Cell Junctions, Cell Adhesion & Extracellular Matrix

Question 1

List the 4 types of cellular junctions

Answer 1

1) Tight or Occluding Junctions (Zonula Occludens)
2) Anchoring Junctions
3) Gap Junctions
4) Signal-Relaying Junctions (Synapse)

Question 2

Tight or Occluding Junctions (Zonula Occuldens)

Answer 2

They involve claudin proteins. They are involved in sealing gaps between epithelial cells. They have 3main functions:

1) seal cells together to create a permeability barrier:
2) regulate paracellular transport: they can be regulated to promote leakage between cells or paracellular transport
3) Apical vs. Basolateral protein/lipid sorting: The fence function of tight junctions separates apical from basolateral plasma membrane domains

Question 3

Plaque or Anchoring Junctions

Answer 3

Link cell to cell (typically via transmembrane cadherin proteins) or cell to matrix (typically via transmembrane integrin proteins). They mechanically attach cells and their cytoskeleton to their neighbors or the extracellular matrix to stabilize against mechanical stress.

-2 Main Categories (cell-to-cell or cell-to-matrix)

• 4 Junction types:
  1) Adherens Junction: Use classical cadherins as their transmembrane adhesion protein and are cell-to-cell. They utilize actin filaments as their intracellular cytoskeletal attachment.
  2) Desmosomes: These are cell-to-cell and they use a non-classical cadherin as their transmembrane protein. They also use intermediate filaments as their cytoskeletal attachment.
  3) Focal Contacts/Focal Adhesions: Actin-linked cell-matrix junction: These use Integrin as the transmembrane adhesion protein and actin as the cytoskeletal filament.
  4) Hemidesmosomes: They use integrin as well and involve intermediate filaments. They are also cell-matrix junctions.

Question 4

Gap Junctions

Answer 4

They allow small molecules and electrical signals to pass between interacting cells.
-Connexins are the proteins that constitute gap junctions and they are 4 pass transmembrane proteins. 6 connections form a functional pore, called a connexon. Also, the gap junction permeability varies with connexin composition (21 genes in humans)

Gap Junction Functions:

1) Electrically connect cells because ions can flow through: Heart muscle cells for example
2) Metabolically couples cells by average small molecules throughout a tissue: Liver.
3) More specialized cells uncouple from cells with different cell fates: Embryogenesis

Question 5

Signal-Relaying Junctions: Synapses

Answer 5

Scaffold proteins organize adhesive proteins, ion channels, receptors, etc. Know that these scaffolding proteins are crucial for the organization of these junctions.

• These tend to be very complicated
• Have some features of adhesion junctions because they have actin and some adhesion junctions between them but they are not very robust like a desmosome
• They are a little weaker adhesion but there needs to be a little bit of adhesion for the two membranes to be located near each other
• There is a scaffolding protein that will anchor signaling molecules like ion channels and signal receptors.
• Signal relaying junctions can be very different

Question 6

Explain the function of tight junctions in blood vessels and in the epithelial cells which line the intestinal lumen

Answer 6

The tight junctions in the epithelial cells perform a multitude of functions, most notably their prevention of the leakage of gastric enzymes into the body would could have detrimental effects. Also, they help establish an apical and basal side of the cell such that certain receptors in the cell are on one side and others on another. They also put specific channels on one side or the other for nutrient diffusion out of the intestines so we get nutrient uptake. The tight junctions in the blood vessels prevent blood loss and keep the blood in circulation as well as all for the intake of nutrients from the intestines.

Question 7

Explain the importance of transcellular transport of nutrients across sheets of epithelial cells that are linked by tight junctions

Answer 7

• Transcellular Transport: Active transport of glucose from gut lumen into cells via Na-driven glucose symporter (active transport). Glucose has to go through the cells because of the tight junctions. Has to go through the cell!
• However, if the junctions are disrupted, we get paracellular transport –> Can now go around the cells

It keeps the permeability barriers preserved!

The transcellular transport is crucial to ensure that nutrients can still move across the cell barrier since paracellular transport is not permissible with tight junctions.

