Cell adhesion and cell junctions Flashcards
What would we dissociate cells from their surroundings?
We may use collagenase or trypsin to dissociate cells if we want to use them in vitro, such as to study cell adhesion
What event catalyzed biological interest in cell adhesion?
Called cell sorting:
When scientists discovered that mixtures of different cell types separate on their own into groups of cells of the same type (later found to be due to differing types and levels of cell adhesion molecules).
What was Steinburg’s hypothesis on cell sorting? What experiment supported it?
Hypothesis:
- cells with different types or levels of a single cell adhesion molecule sort based on thermodynamics (minimizing free energy by engaging all of the available binding sites)
- helps tissues organize during development!
Experiment:
- Two cell types expressing different types of cadherins were mixed and observed to separate into two sections
- two cell types expressing different levels of the same cadherin were mixed and the same was observed
What is the difference between cell adhesion molecules and cell junctions?
- cell adhesion molecules are membrane bound molecules that bind to molecules outside the cell and contribute to mechanical adhesion.
- cell adhesion junctions are aggregates of a number of proteins, both transmembrane and intracellular that contribute to mechanical adhesion and other functions, like communication
How do cell junctions and cell adhesion molecules play a key role in evolution of multicellular organisms?
- junctions and adhesion molecules not only structural (mechanical), but help convey signals in the cell
- developmental roles in separation of different cell types
- changing cell shape
- cell migration
What is the role of cell junctions and adhesions in tissue engineering?
for prostheses, growing tissues in culture must be able to stick together
Types of adhesion in epithelia, connective tissue, and ECM?
- cell-cell adhesion: helps epithelial cells remain connected to each other, each of which bears mechanical stresses via cytoskeletal filaments anchored to adhesion sites
- cell matrix adhesion; helps cells stick to connective tissue (includes collagen fibers) which bears the mechanical stresses of tension and compression
What are the biomedical applications of cell adhesion and cell junctions?
- role in cancer: metastasis, extravasation
- invasion of tissues by pathogens (cytomegalovirus, helicobacter pylori)
- autoimmune skin disorders (attack of junctions - pemphigus vulgaris)
Benign vs malignant tumors.
- benign: tumor cells grow only locally and cannot spread by invasion or metastasis
- metastatic: cells invade neighboring tissues, enter blood vessels, and metastasize to different sites (cell junctions in epithelia are compromised and adhesive properties are changing)
How do tumor cells mimic the process of white blood cells?
During metastasis, they adhere to vacular epithelia of distant tissues to perform extravasation (leaving the blood vessels)
What allows malignant neoplasm to invade intravascular space?
Upregulation of ECM receptors
Describe the adhesion process involved in cell differentiation from epithelium.
- cells initially stick to epithelium
- when dividing, cells lose adhesions
- cells may change association from its progenitor cell after dividing
- e.g. neural development
How does adhesion properties vary between groups of membrane glycoproteins? (e.g. cadherins, integrins, selectins, Ig-CAMS).
- strength of adhesion asnd participation in junction (cadherins are strong, selectins are not)
- length of time of the adhesion
- speed of modulation of adhesion
- up- and down-stream signaling mechanisms that activate/deactivate adhesion
- signaling cascades that are activated by adhesion
- type and degree of cytoskeletal associations (some bind directly to cytoskeleton, some do not)
List the functional classifications of cell junctions.
- anchoring junctions (strong mechanical adhesions)
- occluding junctions (epithelial cell barriers)
- channel-forming junctions (only small molecules can pass)
- signal-relaying junctions
examples of anchoring junctions.
1. actin filament attachment sites
- cell-cell junctions (adherens junctions)
- cell-matrix junctions (actin-linked cell matrix adhesions)
2. intermediate filament attachment sites
- cell-cell junctions (desmosomes)
- cell-matrix junctions (hemidesmosomes)
examples of occluding junctions.
- tight junctions (vertebrates)
- septate junctions (invertebrates)
examples of channel-forming junctions.
- gap junctions (animals)
- plasmodesmata (plants)
examples of signal-relaying junctions.
- chemical synapses (nervous system)
- immunological synapses (immune system)
- transmembrane ligand- receptor cell- cell signaling contacts (delta-notch)
Which cell junctions do classical cadherins associate with?
adherens junctions, synapses
Which cell junctions do desmosomal cadherins associate with?
desmosomes
Which cell junctions do Ig family members associate with?
neuronal and immunological synapses
Which cell junctions do selectins associate with?
none
which cell junction do integrins associate with?
