Lecture 15

Question 1

How are dyneins used?

Answer 1

Flagella and cilia use “axonal dyneins” )there are 15 genes encoding the heavy chains for axonemal dyneins in humans).

Question 2

What do dyneins power?

Answer 2

They power the binding of the flagella. 1 additional heavy chain gene to move material within the axoneme/flagellum (retrograde movement).

Question 3

Is dynein in the cytoplasm?

Answer 3

Yes, it is in the cytoplasm (called cytoplasmic dynein 1; yes, another gene in humans… for a total of 16 human dynein heavy chain genes)

Question 4

Do dyneins use ATP hydrolysis?

Answer 4

Use ATP hydrolysis to move only towards the - ends of the microtubules.

Question 5

What is another name for cytoplasmic dynein?

Answer 5

Cytoplasmic dynein is also called “dynein-1”. It is also found in the cytoplasm of eukaryotic cells (dynein-2 is in the cytoplasm of non-eukaryotic cells)

Question 6

How was dynein discovered?

Answer 6

Gibbons, by himself, took axonemes from Tetrahymena pyriformis and developed a procedure to fractionate their components.
Axonemal components were thought to be insoluble
But,
When Gibbons removed their membrane he could solubilize the axonemal structures.

Question 7

What happened to the microtubules?

Answer 7

The central pair of microtubules dissolved (as did the radial spokes), but importantly, the arms on the other microtubules were also dissociated.

Question 8

What did Gibbons conclude from the microtubules dissociating?

Answer 8

Gibbons ultimately found that when the arms were absent, then ATPase activity was present in the soluble fraction. That is axonemal dynein

Question 9

What did Richard Vallee find in his lab?

Answer 9

Richard Vallee found that dynein was needed for “retrograde transport”. The movement of material from the cell periphery towards the cell body. This towards the - ends of microtubules.

Question 10

What was the retrograde transport caused by?

Answer 10

This was caused by a microtubule-associated protein called MAP1C that they isolated from the brain. MAP1C was very similar to the known axonemal dynein and was then referred to as cytoplasmic dynein.

Question 11

How did they differentiate MAP1C from axonemal dynein?

Answer 11

1) They got purified MAP1C (cytoplasmic dynein) from cow brains (they did this for kinesin too as a control)
2) Added it to coverslips and then added axonemes from Chlamydomonas (that is an alga) to the top
3) Chlamydomonas flagellar axonemes have a uniform microtubule polarity and are frayed at their + ends, so you can tell the polarity of the observed ends
4) They removed the axonemal dynein from the axonemes prior to adding them to the MAP1C coated glass

Question 12

Where does AAA ATPase move towards?

Answer 12

Moves to the - end of microtubules

Question 13

What is the structure of Cytoplasmic Dynein?

Answer 13

AAA ATPase
6 ATPase domains in a large (500kD) Heavy Chain
Globular head is formed by the 6 ATPase domains
N-terminus associates with light and intermediate chains as well as cargo (there are 2 of them total)
Size is ~1.4 MegaDaltons

Question 14

What are the 6 ATPase domains?

Answer 14

AAA1: Main domain for ATP hydrolysis
AAA2: No ATP hydrolysis. Does not have the amino acids needed for ATP hydrolysis
AAA3 and AAA4: Hydrolyses ATP
AAA5 and AAA6: No ATP hydrolysis. Has a structural function. Transmits conformational changes to the AAA ring as a whole

Question 15

What is the function of the stalk in Cytoplasmic Dynein?

Answer 15

Comes out of AAA4
Microtubule binding domain:
It is a helical domain
Binds to the cleft between alpha and beta tubulin

Question 16

What are the 3 Light chains in Cytoplasmic Dynein?

Answer 16

Tctex-1: likely promotes self-association of the intermediate chains
LC7/ roadblock: actually pushes the intermediate chains apart, but when in complex with each other proteins it is part of a sequence of events to dimerize Dynein
LC8: …

Question 17

What is LC8?

Answer 17

Used to dimerize dynein
Also acts as a hub to bind to many proteins

Question 18

How does Cytoplasmic Dynein move?

Answer 18

It all has to do with conformational changes in the molecule
The linker arm (or stalk) bends and rotates the head domain
Pointing the microtubule binding site further towards the - end of the microtubule
Brownian motion (jiggling around) allows the microtubule binding site to find a good place to bind to… this can be 8nm-24nm away from the last binding site

Question 19

How is Dynein considered both non-processive and processive?