Question 8

Explain the process of paracellular transport

Answer 8

Paracellular transport is simply the ability of molecules to move in between cells since they are not as tightly linked to one another. There is a gap between the cells for molecules to pass through. It is the “leakage” between cells.

Question 9

List the 4 different types of anchoring junctions

Answer 9

1) Adheren Junctions
2) Desmosomes
3) Focal Contacts/Focal Adhesions: Actin-linked cell-matrix junction
4) Hemidesmosomes

Question 10

For each of the 4 types of anchoring junctions, identify the class of transmembrane adhesive proteins and the cytoskeletal proteins to which each adhesive proteins attach

Answer 10

1) Adheren Junctions:
- Class of transmembrane adhesive protein: Classical Cadherins
- Cytoskeletal proteins to which each adhesive proteins attach: Actin filaments
- The adapter proteins are catenin

2) Desmosomes:
- Class of transmembrane adhesive protein: Non-classical Cadherins
- Cytoskeletal proteins to which each adhesive proteins attach: Intermediate Filaments
- The adapter proteins are a dense plaque of adapter proteins.

3) Focal Contacts/Focal Adhesions: Actin-linked cell-matrix junction:
- Class of transmembrane adhesive protein: Integrin
- Cytoskeletal proteins to which each adhesive proteins attach: Actin Filaments

4) Hemidesmosomes:
- Class of transmembrane adhesive protein: alpha6-beta4 Integrin, type XVII Collagen
- Cytoskeletal proteins to which each adhesive proteins attach: Intermediate Filaments

Question 11

Illustrate the importance of desmosomes using a clinical example

Answer 11

Pemphigus is an autoimmune disease where auto-antibodies against desmosomal cadherins cause the loss of cell-cell adhesion and get severe blistering

Question 12

Explain the role of selectins with the interactions of white blood cells and endothelial cells

Answer 12

Selectins are Lectins: Carbohydrate binding proteins
Transient, calcium-dependent adhesion
Heterophilic. Cooperate with integrins
E Selectin: Activated endothelial cells

Selecting are critical for allowing the carbohydrate markers of white blood cells to bind to the selectins via the lectin domain. The selectins are crucial for allowing the WBCs to “roll” in the vein. Then, when integrin begins to have a larger role, the WBC can bind tightly and go through the epithelial cells.

• Weak adhesion and rolling is selection-dependent
• Strong adhesion and emigration is integrin-dependent

Question 13

Cadherins

Answer 13

Ca2+-dependent transmembrane adhesion proteins. Involved in Adheren Junctions and Desmosomes for cell-cell junctions.

• Cadherins are extremely important adhesion molecules
• They are calcium dependent
• They have a variety of cadherin repeats in them and the presence of calcium allows the cadherins to become very stiff and then reach out to encounter one another at the amino terminus.
• The two amino terminuses interact.
• To detach cells, you have to wash them with calcium to stop them from binding to each other
• Need calcium in order to function
• It is homophilic binding: Different types of cadherins so it will only bind to another cell with the SAME cadherin on it because it is homophilic.
• These cadherins being homotypic allowed for a self sorting adhesion so that cells with a similar fate will adhere to one another to get appropriate development
• Cadherin forms adhesion junctions that link adjacent cells
• When cells are not touching each other, the cadherin is not very stable and the turnover number is much greater than when they are touching and interacting with cadherin on other cells
• Homophilic binding is important during embryogenesis

Question 14

Integrins

Answer 14

Transmembrane ECM binding proteins. They are a dimer and they bind indirectly to the actin filaments via adapter proteins.

Question 15

Identify the transmembrane protein that forms gap junctions

Answer 15

Connexins

• 4 pass transmembrane proteins
• 6 connexins form a functional pore: Connexon
• Permeability varies with connexin composition

Connexins are the transmembrane proteins which form the connexon or the gap junction (pore). 6 connexins make up a connexon and they are 4 pass transmembrane proteins.

Question 16

Identify specific types of molecules that are able to pass through gap junctions and those that are unable to pass through gap junctions

Answer 16

In terms of molecular weight, those with a molecular weight below 1000 tend to be able to pass through gap junctions. More specifically, Ions, Sugars (mono/disaccharides) and Nucleotides can pass through the gap junctions. What can pass through a specific gap junction or pore depends on the composition of the connexins that make up the connexons.