- immunological synapses (cell-cell)
- focal adhesions (cell-matrix)
- hemidesmosomes (cell-matrix)
Why is it important for extra-junctional adhesion molecules to contribute to cell adhesion, thus allowing cells to stick to one another without formation of junctions?
non-junctional adhesion mechanisms such as selectins and transmembrane proteoglycans act transiently so adhesions can quickly be formed and broken later on when needed.
Describe how to measure the strength of cell adhesion via flipping assay.
Cells are fixed to a plate. Different, fluorescent cells are added, then the plate is flipped over. Gravity lets non-adherent cells fall. And those that remain are imaged. The intensity of the fluorescence is proportional to the strength of the adhesion.
Describe how to measure the strength of cell adhesion via dual pipette assay
Two cells attached to one another are each attached to a micropipette. Aspiration is applied from each pipette, and the instensity of the aspiration needed to pull the cells apart is proportional to the strength of their adhesion to one another.
Describe how to measure the strength of cell adhesion via atomic force microscopy assay.
One cell is fixed to a fibronectin substrate, the second to the AFM cantilever. The cantilever measures the force exerted when the two cells separate.
Describe how to measure the strength of cell adhesion via magnetic bead assay.
Add adhesion molecules to the surface of a magnetic bead. Apply the bead to cells on a fixed surface and apply magnetic force. Measure the amount of force needed to roll the bead along the cells.
Describe how to measure the strength of cell adhesion via FRET assay.
Recombinant protein with two interacting adhesion molecules on either side. Between them are fluorescent regions, and an elastic linker between those. If they are close enough, the fluorescence emitted from the first bead upon excitation will excite the second bead and a wavelength will be measured. If they are not close enough, the fluorescence will not be apparant.
Name some of the ways that junctions can be visualized.
- fluorescence
- electron miroscropy (transmission, freeze fracture TEM)
When is freeze fracture TEM vs thin section TEM used for visualizing cell junctions?
thin section is used if you want high resolution, freeze fracture is used if you want an overview of the distribution of junctions on the cell surface.
Describe how tissues are prepared for freeze fracture TEM for characterizing cell junctions.
- tissue is fixated in glycerol and antifreeze to prevent crystals from forming and breaking open the cells
- tissue is transferred to very cold freon and liquid nitrogen
- cold knife shatters the tissue, separating the lipid bilayers
- ice can be sublimated away to reveal different surfaces
- electron dense metal coating (like platinum) is applied to the tissue. a carbon backing holds and preserves the tissue.
- the metal replica can then be observed by the EM
What are the two leaflets separated by freeze fracture?
- E (exoplasmic) face. inside of the monolayer that is closer to extracellular space (outside of cell)
- P (periplasmic) face. inside of the monolayer that is closer to protoplasm (inside of cell)
membrane proteins are visualized in both faces, but more bumps (proteins) on P face than E face – shows more proteins anchored on cytoplasmic (protoplasmic) side.
What are intramembranous particles?
Membrane proteins that appear in freeze fracture faces. We see these as darker, shadowy spots on the micrograph.
Where are tight junctions located?
in the apical region of junctional complex.
What do tight junctions and the junctional complex look like in a TEM?
tight junctions appear at the most apical region of the junctional complex
How does the junctional complex appear in light microscopy?
It is called the “terminal bar” because it appears as a line between cells.
What are occluding junctions?
They are the same as tight junctions, and don’t allow materials to pass between cells.
Describe the model of protein organization in tight junction sealing strands.
- occludin and claudin on both cell membranes “stitch” together with each other (claudin does the actual blocking)
- accessory ZO proteins bind claudins and occludins to the actin cytoskeletons.
- JAM proteins contain peptide sequences which allow for the passage of calcium ions in order to change membrane permeability.
How do occluding junctions help cells maintain polarization of membrane proteins? Why is this important?
It prevents movement of proteins from apical to basal membranes and vice versa. This is important for the unidirectional movement of certain molecules, such as glucose only into the gut epithelia and not out. For example, sodium-driven glucose symporters are only on the apical membrane of enterocytes.
How do occluding junctions appear in freeze fracture TEM and thin section TEM?
P face is present on ridges of transmembrane particles forming sealing strands, or focal connections. The E face appears smoother.
What is the general role of anchoring junctions?
They link the cytoskeleton to adjacemt cells or to the ECM to promote strong attachment. Interaction with the cytoskeleton is important so membrane proteins are not pulled right out of the membrane upon attachment.
What are the two categories of proteins involved in anchoring junctions?
- intracellular adaptor proteins: bind proteins that span the membrane to the cytoskeleton (either actin or intermediate filaments)
- transmembrane adhesion proteins: cross the membranes and bind between cells to one another, and within cells to intracellular adaptor proteins.
What type of proteins are cadherins?
