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BIOC 212 > Extracellular Matrix > Flashcards

Extracellular Matrix Flashcards

1
Q

What are the 2 different forms of direct binding between cells?

A

Cell-cell junctions
Cell adhesion

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2
Q

What difference type of tissues are found in the intestinal wall?

A
  1. Epithelium → sits on basement membrane
  2. Connective tissue (fibroblasts)
  3. Smooth muscles: Circular fibers, Longitudinal fibers (crossing each other making leaf)
  4. Connective tissue
  5. Epithelium
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3
Q

What are the 3 major classes of cell junctions (and their main function)?

A
  1. Occluding junctions → Seal cells together in an epithelium, no leaking from one side to the other
  2. Anchoring junctions → Mechanically attach cells to the neighbours or to the extracellular matrix
  3. Communicating junctions → Mediates the passage of signals from one cell to the next
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4
Q

What are the 2 types of occluding junctions?

A
  • tight junction (vertebrates only)
  • septate junctions (invertebrates mainly)
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5
Q

What are the different types of anchoring junctions?

A

Actin filament attachement sites:
- cell-cell junctions (adherens junctions)
- cell-matrix junctions (focal adhesions)

Intermediate filaments attachement sites:
- cell-cell junctions (desmosomes)
- cell-matrix junctions (hemidesmosomes)

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6
Q

What are the 3 types of communicating junctions?

A
  • Gap junctions
  • Chemical synapses
  • plasmodesmata (plants only)
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7
Q

What organisms/organelles can you see with a light microscope?

A

Light wavelength ~ 400-700 nm

  • Chloroplast (1um)
  • Most bacterias (1-10 um)
  • Plant/animal cells (10-100 um)
  • Fish egg (> 1mm)
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8
Q

What is the range of molecules you can see with an electron microscope?

A

<1 nm - 100 um

  1. small molecules (< 1nm)
  2. lipids (~2-4 nm)
  3. proteins (5-10 nm)
  4. T2 phage (70-80 nm)
  5. Chloroplast (1 um)
  6. Most bacteria (1-10 um)
  7. Plant/Animal cells (10-100 um)
    *um = micron
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9
Q

How does an eletron microscope work?

A

From top → bottom:
1. Electron gun (Cathode = e- donor, Anode = acceleration)
2. Condenser lens
3. Specimen
4. Objective lens
5. Projector lens
6. Viewing screen/photographic film

*Ultralow vacuum necessary for electrons to become scattered by collision with air molecules

*Wavelength of an electron decreases as velocity increases → accelerating voltage = 100 000V → 0.004 nm wavelength

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10
Q

What is Osmium tetroxide?

A

It is an electron dense (atomic number = 76) staining agent for the specimen to get contrast in image of electron microscopy

It binds to lipid bilayer and proteins
*Dark areas = dense materials

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11
Q

How can we use a tracer to visualize tight junctions?

A
  1. Inject a tracer molecules in gut lumen
  2. Follow the molecules → goes in microvilli
  3. Tracer molecule stops at the tight junction

*Occulins and Claudins bind to hold adjacent plasma membranes together with intercellular spaces between seals

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12
Q

What are the tight-junction proteins formin the sealing strands (and their properties)?

A

Claudin: (Primary component)
- 20-27 kDa
- 24 homologous members (24 genes)
- Specificity: claudin in kidney epithelia → Claudin-16 required for Mg2+ to be reabsorbed from the urine into the blood → mutation causes excessive loss of Mg2+ in the urine

Occludin: (Secondary)
- 65 kDa (bigger)
- 2 isoforms (alternative splicing)
- Localization to tight junctions is regulated by phosphorylation in epithelial and endothelial cells
- Not as essential as claudins

*Both have 4 alpha-helical TM segments with 2 extracellular loops

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13
Q

What phenotype does an Occludin deficient mice show?