Question 17

Identify the cells that secrete most of the matrix molecules of connective tissue

Answer 17

1) fibroblasts
2) chondroblasts
3) osteoblasts
4) epithelium

Question 18

The Extracellular Matrix

Answer 18

A substantial volume of tissues consists of extracellular space, which is filled by an intricate network of proteins and carbohydrate-containing molecules of the extracellular matrix. The components of the extracellular matrix include proteoglycans and glycosaminoglycans (GAGs), structural proteins, and adhesive proteins.

• The ECM is a Hydrated network of protein and carbohydrate containing molecules and it is very abundant in connective tissues.
• Its components are secreted by fibroblasts, chondroblasts, osteoblasts, epithelium
• The ECM bears most of mechanical stress

-It is a hydrated network of protein and carbohydrate containing molecules
The ECM is a whole group of different types of molecules like proteins and sugars and makes up the most of the connective tissue
Most of the components of the ECM will absorb the force that was put on the epithelium above them
There are some specialized cells
It is a contribution of multiple cell types that make it up.

Question 19

Give examples of the major types of components that comprise the extracellular matrix

Answer 19

1) Glycosaminoglycans
2) Hyaluronan (Hyaluronic Acid, Hyaluronate)
3) Proteoglycans
4) Collagen
5) Elastin
6) Fibronectin

Question 20

Identify the characteristics of Glycosaminoglycans (GAGs)

Answer 20

• Unbranched polysaccharide chains composed of repeating disaccharide units (N-acetylglucoamine and glucuronic acid)
• One amino sugar is usually sulfated and the other is a uronic acid
• Absorb large amounts of water: Occupy space
• The high negative charge from the sulfates attracts Na+ ions and water.
• Uronic acid is where the OH is a COO- to get the acid group which is seen on C6

Question 21

Hyaluronan (Hyaluronic Acid, Hyaluronate)

Answer 21

• This is the simplest GAG and none of the sugars are sulfanated in this which is unusual. It is very long, containing up to 25,000 disulfide units.
• Synthesized from the basal side of an epithelial sheet
• Creates a cell-free space into which cells can migrate: Embryogenesis
• Diffusion of nutrients, metabolites, etc.
• Resists compressive forces
• Degraded by Hyaluronidase – Wound Healing
• Think of it like a sponge to create a gradient

Question 22

Proteoglycans

Answer 22

• These are glycans so they are a sugar based molecule with a protein associated with it
• Glycosaminoglycans covalently attached to a core protein
• These contain at least 1 GAG, so any protein with at least one GAG is referred to as a proteoglycan
• Very high carbohydrate content
• Long, unbranched side chains
• Regulate movement of molecules and cells
• Chemical signaling between cells
• Proteoglycans have a core protein and then GAGs attached to that core protein.

-Glycoproteins have few, short, branched carbohydrate chains and a low carbohydrate content, this is a protein with a sugar group attached to it. They tend to me much smaller than proteoglycans. They are proteins at their root, meaning they are proteins with associated sugars.

Question 23

Explain the relationship of Glycosaminoglycans (GAGs) to proteoglycans

Answer 23

GAGs are completely composed of sugars. They are not associated with a protein when they are referred to as GAGs. However, GAGs can associated with proteins at which point they are referred to as proteoglycans.

Question 24

Collagen

Answer 24

Collagens are the most abundant proteins of the ECM and are primarily responsible for the strength of the matrix. Three alpha chains arising from combinations of over 40 collagen genes assemble to give rise to distinct structures and functions. Individual collagen polypeptide chains are synthesized as larger precursor molecules, which undergo several modifications before secretion and assembly into the triple helix.

