Weisel - Cell Matrix

Question 1

Predominant cell type in connective tissue?

Answer 1

fibroblast, and it secretes abundant ECM

Connective tissue is a loose meshwork of cells, proteins, and carbohydrates, usually quite complex

Question 2

2 major classes of macromolecules that make up the ECM?

Answer 2

1. GLYCOSAMINOGLYCANS, in the form of proteoglycans

2. Fibrous proteins - collagen, fibronectin, elastin, laminin, fibrin

Question 3

GAG chains

Answer 3

Glycosaminoglycans - these occupy large amounts of space - they are unbranched polysaccharide chains, stiff, hydrophilic, and form gels at low concentrations. They cat the surface of most cells.
Hyaluronan, chondritin sulfate, dermatin sulfate, heparan sulfate are also unbranched polysaccharide chains that are highly negatively charged.
All attract a cloud of sodium and lots of water.
Too stiff to be folded and strongly hydrophilic, which causes they to form gells

Most GAGs are attached to protein in the form of proteoglycans.

Question 4

Hyaluronan

Answer 4

facilitates cell migration during morphogenesis
a repeating sequence of up to 25,000 non sulfated disaccharide units.
Resists compressive forces in joints and tissues
A spacer during embryogenesis
Creates cell free space through which cells can migrate during organogenesis.

Question 5

Proteoglycans

Answer 5

More carbohydrate than protein. The thing with more is always at the end i think.

Question 6

Aggrecan

Answer 6

Major component of cartilage and has a mass of 3E6 daltons.

Question 7

How to image?

Answer 7

Atoms making up biological macromolecules have intrinsically low contrast to electrons which are not scattered much. Solutions:

1. gold labeled antibody
2. metal shadowing
3. negative contrast - embed molecules in layer of heavy metal, and then evaporate metal at a low angle - i guess this leaves a hole where the protein bump is?? or rotary shadowing, evap of metal while rotating specimen
4. Intrinsic contrast - particles in thin film of vitrified ice plus computer processing

Question 8

Aggrecan

Answer 8

Made of 100 monomers - can occupy a tremendous volume. 100 monomers may bind to a single hyaluronan chain. Such aggregates can occupy a volume equal to that of a bacterium. Proteoglycans can also bind collagen or to the basal lamina.

Question 9

Collagens

Answer 9

Major proteins of the ECM - most abundant proteins in mammals.
There are more than 30 types of collagen. They align precisely to form a band pattern. Covalent corsslinks form within and between molecules to stabilize the fibrils. This is crucial for tensile strength of tendons.
Triple helices.

Question 10

How do collagen secreting cells help to organize fibrils?

Answer 10

They guide formation in close association with infoldings of the plasma membrane; in bone, cornea, and skin, fibrils are laid down in parallel layers perpendicular to each other.
In tendons, the fibrils are parallel to provide tensile strength.

Fibroblasts tug on collagen to draw it into sheets and cables.

Question 11

Elastin

Answer 11

Gives tissues elasticity

Highly HYDROPHOBIC protein, assembled in extracellular space into crosslinked fibers. Dominant protein in the ECM of arteries, giving them their elasticity. May comprise up to 50% of arterial dry weight.

Can be folded, and forms very highly elastic tissues.

Question 12

Fibronectin

Answer 12

ECM protein that helps cells attach to the matrix.

Has type III fibronectin repeats with RGD sequence. (ARG, Gly, Asp). This is an integrin binding domain I think.

2 subunits are connected at c terminal end by disulfide bonds.

Fibronectin is present in fibrils at the cell surface and we can see it present at the end of actin filaments.

Question 13

Type III fibronectin repeat

Answer 13

One of the most common domains in vertebrate proteins, contains RGD sequences that bind to integrins.

Question 14

How does fibronectin fibrillogenesis occur?

Answer 14

1. Fibronectin exists in relatively compact globular state in plasma
2. The interdomain interactions are disrupted by cell-generated tension
3. The n-termini of 2 extended fibronectin molecules form a tight complex, creating fibronectin protofibrils.

Question 15

3 ways that basal lamina can be organised

Answer 15

1. muscle (surrounds muscle)
2. epithelium(beneath epithelial cells)
3. glomerulus - gaps/permeability barrier

Question 16

What are basal laminae composed of?

Answer 16

laminin
type IV collagen
Nidogen
Heparin sulfate proteoglycan

Question 17

Laminin

Answer 17

An important component of the basal lamina
- comprised of 3 polypeptide chains with globular domains, disulfide bonded onto a cross-like structure
LOOKS LIKE A CROSS.
There are lots of binding sites on laminin - this allows you to interact with several proteins simultaneously.

Question 18

How are plant cell walls composed?

Answer 18

Built from cellulose microfibrils interwoven with polysaccharides.

Question 19

Anchorage dependence refers to what?

Answer 19

Most cells need to attach to surface to grow, proliferate, and survive.
In one experiment they put the same amount of matrix components on a surface over the same area but the greater extent of spreading promoted survival of the cell.

Question 20

UPA?

Answer 20

Prostate cancer cells make and secrete UPA, a protease which binds to specific receptors. If you transfect these cells with inactive UPA which bind to receptors but has no protease acivity you make them incapable of metastasis.

Question 21

What can integrins do?

Answer 21

They can activate intracellular signalling pathways. (SPECIFICALLY, the clustering of integrins at site of contact with ECM activates the pathways). Many of the signalling functions of integrins depends on focal adhesion kinase (FAK).

Question 22

Focal adhesion kinase

Answer 22

Responsible for phosphorylation of specific tyrosine residues, activated through integrins.

Question 23

FAK-deficient fibroblasts do what?

Answer 23

They actually form excess numbers of focal adhesions. Normal fibroblasts have fewer focal adhesions and have spread. FAK deficient fibroblsts have more - they do not spread out properly.

has something to do with phorphorylation of specific tyrosine residues??

Question 24

How does integrin signaling work?

Answer 24

Extracellular signal –> intracellular signaling cascade –> altered conformation of the integrin –> extracellular binding site now mediates cell adhesion.

Integrin signals are directly proportional to the number of integrin-ligand bonds.
alpha5beta1 fibronectin binding initiates FAK Y397 phosphorylation.

Question 25

What happens when you switch integrins?

Answer 25

It can change adhesion and cell migration.

Question 26

Immunologial synapse/APC integrin?

Answer 26

APC –> weak binding that activates integrin –> stronger binding of integrin to ICAM. (this is in t lymphocytes)

Question 27

Cytoskeleton puts force on focal adhesions and integrin?

Answer 27

Yes. Forces outside can be transmitted inside.

Question 28

What happens when you switch integrins?

Answer 28

It can change adhesion and cell migration.

Question 29

Immunologial synapse/APC integrin?

Answer 29

APC –> weak binding that activates integrin –> stronger binding of integrin to ICAM. (this is in t lymphocytes)

Question 30

Cytoskeleton puts force on focal adhesions and integrin?

Answer 30

Yes. Forces outside can be transmitted inside.

Question 31

What is vinculin?

Answer 31

Vinculin binds talin – tail binds actin. Tension sensor occurs between head and tail.

Question 32

What experiment did they use to determine that vinculin – actin is a tension sensor?

Answer 32

FRET – can use to measure mechanical tension across vinculin.

Question 33

How can you analyze force on a surface?

Answer 33

Displacement of fluorescent beads embedded in a polyacrylamide gel. Use acrylamide gels to control the stiffness of surfaces.