Weisel - Cell Adhesion

Question 1

Functions of cell junctions:

Answer 1

Tight junctions - selective permeability
Anchoring junctions - cytoskeleton-cytoskeleton connections
Gap junctions - cell cell communication
Selective cell-cell adhesion and cell sorting?
Cell adhesion receptor clustering?

Question 2

Anchoring junctions

Answer 2

Cell-cell: e.g. cadherins

Cell-matrix: e.g. integrins

Question 3

Occluding junctions

Answer 3

Between epithelial cells - e.g. claudins

Question 4

Channel forming junctions

Answer 4

Connexins or innexins

Question 5

Actin attachment sites

Answer 5

Adherens junctions - cellcell

Focal adhesions - cellmatrix

Question 6

Intermediate filament attachment sites

Answer 6

Desmosomes - cellcell

Hemidesmosomes - cellmatrix

Question 7

Occluding junctions

Answer 7

Tight junctions - vertebrates only

Septate junctions - invertebrates only

Question 8

Channel forming junctions

Answer 8

Gap junctions - animals

Plasmodesmata - plants

Question 9

Tight junction

Answer 9

Seals gap between epithelial cells, at the top

These form a selective permeability barrier across epithelial cell sheets, helsp cells withstand stresses

Question 10

Adherens junction

Answer 10

Next one down, connects actin filament bundle in one cell with that in the next cell

A CONTINUOUS ADHESION BELT PRESENT IN EPITHELIAL CELLS - CONNECTING BUNDLES OF ACTIN FILAMENTS FROM CELL TO CELLS.
Adhesion proteins may be cadherins.

Question 11

Desmosome

Answer 11

Connects intermediate filaments in one cell to those in the next cell

Question 12

Gap junction

Answer 12

Allows passage of small water soluble stuff

Question 13

Hemidesmosome

Answer 13

Anchors intermediate filaments to the ECM

Question 14

Actin linked cell matrix, anchoring junctions

Answer 14

Actin-linked cell matrix adhesion anchors actin filaments in cell to ecm

Question 15

Which junctions are stable and which are labile?

Answer 15

Tight/occluding junctions are stable
Some anchoring junctions as well
Others are labile

Question 16

How does selective cell-cell adhesion occur ? (binding types

Answer 16

1. homophilic binding
2. heterophilic binding
3. binding thorugh extracellular linker molecule

Question 17

How was the presence of tight junctions shown?

Answer 17

Electron dense tracer was added to the surface of the cell and it did not go beyond where the tight junction was. it could not cross.

You can also visualize using freeze fracture which allows you to see the channels

Question 18

What proteins form tight junctions?

Answer 18

Claudins and occludins - small proteins with 4 transmembrane helices and both N- and O-terminal ends in the cytoplasm and extracellular loops that interact in a homophilic manner.

Question 19

Purpose of anchoring junctions?

Answer 19

Form a strong membrane spanning structure.

Question 20

2 main components of anchoring junctions?

Answer 20

1. Intracellular anchor proteins - form a plaque on cytoplasmic face of the membrane and connect the adhesion membrane protein to the cytoskeleton
2. Transmembrane adhesion proteins - cytoplasmic domain that binds to anchor protein and extracellular domain that binds to another cells adhesion protein or to ECM.

Question 21

Cell adhesion molecules

Answer 21

Cadherins
Ig-superfamily CAMs
Mucin like CAMs
Integrins

Question 22

Cadherins

Answer 22

main adhesion molecules holding cells together in early embryonic tissues, expressed in virtually all cells.

Calcium dependent

Form homodimers in the cell membrane - extracellular domain of one cadherin dimer binds the identical dimer on the adjacent cell. Ca2+ makes them rigid.

5 repeats (extracellularly) separated by Ca2+ binding sites 
Crystal structure of a single repeat resembles an Ig Domain.

Cadherins can be coupled to actin via alpha and beta catenins.

Question 23

Integrins

Answer 23

Cell adhesion proteins that link to the cytoskeleton or cell-cell. Importnat for cell-cell junction. There is an alpha and beta subunit but lots of combinations (and for instance beta 3 integrin can be joined to a different alpha subunit..) lots of combinations!
Platelets can be used to study integrins…
Integrin aIIbBeta3 is missing and does not bind to fibrinogen in glanzmann’s thrombasthenia.
Activation of integrin leads to conformtional change that binds ligand.

Question 24

How can you study integrin?

Answer 24

Make nanodisks, each one with a single integrin in the membrane. Look at different conformations of the integrin. Mostly you see bent and resting conformation but in presence of tailin you see unfolded open form. Resting conformation - bent form. Active conformation - open form.

Question 25

Assay for cell-cell adhesion

Answer 25

Take isolated cells and put them on a plate and see if they aggregate. Block with appropriate antibody first. Melanoma cells have lost E-cadherin and gained N-cadherin.

Question 26

Optical trapping experiment? / optical tweezers

Answer 26

If you shine a beam of light you can actually use force from that light to trap things. This comes in useful if you have a ligand on one surface and receptor molecule on the other surface. You can move the beam and moved the trapped bead and touch it to the pedestal and then measure the bead's position in the trap. If there is a ligand receptor bond, it will take force to break the bond and you can measure the force needed to break the bond.

Question 27

P-selectin

Answer 27

Present on platelets and endothelial cells that have been activated by inflammatory response. This mediates leukocyte adhesion and rolling...

Question 28

Ig Superfamily - CAMS

Answer 28

Mediates calcium independent cell cell adhesion. Weaker than those mediated by cadherins. CAMs used during development and regeneration. There are different forms of N-CAM (neural cell adhesion molecule) present in many cells. they are made of ig domains and fibronectin type iii domains and they interact via homophilic binding.

Question 29

Gap junctions allow for

Answer 29

Electrical coupling Metabolite sharing Embryogenesis - cooperative assembly of cells following same pathway. In early embryo, cells are coupled to each other electrically. Granulosa cells also communicate each other this way.

Question 30

How could we visualize gap junctions

Answer 30

Freeze fracture is one way

Question 31

Can gap junction permeability be regulated?

Answer 31

Yes - in general decreaes of cytosolic ph or increases of ca2+ decrease gap junction permeability which is likely a damage control response.

Question 32

Vinculin

Answer 32

Anchor protein, located at focal adhesions, where actin bundles terminate at the membrane.

Question 33

How do we study clustering of adhesion receptors in the membrane?

Answer 33

We can do it with FRET - 2 fluorescence labels, one excites the other. You can use this to determine if 2 molecules in a cell are close to each other. There has to be overlap between emission wavelength of protein 1 and excitation of fluorophore 2. Or use BRET - which is a fret variation that uses bioluminescent luciferase.

Question 34

Adhesion mediated signaling

Answer 34

- REceptor occupancy - binding cause signal transduction across cell membrane - Receptor clustering - junctional complexes - REceptor tensioning - binding to an immobilized ligand allows myosin contraction to tension linkages from ligand to actin.