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EMC > Adhesive Interactions Between Cells: The Extracellular Matrix L1-3 (Jo Adams) > Flashcards

Adhesive Interactions Between Cells: The Extracellular Matrix L1-3 (Jo Adams) Flashcards

1
Q

What is the extra cellular matrix?

A

In all animals the ECM is a complex assembly of:
glycoproteins
collagens
polysaccharides

Polypeptides > 1000amino acids long
The proteins are secreted by cells and assembled extra cellularly into insoluble, large-scale networks by molecular interactions that include covalent cross-linking

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2
Q

How many ECM proteins are there in humans?

A

About 300ECM proteins in humans

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3
Q

What is the most abundant protein in the human body?

A

Collagen

ECM forms about 80% by mass of protein in many tissues of mammals

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4
Q

What are the essential part of an ECM?

A

1) Fibril systems - collagen and fibrin are the most ancient fibril-forming EMC proteins
2) Extracellular proteases or accessory proteins so that fibril systems can be remodelled
3) Receptors to attach the ECM to the cell surface

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5
Q

What are the two main forms of ECM in vertebrates?

A

1) Connective ECM

2) Basement membrane

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6
Q

Connective ECM

A

3D meshwork that surrounds mesenchymal cells or neutrons. Major components are
COLLAGEN FIBRILS - gives high tensile strenght
FIBILLIN AND ELASTIN microfibrils - gives elasticity
FIBRONECTIN fibrils
EXTRACELLULAR PROTEOGLYCANS eg docarin, that bind to fibril components or aggregate with hyaluronan
HYALURONAN, a glycosaminoglycan polymer (BINDS WATER to ECM to provide volume and strength of tissue)

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7
Q

How many collagen genes in humans?

A

29, many tissue specific collagens

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8
Q

Proteoglycans

A

protein cores substituted with long complex carbohydrate chains

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9
Q

Basement membrane/Basal lamella

A

Thin sheetlike network of
LAMININ
COLLAGEN IV (large long thin)
NIDOGEN (small protein)
PERLECAN (secreted proteoglycan)
Supports epithelia and endothelial cell layers, surrounds skeletal muscle fibres
Acts as a barrier and support for endothelial layers

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10
Q

Collagen fibre

A

made up of individual fibrils
fibres are formed from collagen molecules secreted by the fibroblasts
These structures run in diff orientations of the tissue-important for tissue strength in every dimension
Exist in fibroblasts
FIBROBLASTS exist throughout the lifespan of the individual and are factories to produce collagen

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11
Q

What produces collagen?

A

Fibroblasts

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12
Q

Types of collagen

A

Collagen I- in dermis/skin

Collagen II- in cartilage and tendon

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13
Q

How is collagen synthesised?

A

Pro collagen-globular N and C terminal domains
These must be cleaved to give mature collagen - can then form fibrils
3 polypeptides per collagen molecule.

Molecules of collagen interact in a particular way in the fibrils-each collagen molecule interacts with FOUR other molecules at their N and C terminals

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14
Q

What type of aa repeats exist in the middle of collagen?

A

Helical central domain - GXY repeats
Form a left handed helix
3 residues/turn
G=glycine
X=often proline
Y= often hydroxy-proline (greater capacity to form H bonds than proline itself)
hydroxy-prolines in the different chains of the triple helix are essential for this structure to be stable

3 helices together form RH super helix. 10 to 10.5 residues/turn (TRIPLE HELIX)

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15
Q

What enzyme cross-links fibrils into fibril bundles and then fibres in collagen?

A

Lysyl oxidase

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16
Q

Fibrillin

A

Forms thin MICROfibrils compared to collagen
7 TM domains
Enormous array of binding sites for other ECmatrix proteins (near N terminus and centre),- links diff fibril systems into a super system
Many EGF domains
Has binding sites for the TGF beta family of growth factors - crosses their localisation and activity state
Additional function in vertebrate blood vessels and other tissues that need to stretch-template assembly of ELASTIN to form elastic fibres

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17
Q

Elastin

A

intrinsically disordered molecule-exists in a globular state
microfibrils+elastin=elastic fibre
Elasticity used in blood vessels/aorta to allow them to deform and relax as the heart pumps blood around the body

Elastin elongates under mechanical force, then undergoes elastic recoil when relaxed.