• The most abundant proteins of the extracellular matrix
• 25% of protein mass in mammal
• Responsible for the strength of the ECM

Question 25

Collagen Structure

Answer 25

• There is approximately 43 collagen genes in the human genome and three of these come together to form the collagen molecule. –> It is a triple helix!!!
• It is a Gly-X-Y where X is usually proline and Y hydroxyproline and proline introduces a kink so get a very tightly wound helix. Get a turn-turn-glycine and glycine is on the inside of the helix because it is small enough
• Get a tightly wound triple helix and the structure is reinforced by the many different bonds –> Intrastrand H-bonding via OH-Pro and OH-Lys
• There can also be modifications to the lysines to get lycine aldehydes to get covalent intra-molecule and inter-molecule cross-linking. Two different collagen molecules can be cross-linked.
• Hydroxylation of proline is done via Vitamin C and if you don’t have this then collagen is not made as strongly and this results in scurvy –> Hydroxyproline helps provide more strength to the collagen!!!
• Triple-stranded, a-chains
• ~1000 amino acids long
• Gly-X-Y repeat
  
  • Glycine – smallest amino acid
  • X: Proline
  • Y: Hydroxyproline
    - Vitamin C (Scurvy)
• OH-Pro, OH-Lys
  - H-bonding
• Lysine aldehydes
  
  • covalent intra-molecule and inter-fibril cross-linking so get more strength in the collagen.

Question 26

Identify three principle types of collagen

Answer 26

Types I, II, and III compose 80-90% of the collagen in the body. They are fibril in their polymerized form

-Fibril associated collagens bind to the fibrous collagen and help organize their shape and structure thus a mutation in one of these can give severe structural defects.

Question 27

Synthesis of Collagen Fibrils and Fibers

Answer 27

• It is synthesized by being assembled in the cell as procollagen
• Procollagen has these propeptides on the C and N termini which prevent collagen from forming a full collagen molecule and from interacting with other collagen
• Once it is secreted from the cell these propeptides are cleaved and this now allows the collagen molecules to assemble into a collagen fibril
• This gives the striated pattern
• The rod shaped molecules interact with each other to get a strong cable like structure.
• Collagen fibrin is 10-300nm where the collagen fiber (many fibrils associating with each other) is 0.5-3um

Question 28

Osteogenesis Imperfecta

Answer 28

It is a defect in Type I collagen –> get fragility

Question 29

Ehlers-Danlos Syndrome

Answer 29

Mutations in collagen Types I, III,V Collagen maturation/secretion –> Get a lot of elasticity

Question 30

Identify the characteristics of elastin fibers that give them their elasticity

Answer 30

Elastin imparts elasticity and flexibility to tissue.

• Elastin: Imparts elasticity to tissues
• It is important in vessels and arteries that need to resist the expansive form of blood being pushed in.
• It is highly cross-linked and kinked.
• It can go through this stretch and release cycle!
• Fibrillin is very important for its elasticity too! It is important for the integrity of the elastic fibers.
• People with Fibrillin mutations tend to be very high in stature and such

Question 31

Explain the consequences of a mutation in the fibril gene, i.e. Marfan’s Syndrome

Answer 31

Fibrillin is essential for the integrity of the elastic fibers. People with these mutations tend to have aorta rupture due to the lack of flexibility in the aorta as well as skeletal abnormalities.

Question 32

Fibronectin

Answer 32

• Adhesive ECM Glycoprotein that binds cells to the matrix
• Binds cells to the ECM and guides cellular migration
• Arg-Gly-Asp (RGD) binds integrins!!!
• Directs cell migration during development
• Fibronectin is a glycoprotein.
• It is an adhesive EMC glycoprotein and thus allows cells to bind to the matrix
• It is a dimer structurally with a bunch of fibronectin repeats
• Arg-Gly-Asp (RGD) binds to the integrins
• Many times the matrix proteins that integrins bind to are fibronectin at this three amino acid sequence. It is required for this strong binding
• RGD sequence is found in many other adhesive matrix proteins and thus can be bound by different integrins

Question 33

Identify the tripeptide sequence in the fibronectin repeat that serves as a binding site to integrins

Answer 33

RGD sequence –> Arg-Gly-Asp binds to integrins

Question 34

Identify the major glycoprotein and proteoglycan components of basal lamina

Answer 34

1) Laminin: Laminin is a major component of basement membrane and can bind to many different cell –> acts as a glue to hold everything together
- Adhesive protein which binds cells, collagen, GAG, PG
2) Fibronectin
3) Type IV Collagen
4) Perlecan (proteoglycan)
5) Nigoden (glycoprotein)
6) Heparin Sulfate (glycosaminoglycan)