Answer 19

Dynein is not-processive in most cells on it’s own (it is processive in yeast)
By interacting with another complex (the dynactin complex) it becomes very processive (it can travel long distances without detaching), but it does not change its speed

Question 20

What is Dynactin?

Answer 20

Dynein Activator
11 protein (containing 23 subunits), 1-2 mega dalton protein complex

Question 21

How does Dynein bind to the dynactin complex?

Answer 21

Dynein binds to the dynactin complex through the p150(glued) subunit and dynein intermediate chain

Question 22

How many steps does it take under no load?

Answer 22

Under no load, it takes 24-32nm steps

Question 23

How many steps does it take under load?

Answer 23

Under load it takes 8nm steps (but stronger steps)

Question 24

What directions can dynein move?

Answer 24

Dynein can take backwards and sideways steps

Question 25

How can dynein binding site of p150 (of dynactin) itself enable dynein processivity?

Answer 25

The dynein binding site of p150 (of dynactin) itself can enable dynein processivity.
How?
It increases dynein’s step size, while inhibiting dynein’s lateral stepping and microtubule release

Question 26

What does Arp1 have to do with dynactin?

Answer 26

Arp1 binding to spectrin allows for dynein-dynactin binding to phospholipids to membranous cargo

Question 27

What does Ankyrin have to do with dynactin?

Answer 27

Ankyrin binds to dynactin

Question 28

What does p62 have to do with dynactin?

Answer 28

p62 binds to phosphatidylinositol 3-phosphate in cargo membranes

Question 29

Can dynactin recruit more than 1 dynein?

Answer 29

Yes, dynactin can recruit more than 1 dynein
This is thought to increase processivity
Can increase the “collective force production”
Favours unidirectional movement of the material being transported

Question 30

What end does dynein move cargo to?

Answer 30

To the - ends of microtubules, so it usually has to get to the microtubule + ends to dock with vesicles and start their journey. How does it do this?

Question 31

How does dynein use kinesins (eg. Kip2 in yeast) to move?

Answer 31

CLIP170 (a microtubule + end tracking protein) and the Dynein-binding protein Lis1 (AKA NudF, a nuclear distribution [Nud] family protein).
It doesn’t just go for the ride, it fights kinesin all the way, but the kinesin wins as its recruitment of CLIP170 and EB1 enhances Kip2’s processivity.

Question 32

How does dynein use CLIP170 and EB1 to move?

Answer 32

Use CLIP170 and EB1, but not Kip2, to directly recruit the p150 subunits of the dynatic complex to the + ends of microtubules
Dynein then binds to dynactin

Question 33

What is a problem of movement?

Answer 33

The cell is packed with roadblocks.
Microtubules and other structures intersect, not allowing enough space for the cargoes (vesicles) carried by motors to be able to pass
On average there will be 1-2 intersections per um and the distance from the cell nucleus to the periphery is tens of um.

Question 34

What happens if you “pause” the movement?

Answer 34

If you “pause” the movement you can get physiological disorders and accumulation of cargoes in locations they should not be and can block the movement of other things in the cell
Meaning that the motors are not strong enough to push through the pinch points

Question 35

So, what happens to the vesicles and how is this problem dealt with?

Answer 35

Endosomes can undergo fission
This makes them smaller
This can also further sort the material in the endosomes (eg. those materials destined for degradation vs. those for recycling)
Vesicles can switch microtubules to get around the intersections

Question 36

What is wrong with dynein heavy chain null mice?

Answer 36

They are embryonically lethal

Question 37

What can alterations to dynein itself or its regulators cause?

Answer 37

Neurodevelopmental diseases:
Peripheral neuropathy (Charcot-Marie-Tooth disease)
Spinal muscular atropy
Malformations of cortical development

Axonal transport defects also lead to:
Alzheimer’s disease
Parkinson’s disease
Huntington’s disease

Question 38

How does cytoplasmic dynein get to the cell nuclei?

Answer 38

Cytoplasmic dynein is also routinely hijacked by viruses, like Herpes simplex virus (HSV-1) and Human immunodeficiency virus type 1 (HIV-1), to transport them to the cell nuclei.