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18
Q

What do matrix metalloproteases do?

A

These naturally cleave fibrillin. This turns over and destabilises fibrillin fibrils so many destroy binding sites for TGFbeta complexes so then they get released from the fibril
Release of TGFbeta complex from the fibril is a trigger for the latency partner protein to dissociate and the activity of TGFbeta to be released

Proteases may also release small fragments of fibrillin that actually contained a TGFbeta binding site-compete for intact fibril for the binding of the TGCbeta complex

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19
Q

TGFbeta

A

Microfibrils bind inactive TGFbeta complexes and other TGFbeta family growth factors (BMPs)
The fibrillin fragments compete for TGFbeta binding, this releases active TGFbeta from ECM

This system provides spatial and temporal control on the release of active TGFbeta to bind to its receptor and activate cells

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20
Q

Marfans syndrome

A

Inherited disorder
Caused by mutations in Fibrillin-1 (effects the connective tissue)
Autosomal DOMINANT
affects 2/3 induviduals/10,000
Affects ECM organisation and strength in the eye, musculoskeletal system and cardiovascular system. Problems can arise-weakens walls of aorta over time
Mutations occur throughout the molecule
Mutations effect microfibril organisation and strength, resulting in flexible joints and or/ cardiovascular, skeletal or ocular problems in adult life.
Microfibril disruption results in aberrant local activation of TGFbeta and excess TGFbeta signalling, because of loss of binding sites for the latent TGFbeta complex on the fibrillin microfibril

TGFbeta regulates ECM synthesis > ECM composition altered

Cells respond to excess TGFbeta by over-producing proteases and other ECM proteins > aberrant, weaker ECM (POSITIVE FEEDBACK)

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21
Q

What types of cell surface receptors are there for ECM components?

A

Integrins and membrane-bound proteoglycans

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22
Q

What is the dystroglycan complex?

A

A laminin binding proteoglycan receptor. Its role in skeletal muscle and the molecular basis of muscular dystrophy

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23
Q

Fibronectin
Typical ECMprotein of connective ECM
Soluble disulfide-bonded dimer secreted from cells

Fibronectin line up in similar linear bundles to actin filaments inside cells
These 2 proteins in diff compartments are linked and organised in a similar way (coalignment) fibroblasts secrete a lot of fibronectin of their own

A

Multidomain protein-3 types of domains are repeated many times along the length of the protein
Forms fibrils in the connective matrix on a nano/microscale compared with the collagen fibrils
Fibronectin is much less abundant than the large collagen structures/fibres
C terminal -disulfide bonds form the dimer. Transglutaminases cross-link the dimers into larger assemblies which form the network of fibrils in the connective ECM (transglutaminase sites exist in TYPEI and TYPEII domains)

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24
Q

How long is the typeIII fibronectin domain?

A

90aa long
These have common fold with beta strands and small loops The 10th repeat is more unusual since it has a large loop that extends on one side of the domain and the RGD is positioned at the tip of the loop-Well exposed away from the core of the protein fold, therefore able to interact with integrin cell surface receptors
Each type III repeat folds as an independent domain

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25
Q

RGD

What 3aas are crutial for the fibronectin molecule to interact with other cells?

A

Binds integrin receptor
The central activity for binding cells residues in just one of the type III repeats (repeat 10)
3aas were found to be absolutely crucial for the fibronectin molecule to interact with other cells (ARG, GLY, ASP acid-RGD)
Seqs in neighbouring type III fibronectin domains assist in the affinity of the interaction with integrin (specificity)

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26
Q

Other molecules fibronectin binds

A

Fibrin in plasma-forms clots (wound healing response)
Collagen
Herapan sulfate (proteoglycan)

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27
Q

Plasma fibronectin

A

Abundant in blood (as a dimer)
Involved in clotting when platelets get activated
Also present in the connective matrix in many tissues

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28
Q

Integrin
Structure?
Length?