Question 35

Basal Lamina

Answer 35

Basal lamina are flexible, thin extracellular matrix mats. They underlie epithelial cell sheets and tubes and they surround fat, muscle, and Schwann cells

• Flexible, thin extracellular matrix mats that underlie epithelial cell sheets and tubes, surround fat, muscle, and Schwann cells
• Functions as a molecular filter: Kidney glomerulus
• Functions to orient/polarize cells: Fibronectin fibrils and cytoskeleton
• Cell Migration
• Barrier to cell migration: Cancer
• Important for cell survival: Anchorage dependence

Schwann cells: Myelin producing cells of peripheral nervous system. Basal lamina serves as a scaffold for axon regeneration.
-Basal Lamina is formed by these different adhesive proteins
-It is very specialized
-It is found surrounding tissues
-It is found around blood vessels
-It is a way to separate epithelium or other cells from connective tissue
-It has a lot of different functions:
As a molecular filter everything would have to diffuse through it and thus only some things can!
Functions to polarize cells Epithelial cells are taught from the bottom. This gives the cells a lot of positional and survival clues.
Cell migration
Important for cell survival
Acts as a barrier! Keeps things out and acts as a barrier to cell migration and not allow cells to go in or out. We know this is broken down in cancers and thus contributes to the metastasis of cancers
-It is a very specialized type of matrix
-Cells can move along it (migrate) but prevents cells from moving through it.
-Know that it is able to act as a filter, a barrier from migration, gives cells polarization signals, etc.

Also, the basal lamina holds important information in regards to cell organization, nerve innervation, etc.

• It gives very important positional information
• Muscle has its own special basement membrane and so does nerve.
• The cells are then killed and if you allow the nerve to regenerate, it will re innervate at its same location. Also, the muscle will to the same thing. It helps organize the tissue

Question 36

Integrins

Answer 36

Integrins are cell surface receptors that bind the cell to components of the ECM. They are dimers containing an alpha and a beta chain.

• EMC Receptors (Laminin, Fibronectin, Collagen…)
• Low Affinity, High copy number
• Heterodimers of alpha and beta glycoproteins
• Overlapping specificity
• Cytoplasmic tails of b subunits link to cytoskeleton (usually Actin)
• Integrins are the ECM receptor proteins
• They are dimers consisting of an alpha and beta chain
• Beta chain is linked by the adapters
• They are matrix receptors
• They are low affinity by themselves but when they all come together they are strong
• Divalent cations are very important for their binding affinity
• They are heterodimerizing
• Specificity is determined by which alpha and beta subunit are coming together.
• Beta subunit is what is linked to the cytoskeleton

THE ALPHA AND BETA PAIRS OF INTEGRIN DETERMINE THE SPECIFICITY

Question 37

Cadherins vs. Integrins

Answer 37

Cadherins tend to have a stronger binding affinity in cell-cell and cell-matrix interactions where the integrins are weak interactions.

Question 38

Identify the intracellular proteins to which integrins attach and predict their importance to the strength of the association

Answer 38

The integrins attach to an adapter protein within the cells that connect it to actin and intermediate filaments.

Question 39

Distinguish Between inside-out signaling and outside-in signaling

Answer 39

Inside-Out Signaling:
-Inactive integrins activated by cells
-Platelets activate b3 to bind fibrinogen and aggregate
T lymphocytes activate b2 integrins to bind antigen presenting cells
-Inside-out signaling is when an integrin is expressed on the cell surface and is inactive and something inside the cell stimulates it and activates it, allowing it to now bind its ligand.
-Activated by something inside the cell and now can bind to ligand

Outside-in Signaling:

• Integrin binding to ECM activates intracellular signaling
  
  • Clustering of integrins at ECM contact sites
  • Focal Adhesion Kinase recruitment, clustering, activation
  • Similar to growth factor receptors
• Outside-in signaling is where the integrin is fully active but is just waiting for the matrix protein to come along, activate it and then get an active signal in the cell.
• Integrin binding activated some kind of intracellular signaling pathway
• Activated by something outside the cell and now can bind ligand inside cell.