A
  • Chronic inflammation, gastric epithelium calcification → not easily explained by barrier function

Occludins might be involved in epithelial differentiation

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14
Q

What protein is responsible for anchorage to the cytoskeleton of claudin and occludin?

A

ZO proteins = Zonula Occludens → bind to actin filament

*Tight junction complexes connect to actin filaments

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15
Q

What is the molecular weight of 1 amino acid?

A

0.11 kDa ~ 100 Da

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16
Q

What are the 3 major types of cytoskeleton fibers?

A
  1. Actin filaments → made out of actin → involved in tight junctions
  2. Intermediate filaments → made out of Keratin, Vimentin, Desmin → involved in tight junctions
  3. Microtubules → made out of tubulin → not involved in tight junctions
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17
Q

What is the impact of a mutation on Cldn-1 gene (claudin gene)?

A

Causes Neonatal ichtyosis and sclerosing cholangitis

Neonata ichtyosis → heterogenous family of at least 28 mostly genetic skin disorders → dry, thickened, scaly or flaky skin

Cholangitis → infection of the common bile duct (tube that carries bile from the liver to the gallbladder and intestines)

*Tight junction-associated hereditary disease

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18
Q

What is the impact of a mutation on Cldn-19 gene (claudin gene)?

A

Hypomagnesemia (low Mg in blood) with hypercalciuria (high Ca in urine) + nephrocalcinosis (disorder causing excess Ca deposited in the kidneys) with visual impairment

*Tight junction-associated hereditary disease

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19
Q

What is the impact of a mutation on ZO-2 gene (ZO-protein gene)?

A

Familial hypercholamenia (FHC) → elevated serum bile acid concentrations, itching and fat malabsorption

*Tight junction-associated hereditary disease

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20
Q

Why is it important to have anchoring junctions?

A
  • Cell is flimsy
  • Transmission of force
  • Tension bearing cytoskeleton
    *Anchoring provides stability to the cell
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21
Q

In what tissues are anchoring junctions more abundant?

A

Widely distributed in animal tissues → most abundant in muscle, heart, epidermis, epithelia (intestinal, skin cells)

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22
Q

What are the 2 main protein classes involved in anchoring junctions?

A
  1. TM adhesion proteins:
    - Cytoplasmic tail + TM + extracellular domain
    *The extracellular domain can bind to another TM adhesion protein or the ECM
  2. Intracellular anchor proteins:
    - connect to actin and intermediate filaments
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23
Q

What anchoring junctions are responsible for Cell-cell vs Cell-ECM interactions?
(Which proteins?)

A
  1. Cell-cell:
    - Adherens junctions → joins actin filaments of neighbouring cells together
    - Desmosomes → joins intermediate filaments together (stronger)
    → CADHERINS as the TM adhesion protein
  2. Cell-ECM:
    - Focal adhesions → joins actin filaments to ECM
    - Hemidermosomes → joins intermediates to ECM
    → INTEGRINS as TM adhesion proteins
24
Q

What are all the proteins involved in Adherens junctions (and their properties)?

A
  1. Intracellular anchor proteins (adaptor proteins): catenins (alpha, beta), vinculin
  2. TM protein: Cadherins (E-Cadherins) → homophilic interactions, homodimers, calcium dependent

Structure:
Intracellular domain of cadherin interacts with p120-catenin + b-catenin → b-catenin interacts with a-catenin (longer bridge) → a-catenin interacts with vinculin which binds to an actin filament
→ a-catenin also binds to an actin filament (not the same)
*In 1 subunit of adherens junction → 2 actin filaments eahc bound to a set of adaptors → each bound to a cadherin that dimerizes at their extracellular domains

25
Q

What is the organization and importance of actin filaments in adherens junctions

A

*Adherens junctions = Cell-cell anchoring junctions binding actin filaments in neighbouring cells

Contractile Actin filament running along cytoplasmic surface of the junctional plasma membrane, attached to adhesion belt → Contraction of actin network by myosin motor proteins (fundamental in morphogenesis)

*Required for formation of tight junctions

26
Q

What allows folding of the epithelial sheet in embryogenesis (What junctions/organization)?

A
  1. Organized tightening along adhesion belts in selected regions of the cell sheet → epithelial cells narrow at their apex
    *Adhesion belt = continuous adhesion of cells by interaction of actin filaments and cadherins (adherens junctions) within the epithelial layer
  2. Epithelial tube piches off from overlying sheet of cell → neural tube formation for example (1st structure of CNS)
27
Q

What system provide contractility to non-skeletal muscle cells (ex: epithelial cells)?

A

Actin-myosin II contractility system (ATP-dependent):
*Responsible for folding of the epithelial sheet

Myosin II:
- Responsible for contractility of actin structures
- 2 heavy chains + 4 light chains
- Dimerizes (heavy chains for coiled-coil helix)
- Self-assembly into bipolar filaments (tails against tails, heads on both ends 15-20 molecules)
- Inactive-state: light chains not phorphorylated → blocked actin-binding site
- Active state: light chains ATP phosphorylated by MLCK → actin-binding site exposed
- Power stroke: Hydrolysis of ATP → ADP

28
Q

What are the intermediate filament proteins involved in formation of desmosomes?

A

Desmosomes = cell-cell junctions attaching intermediate filaments from neighbouring cells together (Anchoring junctions)

*intermediate filaments are linked to a network
Keratin filaments → Epithelial cells
Desmin filaments → Heart muscles

29
Q

What proteins of the cadherin family are involved in desmosomes anchoring junctions?

A

*Cadherin family → extracellular, TM, intracellular domains, dimerize and interact with cadherins of neighbouring cells

  • Desmoglein
  • Desmocollin
30
Q

What is the structure of desmosomes anchoring junctions (different poteins and their functions)?

A
  1. At the surface of the cell → formation of a dense cytoplasmic plaque made of intracellular anchor protein → Desmoplakin + Plakoglobin + Plakophilin
  2. Keratin filaments (IF) are anchored to cytoplasmic plaque (laterally) → bind to desmoplakin
  3. Cadherins: Desmoglein + desmocollin → dimerize and interact with neighbouring cells

Cytoplasmic tails of cadherins bind plakoglobin (g-catenin) → binds desmoplakin → binds intermediate filaments

31
Q

What is Pemphigous vulgaris?

A

*Autoantibodies to desmoglein
Autoimmune disease that affects the skin and mucous membrane. When autoAb attack desmogleins → cells become separated from each other (no anchoring junctions, desmosomes) → epidermis becomes “unglued” → called acantholysis

32
Q

What is the definition of a homophillic interaction?

A

When the same proteins bind together and different proteins don’t
Ex: for desmosomes, demogleins bind desmoglein from neighbouring cells and desmocollin bind desmocollins from neighbouring cell (all cadherins)

33
Q

What are the MW and characteristics of plakoglobulins and desmoplakins?

A

*intracellular anchor protein in desmosomes

Plakoglobins (g-catenin) ~ 80 kDa →mediates binding of desmoplakin to cadherins

Desmoplakin ~ 260/330 kDa → bigger because needs to bridge between both plakoglobulins and IF
- work as dimers → coiled-coil region (1st, 4th AA in same region; 7AA/2turns → 1 and 4 hydrophobic AA of one interacts with 4 and 1 hydrophobic from other one) + ionic interactions of AA next to 1 and 4 + hydroxyl groups to stabilize coiled-coil helix

*Cadherins → Plakoglobin → Desmoplakin → IF

34
Q

What diseases can be caused by mutations in plakoglobin and desmoplakin (in humans)?

A

Cardiomyopathy
Skin diseases

35
Q

What are focal adhesions? (proteins involved)

A

Achoring junctions attaching actin filaments in a Cell-ECM fashion

  1. Integrins interact with ECM as heterodimers:
    - 18 alpha subunits + 8 beta subunit → 24 integrin heterodimers (alpha and beta are TM proteins)
  2. alpha-actinin, talin or filamin binds the intracellular tail of the beta-subunit + binds vinculin
  3. Actin filament are attached to integrins via a-actinin, filamin, talin, vinculin
36
Q

How does immunofluorescence imaging differ when visualizing Actin and Vinculin between WT and integral alpha subunit KO?

A

*Focal adhesion
In WT, vinculin is localized to periphery of the cells vs a2-/- not specifically localized

By looking at both expression in an overlapping manner → WT shows end of actin filament in region that is stained for Vinculin

*2nd Ab for signal amplification

37
Q

What proteins are involved in hemidesmosomes?

A

*Achnoring junctions cell (IF) - ECM
1. Keratin (IF) end in hemidesmosomes (vs lateral attachements in desmosomes)
2. Plectin (intracellular anchor protein)
3. a6b4 integrin
4. Laminins + Collagen IV in the basement membrane (ex: in the epidermis of the skin)

38
Q

What is Epidermolysis bullosa?

A

Caused by a genetic mutation making the skin more fragile in

from bottom → top (more → less severe):
Type VII Collagen → dystrophic EB
Laminin 5 → Junctional EB
a6b4 integrin → Junctional EB
Plectin → Hemidesmosal EB
Keratin 5 and 14 → Simplex EB

From less severe to more severe:
SHemiJunDy
KPALT

39
Q

What are the TM adhesion proteins responsible for the different anchoring junctions?

A

Adherens junctions (cell-cell, actin) → cadherin (E-cadherin)
Desmosome (cell-cell, IF) → cadherin (desmoglein, desmocollin)

Focal adhesion (cell-ECM, actin) → integrins
Hemidesmosomes (cell-ECM, IF) → integrin a6b4, BP180

40
Q

What molecules can and cannot pass freely through Gap junctions?

A

Gap junctions important for cell communication

Small molecules < 1000 Dalton (not charge dependent):
ex: Water, sugars, amino acids, vitamins, second messengers, ATP, protons (pH),
ex of communication: electric coupling, pH
NOT: macromolecules, proteins, enzymes, RNA, large compounds

41
Q

What is the structure and the composition of Gap junctions?

A
  • Channel = 1.5 nm in diameter
  • gap of 2-4 nm between cells
  • Continuous, aqueous channel between 2 cells
  • Channels can be homotypic or heterotypic

Proteins = connexins → 6 connexins form 1 connexon (homomeric/heteromeric) → 2 connexons assemble to 1 form intracellular channel (homotypic/heterotypic)

1 Channel = 12 connexins = 24 helices/connexon * 2

42
Q

What is the structure/characteristics of connexins?

A
  • 4 pass TM proteins
  • 21 different connexins → different genes, different tissue distribution
  • 20-60 kDa (humans)

*involved in Gap junctions

43
Q

How does Gap junction regulation occur?
Why is it important?

A

Gap junctions = dynamic structures
Closed = high cytosolic Ca2+, low pH
Open = low cytosolic Ca2+, high pH

*Ca2+ direct binding and Calmodulin-bound → closes the gap junction

Important in cell death → don’t want everything to diffuse in neighbouring cells (ex high Ca influx to flow in neighbouring cells)
Also would not want free diffusion of H+

44
Q

What are intracellular and extracellular physiological concentrations of calcium?

A

Intracellular = 0.1 uM
Extracellular = 2 mM (ex: blood, ECF)

45
Q

What are the functions of Gap junctions?

A
  1. Signals spread rapidly (nerve, muscles)
  2. Electric coupling invertebrates → synchronization of contractions of heart muscle cells and smooth muscles (intestine)
  3. Non excitable cells → sharing of the small metabolites and ions
    Ex: in the liver, noradrenaline/glucose/glycogen shared between cells
46
Q

What are large and small Gap junctions?

A

Large Gap Junctions → 200-400 nm (many channel in 1 patch)

Small Gap Junctions → 40-50 channels

*In electron microscope associated exclusively with the cytoplasmic fractur face (P face) of the plasma membrane

47
Q

What is the structure of classical cadherins? By which technique can it be visualized?

A
  • Dimer or large oligomers
  • Extracellular domain folded in 5-6 (or more) cadherin domains (structurally related to Immunoglobulin domains)
  • 2 Ca binding sites between each cadherin domain
  • 3 Ca binding sites before the N-terminus Cadherin domain/repeat
  • Cytosolic C-term

Visualized by X-ray crystallography or Nuclear magnetic resonance spectroscopy

48
Q

What is the importance of calcium in cadherins?

A

Calcium binds to sites between Cadherin repeats → locks the repeats, make them more stiff and rod-like (More Calcium → more rigid)
→ Calcium is needed for homotypic interations

If Ca is removed (EDTA) → extracellular part beomces floppy → rapidly degraded by proteolitic enzymes (recognize)
Kd of Cadherins for Ca ~ mM range → >1mM Ca concentration cadherins are saturated with Ca

49
Q

What characterizes the binding of cadherins with each other in cell-cell adhesions?

A
  • At the N-terminal of cadherins (seen by X-ray diffraction) → terminal cadherin domain (different from other ones, has 3 Ca binding sites)
  • Homophilic binding between terminal knob and nearby pocket both molecules (knob in pocket in a lock and key model)
  • Creates a characteristic Gap between cells (40 nm)
  • Weak individual attachement (low affinity, high Kd) by strong cumulative attachement (high avidity)
50
Q

What are the definitions of avidity and affinity?

A

Affinity → interaction of 2 components (Kd) → low for cadherins
Avidity → accumulated strength of multile affinities → high for cadherins

51
Q

When do the cells in the mouse embryo start expressing E-cadherin? What is the effect?

A

~ 8 cell stage → cells stick together much more strongly, close adherence between cells

52
Q

How does expression of different classical cadherins vary in the embryonic mouse brain?

A

In nerve tissues → many different cadherins with distinct, overlapping expression patterns → roles in synapse formation and stabilization
E-cadherin → localized mainly in the
R-cadherin → Forebrain + spinal cord
Cadherin-6 → Sides of spinal cord and forebrain

53
Q

What is the importance and specific characteristics of protocadherins (non-classical)?

A

They differ in their N-terminus, but are all identical in their C-terminal
Gene organized as a protocadherin gene clusters
In the cluster the variable exons are each preceeded by their own promotor → 1 variable exon/protocadherin + constant region for all of them
Variable exons encode for the extracellular domains, 3 constant exons encode for the intracellular domain (constant because interaction with actin filaments for all)

54
Q

What is the effect of a mutation in Cldn-14 and tricellulin

A

non-syndromic deaffness (DFNB29)

55
Q

You have cells on a plate bound to a support. How would you treat these cells to detach from the plate, to resuspend them?

A
  1. EDTA chelates Calcium
  2. Add trypsin (protease) → denatures cadherins?
56
Q

What is the process of transport of glucose from the blood the the lumen of the gut?

A

*In invertebrates
Glucose concentration: Lumen/low → Cell/high → Blood/Low
Na+ concentration: Lumen/high → Cell/low

Glucose is transported from lumen of the gut by Na+ driven glucose symport → to the blood down the concentration gradient through passive glucose carrier protein

Tight junctions between cell → not diffusion of glucose from cell to cell, tight junctions are between tip of microvili and intercellular space → increases basolateral surbace of ECF for more glucose diffusion

*Tight junctions important for controlled transport through channels/transporters only, through cell

57
Q

What are the anchoring proteins for each anchoring junction?

A

Adherens junctions: p120, a-, b-catenins, vinculin

Desmosomes: Plakoglobin, desmoplakin, plakophilin

Focal adhesions: a-actinin, filamen, talin, vinculin

Hemidesmosomes: plectin

BIOC 212 (15 decks)

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