A

alpha/beta heterodimers
Extend more than 20nm outside form the lipid bilayer
Non covalently associated-come together at the cell surface
The alpha/beta chains are both glycosylated held together by non-covalent bonds. The receptor complex assembles co-translationally

29
Q

Where does talin and alpha-actinin bind in the integrin structure?
Integrins bind fibronectin, collagen, laminin, leukocyte-specific receptors(LSR-these act in cell-cell adhesion, direct interactions, don’t interact with the ECM-beta2/7)

A

beta1

30
Q

How many combinations of alpha and beta interns exist?

A

24

31
Q

What integrin receptor is prevalent in fibronectin?

A

alpha5beta1

32
Q

Mechanism for binding ECM ligand

A

1)RGD INTEGRINS
RGD of fibronectin binds to integrin and interacts with the “head” of both integrin subunits-RGD docks into the cleft b/w the subunits
2) THE I DOMAIN INTEGRINS (COLLAGEN-BINDING INTEGRINS and the LEUKOCYTE-SPECIFIC INTEGRINS)
Alpha subunit contains an EXTRA DOMAIN, the I domain=inserted domain-binds to collagen ligands
Collagen triple helix motif GFOGER (O=hydroxyproline)
This motif interacts with a wide region on the outerface of the I domain through a lot of electrostatic and van de waals interactions (alpha1beta1/alpha2beta1)

33
Q

Active integrins set up INTRACELLULAR signals

A

1) responses to MECHANICAL forces are exerted through the ECM/integrity/cytoskeleton linkage (helps tissues move in a coordinated way)
2) CHEMICAL signalling occurs via Kinases and second messengers
- these are activated downstream of talin(beta chain) that bind to talin and integrin alpha cytoplasmic domain

34
Q

Importance of cytoskeletal linkage for mechanical force transduction in cell-ECM adhesion, regulates diff kinds of cell behaviours

A

EG
Mechanical force transmitted by integrin-ECM attachment controls the orientation of the MITOTIC spindle (mitotic spindles always align in direction of highest mechanical force)

35
Q

Cellular/chemical signalling

A

Talin links to focal adhesion kinase (FAK) and the Ras pathway
Talin phosphorylates FAK, FAK is recruited to the p.memb
FAK phosphorylates Grb2 (activator of Ras pathway)-activates Ras dependent kinase cascade leading to G1 phase and cell proliferation

36
Q

Integrin intracellular signalling is necessary for cell survival and proliferation…how?

A

Lack of integrin signalling activates p53 —> Bax—> apoptosis
p53 inhibited by Bcl2 (pro survival factor)
Lack of integrin signalling activates Effector caspase

Integrin signalling via talin activates FAK—-> PI3K—>cell survival
Integrin signalling via talin activates shc/Grb2/sos—>ras—> raf—>MAPK—>cell survival

37
Q

Proteoglycans-p.memb proteins modified by O-linked carbohydrate chains example is dystroglycan

A

Dystroglycan complex important for cell attachment to the BASEMENT MEMB of skeletal muscle
Dystrophin connects dystroglycans to actin filaments (analogous to talin)
Dystroglycan binds to laminin-a component of the basement membrane form of the ECM
This protein complex withstands changes in mechanical forces as muscles contract and relax

38
Q

Muscular dystrophy

A

Inherited disorder
Muscle weakness and loss of muscle tissue
>20 diff types of severity - onset in children is more severe
Mutations in:dystrophin/dystroglycan/laminin/other associated proteins
Disrupt integrity of the complex-loss of mechanical force balance and loss of link to cytoskeleton-cell death-disruption of muscle tissue and appearance of muscle wasting through cell death

39
Q

Epithelial cells

A

Form a covering over the internal and external surfaces of the body
e.g. skin, inner linings of the mouth, lungs or digestive track
Also form GLANDS where cells sink under the surface of the adjacent epithelium

40
Q

What is endothelia?

