have a long way to travel before reaching synapses else where
They can be over 1 metre long

1. BIOC 1301 S2 (23 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions