Lecture 16 Flashcards
What are the different ways for cells to move?
1) Actin-based movements (Lamelipodia, filopodia)
2) Cilia and Flagella
What uses flagella and cilia for motility?
Spermatozoa/paramecia
What is the similarity and difference between spermatozoa and paramecia?
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.
What kind of arrangement are microtubules in?
The microtubules are arranged in a 9+2 doublet arrangement
How many protofilaments are in the A and B-tubule?
13 protofilaments in the A-tubule, 10 in the B-tubule
How many proteins reinforce the 9+2 microtubules?
More than 200 proteins reinforce the 9+2 microtubules
Where are the + and - ends of microtubules located?
+ ends of microtubules are at the tip of the flagellum, - ends are towards the head of the sperm
What is flagellar dynein attached to?
Flagellar (or axonemal) dynein attached to only the A-tubule of the microtubule doublets
How many repeats of dynein are there?
There are 7 diffeent dyneins in the axoneme repeated every 96 nm on the A-tubule
What does altering of dynein do?
Altering even 1 of the dynein types can alter the beating
What happens when ATP is added to dynein?
With the addition of ATP the dynein engages with the neighbouring microtubule doublet and slides
What does the addition of ATP do to the flagellum?
This bends the flagellum
What would happen if there were no doublet microtubules?
Without the doublet microtubules, the microtubules would just slide across each other without anything to anchor them
What is also needed?
Also needed are links between the B-tubule and the membrane
What is intraflagellar transport?
Material needs to be moved to the end of the flagellum/cilium for growth (it grows from the tip)
What does Kinesin-2 do?
Kinesin-2 brings material (using “intraflagellar trains”) to the tip using the B- tubules
What does IFT dynein do?
IFT dynein (similar to cytoplasmic dynein) brings materials
What are intraflagellar trains?
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
Where do the trains start?
Trains start at the basal body and moves towards the tip
What happens at the tip?
At the tip many cargoes are released, the trains are reorganized and are returned to the cell body
Are train cargo interactions strong or weak?
Train cargo interactions are thought to be weak
(have never been isolated)
How is flagella used in spermatozoa and bacteria?
Spermatozoa and bacteria both use flagella for propulsion
Although they look morphologically similar, they are molecularly very different
What is the structure of a filament in bacterial flagellum?
Rigid helical filaments made from the protein flagellin
What is the assembly of flagellum?
Flagellin monomers self-associate
Flagellum grows from the tip downwards
Flagellin is secreted through the basal body
What is the mechanism of movement for flagellum?
Flagellum acts analogously to a boat propeller
ATP is NOT required
H+ gradient across the plasma membrane
How fast can flagellum rotate?
300-1000 rotations/sec
In order for a cell to move, what are 3 key steps to remember?
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
What forms as the cell moves and trailing ones release?
New (artificial) junction called focal adhesion (AKA. focal contacts) form as the cell moves and trailing ones release
What is coordination likely due to?
Coordination is likely due to tension in the cell forcing the release
What are focal adhesions?
Formed by over 50 proteins
Most are actin-associated proteins
What are intermediate filaments at focal adhesions?
In non-epithelial cells (eg. fibroblasts; these are more like mesenchymal cells) focal adhesions can bind to vimentin and desmin-based intermediate filaments
What can vimentin directly bind to?
Vimentin can directly bind to the cytoplasmic region of BI integrin
But, vimentin intermediate filaments can also associate with focal adhesions through plectin
What do Kertain 5 and 14 intermediate filaments bind to?
Keratin 5 and 14 intermediate filaments can bind to focal adhesions in epithelial cells through plectin
What can focal adhesions act as?
Focal adhesions can act as nucleation sites for keratin intermediate filaments (hasn’t been shown yet for the other intermediate filament proteins)
What happens in plectin KO cells?
Intermediate filaments extend beyond the focal adhesion
There are less focal adhesions
(Vimentin KO cells also have fewer focal adhesions)
How do intermediate filament associations with focal adhesions increase?
Intermediate filament associations with focal adhesions increase when endothelial cells undergo shear stresses
Everything about focal adhesions is discussed in the context of?
2D cultures
What are focal adhesions there for?
Focal adhesions are there to fight against Brownian motion. Without focal adhesions the cells would never be able to grasp the substratum.
What about in 3D cultures (or when cells are confined)?
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”
What happens if you block or knock-out integrins?
There is no effect on cell motility in 3D!
So what do they use to produce force in 3D?
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)
So, are focal adhesions real?
They have never been seen in vivo