A

Epithelial lining the internal blood vessels

41
Q

Two types of epithelia?

A

Simple (single cell layer)

Stratified (multiple cell layers)

42
Q

Types of interactions b/w cell cell and cell-ECM junctions

A

Between cells:
Tight junction (actin)
Adherens junction
Desmosome (intermediate filaments)

Between cells and ECM:
Both depend on integrins
Focal adhesion
Hemidesmosome

43
Q

How thick is the basement membrane?

A

100nm
Dense thin protein sheet
Set of proteins secreted by cells to assemble to form this dense extracellular structure
Collagen IV and laminin together produce a dense sheet. Prelecan and nidogen add more protein-protein interactions that stabilise the structure

44
Q

What cells are focal adhesions present in?

A

Epithelial cells

Fibroblasts

45
Q

Functions of the basement membrane

Essential for life

A

1) support to sheets of epi/endothelial cells
2) barriers b/w epithelial cells and other tissue layers
3) If degraded by excess proteases, invasion of cancer cells can take place-metastasis
4) Rare mutations can give rise to SKIN BLISTERING diseases
5) Mechanisms: Basement memb is made from a special set of ECM glycoproteins

46
Q

Laminin

A

3 subunits encoded by 3 diff genes (3 stranded coil alpha/beta/gamma)
Within each peptide structure there are many diff domain regions
Cross-like structure binds collagen IV
C terminus =main integrin binding site and site for dystroglycan
Length of laminin defines the basement membrane
Middle of cross binds NIDOGEN
Lots of herapin/herapin sulphate binding domains

47
Q

Perlecan

A

array of proteinaceous domains and glycoseaminoglycans substituted onto the protein core

48
Q

Collagen IV molecules assemble into a planar meshwork
Gives basement membrane sheet like properties
PROPERTIES OF COLLAGEN IV?

A

Non-collagenous (NC1) domain is NOT cleaved off (restricts types of structures this collagen molecule can assemble into outside the cell)
Each TRIMERIC coll IV molecule forms TETRAMERS through their C terminal domains-“the spider”
Binding affinity b/w extreme C terminal regions of the molecules
Two NC1 domains bind together head-to-head to form flat meshwork -binding affinity for N terminal globular regions

49
Q

What integrin molecules are in focal adhesions?

hemidesmosomes?

A

focal adhesions-beta1 and beta3

hemidesmosomes-alpha6beta4

50
Q

Which integrins binds collagen IV in the basement membrane?

A

alpha1beta1

alpha2beta1

51
Q

How many main laminin receptors are there in cells?

A

4
3 partner with beta1
1 partners with beta4

52
Q

Focal adhesion
Present in many cell types
Cell isn’t uniformly stuck to matrix all over its surface

A

beta1/beta3 intergrins are clustered-useful feature for the intensity of signalling or rapid adaptation of signalling to change the ECM environment
integrins binds talin and other linker proteins to link to the actin cytoskeleton and activate signalling molecules.

53
Q

The hemidesmosome-whats unusual about the integrin?
This molecular assembly DOESN’T connect to the actin cytoskeleton like other integrins
Hemidesmosome-more specific to Epithelial cells

A

alpha6beta4
the beta4 domain has an unusually long cytoplasmic domain-used to bind PLECTIN (intracellular protein) which binds to KERATIN IFs
These structures are more stable than focal adhesions
Keratin is less dynamically turned over and mechanically more rigid than actin

54
Q

What co receptor is involved with alpha6beta4 integrins of the hemidesmosomes?

A

BP180 binds to BP230

55
Q

Extra: What collagen molecule links basement memb to connective ECM?

A

Collagen VII anchoring fibrils

56
Q

What cell-cell interactions involve actin?

