Lecture 16

Question 1

What are the different ways for cells to move?

Answer 1

1) Actin-based movements (Lamelipodia, filopodia)
2) Cilia and Flagella

Question 2

What uses flagella and cilia for motility?

Answer 2

Spermatozoa/paramecia

Question 3

What is the similarity and difference between spermatozoa and paramecia?

Answer 3

They are different words
They look like different structures
One is longer
One is shorter
…but they are very similar molecularly
Their main difference is in the way that they beat.

Question 4

What kind of arrangement are microtubules in?

Answer 4

The microtubules are arranged in a 9+2 doublet arrangement

Question 5

How many protofilaments are in the A and B-tubule?

Answer 5

13 protofilaments in the A-tubule, 10 in the B-tubule

Question 6

How many proteins reinforce the 9+2 microtubules?

Answer 6

More than 200 proteins reinforce the 9+2 microtubules

Question 7

Where are the + and - ends of microtubules located?

Answer 7

+ ends of microtubules are at the tip of the flagellum, - ends are towards the head of the sperm

Question 8

What is flagellar dynein attached to?

Answer 8

Flagellar (or axonemal) dynein attached to only the A-tubule of the microtubule doublets

Question 9

How many repeats of dynein are there?

Answer 9

There are 7 diffeent dyneins in the axoneme repeated every 96 nm on the A-tubule

Question 10

What does altering of dynein do?

Answer 10

Altering even 1 of the dynein types can alter the beating

Question 11

What happens when ATP is added to dynein?

Answer 11

With the addition of ATP the dynein engages with the neighbouring microtubule doublet and slides

Question 12

What does the addition of ATP do to the flagellum?

Answer 12

This bends the flagellum

Question 13

What would happen if there were no doublet microtubules?

Answer 13

Without the doublet microtubules, the microtubules would just slide across each other without anything to anchor them

Question 14

What is also needed?

Answer 14

Also needed are links between the B-tubule and the membrane

Question 15

What is intraflagellar transport?

Answer 15

Material needs to be moved to the end of the flagellum/cilium for growth (it grows from the tip)

Question 16

What does Kinesin-2 do?

Answer 16

Kinesin-2 brings material (using “intraflagellar trains”) to the tip using the B- tubules

Question 17

What does IFT dynein do?

Answer 17

IFT dynein (similar to cytoplasmic dynein) brings materials

Question 18

What are intraflagellar trains?

Answer 18

Supramolecular protein arrays
They are the primary cargo being transported within flagella
But, they can also be used as adaptors for other proteins to hitch a ride

Question 19

Where do the trains start?

Answer 19

Trains start at the basal body and moves towards the tip

Question 20

What happens at the tip?

Answer 20

At the tip many cargoes are released, the trains are reorganized and are returned to the cell body

Question 21

Are train cargo interactions strong or weak?

Answer 21

Train cargo interactions are thought to be weak
(have never been isolated)

Question 22

How is flagella used in spermatozoa and bacteria?

Answer 22

Spermatozoa and bacteria both use flagella for propulsion
Although they look morphologically similar, they are molecularly very different

Question 23

What is the structure of a filament in bacterial flagellum?

Answer 23

Rigid helical filaments made from the protein flagellin

Question 24

What is the assembly of flagellum?

Answer 24

Flagellin monomers self-associate
Flagellum grows from the tip downwards
Flagellin is secreted through the basal body

Question 25

What is the mechanism of movement for flagellum?

Answer 25

Flagellum acts analogously to a boat propeller ATP is NOT required H+ gradient across the plasma membrane

Question 26

How fast can flagellum rotate?

Answer 26

300-1000 rotations/sec

Question 27

In order for a cell to move, what are 3 key steps to remember?

Answer 27

1) Extend a lamellipodium 2) Bind to the surface at the leading edge of the movement 3) Release from the training end of the cell

Question 28

What forms as the cell moves and trailing ones release?

Answer 28

New (artificial) junction called focal adhesion (AKA. focal contacts) form as the cell moves and trailing ones release

Question 29

What is coordination likely due to?

Answer 29

Coordination is likely due to tension in the cell forcing the release

Question 30

What are focal adhesions?

Answer 30

Formed by over 50 proteins Most are actin-associated proteins

Question 31

What are intermediate filaments at focal adhesions?

Answer 31

In non-epithelial cells (eg. fibroblasts; these are more like mesenchymal cells) focal adhesions can bind to vimentin and desmin-based intermediate filaments

Question 32

What can vimentin directly bind to?

Answer 32

Vimentin can directly bind to the cytoplasmic region of BI integrin But, vimentin intermediate filaments can also associate with focal adhesions through plectin

Question 33

What do Kertain 5 and 14 intermediate filaments bind to?

Answer 33

Keratin 5 and 14 intermediate filaments can bind to focal adhesions in epithelial cells through plectin

Question 34

What can focal adhesions act as?

Answer 34

Focal adhesions can act as nucleation sites for keratin intermediate filaments (hasn't been shown yet for the other intermediate filament proteins)

Question 35

What happens in plectin KO cells?

Answer 35

Intermediate filaments extend beyond the focal adhesion There are less focal adhesions (Vimentin KO cells also have fewer focal adhesions)

Question 36

How do intermediate filament associations with focal adhesions increase?

Answer 36

Intermediate filament associations with focal adhesions increase when endothelial cells undergo shear stresses

Question 37

Everything about focal adhesions is discussed in the context of?

Answer 37

2D cultures

Question 38

What are focal adhesions there for?

Answer 38

Focal adhesions are there to fight against Brownian motion. Without focal adhesions the cells would never be able to grasp the substratum.

Question 39

What about in 3D cultures (or when cells are confined)?

Answer 39

You don't have to worry about grasping the substratum because its all around the cell In 3D cultures (like what you would have in vivo) integrin-based adhesion for directed cell movement is not needed It's called "adhesion independent migration"

Question 40

What happens if you block or knock-out integrins?

Answer 40

There is no effect on cell motility in 3D!

Question 41

So what do they use to produce force in 3D?

Answer 41

Little is known, but potentially cadherins or other molecules. Or are cells "swimming" in 3D? Generating constant propelling forces (think of a corkscrew, flagellum or direction motion)

Question 42

So, are focal adhesions real?

Answer 42

They have never been seen in vivo