Transmembrane adhesion proteins involved in anchoring junctions.
Describe cadherin binding to one another.
Cadherins bind one another homophilically at the N-terminal cadherin repeat. They also have a flexible hinge domain at their N-termini to which three calcium ions bind.
How does cadherin structure depend on calcium binding?
When calcium is high and binds to cadherin, it has a straight conformation and can more readily bind across the cell. When calcium is low, it is not bound to cadherin and the protein is no longer compatible with strong adhesion to another cadherin protien.
What is a cadherin zipper?
It is the structure that forms when a cadherin dimer links two cells together by binding to their actin cytoskeletons via intracellular adaptor/anchor proteins.
What proteins link classical cadherins to the actin cytoskeleton?
- p120-catenin
- beta-catenin
- other anchor/adaptor proteins
What is the role of aderens junctions in development? How do we know this?
During key times in development, adherens junctions contract, contributing to formation of cellular invaginations such as tubes and spheres in epithelial cells.
This works by actins and non-muscle myosins pulling on adhesion proteins on the apical side, giving curvature to the epithelial sheet
We can label N-cadherins and E-cadherins during neural tube development and see a differential expression of them in the neural tube being pinched off or in the epithelial sheet doing the pinching (ectoderm)
What is the role of adherens junctions in mechanotransduction?
When tension is applied on one cell from another, cadherin proteins are pulled, allowing catenin adaptor proteins to stretch out and reveal binding sites for other adaptor proteins, such as vinculin, which allows for more actin binding to adaptor proteins. Then, non-muscle myosin can contract with the myosin to elicit a signal inside the cell.
How are adherens junctions characterized in thin section TEM?
They are characterized by the length of the cadherin zipper which holds cells at a distance from one another. Can also see actin filaments on either side of the zipper, which are not as dark and thick as intermediate filaments (which you will see for desmosomes and hemidesmosomes)
How are adherens junctions characterized in freeze fracture TEM?
There are much less IMPs where the adherens junctions are compared to occluding junctions, for example, which have the sealing strands.
How are desmosomes and hemidesmosomes similar? Different?
They are both anchoring junctions that involve anchoring to intermediate filaments. They differ, because desmosomes anchor one cell to another using cadherins transmembrane proteins, and hemidesmosomes anchor the cell to the basal lamina using a different type of transmembrane protein.
Describe the desmosome model.
Again there is a cadherin zipper as is common for cell-cell anchoring junctions. Because these are anchored to intermediate filaments rather than actin filaments, there is a dense plaque of adaptor anchor proteins on the inside of either cell.
Describe desmosomes in thin section TEM.
Dark regions between two cells. Zipper between cells, intermediate filaments on either side of the dark plaque.
How are desmosomes characterized in freeze fracture TEM?
Spotted wells are evidence of desmosomes in freeze fracture TEM.
How do hemidesmosomes appear in EM?
- they appear as half desmosomes
- can see them near little dots of collagen bundles
Describe the model of hemidesmosome attachment to the extracellular matrix proteins.
- integrins span the membrane and binds adaptor proteins collagen XVII, plectin, and dystonin
- adaptor proteins bind to keratin intermediate filaments inside the cell
- on the outside, integrins bind to laminins and collagen of the ECM.
What is this image, and what are the different fluorophores?
This represents the focal adhesions of focal contact associated kinase (red) to to actin stress fibers (green).
Describe the modes of integrin activation (for hemidesmosomes and focal contacts).
- outside-in-activation: strong ligand activation to integrin on outside of cell
- inside-out-activation: talin binds to the inside portion of the integrin dimer and activates it.
Describe the activation of integrins via talin activation.
talin is activated either through GPCRs or RTKs. It then binds to the cytosolic side of the integrin dimer and acts as an adaptor protein to contribute to actin binding.
How do some intracellular adaptor proteins act as mechanotransducers in focal contacts?
Tension on integrins from the ECM can cause talin adaptor protein to be stretched out in the cytosol, revealing extra binding sites to which vinculin can bind. Vinculin binding allows for even more actin to be bound inside the cell.
Describe the model of selectin structure and actin attachment.
selectin is a transmembrane protein that binds to lectin carbohydrates on the outside of the cell, and to anchor proteins directly, and actin filaments indirectly
What are Ig-CAMs?
immunoglobulin family of adhesion molecules
- transmembrane adhesion proteins that take part in cell-cell adhesion.
- have immunoglobulin-like extracellular domains
- can bind each other homophilically or heterophilically
Describe the phases of extravasation.
- selectin adhesion of leukocyte to endothelial cell (slows down rolling)
- integrin adhesion of leukocyte to ICAM of endothelial cell (stops rolling)