A

1) tight junction

2) adherens junction

57
Q

Where is the tight junction positioned in epithelial cells?

A

Apex (top)
Particular protein composition is contained here
P.membs of the two cells come very close together

58
Q

Tight junctions

A

Mediated by head to head interactions of a specific set of cell surface receptors
Tight junction prevent lateral diffusion of memb. proteins in the lipid-bilayer
Maintains apical-basal distribution of memb proteins
2 cells also maintain own different protein composition

59
Q

What is able to pass through tight junctions?

A

Ions and small molecules can pass b/w the epithelial cells through an aqueous pore
Rest of the space is blocked by the TJ proteins (protein barrier forms b/w plane of 2 p.membs of neighbouring cells)

60
Q

What proteins are involved in the tight junctions?

A

Claudin-has 4TM domains with 2 short loops (extracellular domains)
Interact closely with neighbouring claudins to bring the plasma membs close together
occludin-similar topology to claudin yet larger cytoplasmic domain at c terminus
JAM-single TM domain

Each memb. protein interacts with the extracellular domains of the equivalent proteins of the neighbouring cells

All the proteins are linked inside the cell to the ACTIN cytoskeleton

61
Q

What is occludin involved in?

A

Tight junctions

ZO proteins interact with actin microfilaments and cytoplasmic domain to mediate linkage with occludin

62
Q

What protein is involved in adheren junctions?

A

Cadherin molecules on adjacent cells

These have short cytoplasmic domains and are assembled into lateral arrays

63
Q

How many CAD domains do cadherins have in adheren junctions and how many of these contain Ca2+ binding sites?

A

5 CAD DOMAINS (Calcium dependent adhesion molecules)
Three contain Ca2+ binding sites
The ability of the cadherins to interact with each other at the cell surface depends on the calcium being bound to these receptors

64
Q

What peptide motif do cadherins have in adheren junctions to bind to the equivalent cadherin of the neighbouring cell?

A

First domain contains binding site of for another cadherin molecule that includes the peptide motif
HAV

65
Q

What are the catenin linker proteins and where are they found?

A

Catenins are cadherin binding proteins (alpha beta and gamma)
beta interacts with cytoplasmic tail of cadehrin
alpha binds to the beta catenin and also has binding affinity for actin microfilaments
p120
multiple proteins are involved in coupling cadherins and cattiness to actin filaments

66
Q

Cadherins

A

Large protein superfamily
Provide tissue specificity for cell-cell interactions in epithelia and nervous system
Cadherins control/mediate sorting out of epithelial cells
Diff forms of cadherins are present in diff sets of epithelium in the body-differences in expression patterns of cadherins
Preferential hierachy b/w cadherins that allow some to form aggregates and others to be excluded
E-cadherin forms clusters -are able to aggregate together (preferential process) N cadherin is excluded

67
Q

What cells express E-cadherin (epithelial cadherin)?

A

Ectoderm cells
The cells in the neural tube have N-cadherin (neural cadherin) and are separate from the ectoderm-beginning of development of the central nervous system

68
Q

The desmosome-what proteins involved and where is it located?

A

Provide rigidity near the base of the cell
Desmosomal cadherins=desmocollin and desmoglein
mediate cell attachment of the desmosome
Collection of Ifs coming to concentrate at the intracellular face of the junction-links to KERATIN IFs
Set of linker proteins specifically concentrated at the desmosome
SMALL GAP region b/w the p.membs

69
Q

How to epithelial cells communicate by GAP JUNCTIONS?

A

Channels form between cytoplasms by using 6 CONNEXIN proteins that allow direct communication b/w the cytoplasm of the adjacent cells. These 6 proteins interact head-to-head with the connexin proteins on the adjacent cell
The p.membs of the cells are v close together
Molecules >1000Da can diffuse between the cell cytoplasms
Closing and opening of GAP junctions can be regulated by phosphorylation events
Signals can be directly propagated at lower levels to adjacent cells (secondary messengers)

EMC (12 decks)

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