Extracellular Matrix Flashcards
What are the 2 different forms of direct binding between cells?
Cell-cell junctions
Cell adhesion
What difference type of tissues are found in the intestinal wall?
- Epithelium → sits on basement membrane
- Connective tissue (fibroblasts)
- Smooth muscles: Circular fibers, Longitudinal fibers (crossing each other making leaf)
- Connective tissue
- Epithelium
What are the 3 major classes of cell junctions (and their main function)?
- Occluding junctions → Seal cells together in an epithelium, no leaking from one side to the other
- Anchoring junctions → Mechanically attach cells to the neighbours or to the extracellular matrix
- Communicating junctions → Mediates the passage of signals from one cell to the next
What are the 2 types of occluding junctions?
- tight junction (vertebrates only)
- septate junctions (invertebrates mainly)
What are the different types of anchoring junctions?
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)
What are the 3 types of communicating junctions?
- Gap junctions
- Chemical synapses
- plasmodesmata (plants only)
What organisms/organelles can you see with a light microscope?
Light wavelength ~ 400-700 nm
- Chloroplast (1um)
- Most bacterias (1-10 um)
- Plant/animal cells (10-100 um)
- Fish egg (> 1mm)
What is the range of molecules you can see with an electron microscope?
<1 nm - 100 um
- small molecules (< 1nm)
- lipids (~2-4 nm)
- proteins (5-10 nm)
- T2 phage (70-80 nm)
- Chloroplast (1 um)
- Most bacteria (1-10 um)
- Plant/Animal cells (10-100 um)
*um = micron
How does an eletron microscope work?
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
What is Osmium tetroxide?
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
How can we use a tracer to visualize tight junctions?
- Inject a tracer molecules in gut lumen
- Follow the molecules → goes in microvilli
- Tracer molecule stops at the tight junction
*Occulins and Claudins bind to hold adjacent plasma membranes together with intercellular spaces between seals
What are the tight-junction proteins formin the sealing strands (and their properties)?
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
What phenotype does an Occludin deficient mice show?
- Chronic inflammation, gastric epithelium calcification → not easily explained by barrier function
Occludins might be involved in epithelial differentiation
What protein is responsible for anchorage to the cytoskeleton of claudin and occludin?
ZO proteins = Zonula Occludens → bind to actin filament
*Tight junction complexes connect to actin filaments
What is the molecular weight of 1 amino acid?
0.11 kDa ~ 100 Da
What are the 3 major types of cytoskeleton fibers?
- Actin filaments → made out of actin → involved in tight junctions
- Intermediate filaments → made out of Keratin, Vimentin, Desmin → involved in tight junctions
- Microtubules → made out of tubulin → not involved in tight junctions
What is the impact of a mutation on Cldn-1 gene (claudin gene)?
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
What is the impact of a mutation on Cldn-19 gene (claudin gene)?
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
What is the impact of a mutation on ZO-2 gene (ZO-protein gene)?
Familial hypercholamenia (FHC) → elevated serum bile acid concentrations, itching and fat malabsorption
*Tight junction-associated hereditary disease
Why is it important to have anchoring junctions?
- Cell is flimsy
- Transmission of force
- Tension bearing cytoskeleton
*Anchoring provides stability to the cell
In what tissues are anchoring junctions more abundant?
Widely distributed in animal tissues → most abundant in muscle, heart, epidermis, epithelia (intestinal, skin cells)
What are the 2 main protein classes involved in anchoring junctions?
- TM adhesion proteins:
- Cytoplasmic tail + TM + extracellular domain
*The extracellular domain can bind to another TM adhesion protein or the ECM - Intracellular anchor proteins:
- connect to actin and intermediate filaments
What anchoring junctions are responsible for Cell-cell vs Cell-ECM interactions?
(Which proteins?)
- Cell-cell:
- Adherens junctions → joins actin filaments of neighbouring cells together
- Desmosomes → joins intermediate filaments together (stronger)
→ CADHERINS as the TM adhesion protein - Cell-ECM:
- Focal adhesions → joins actin filaments to ECM
- Hemidermosomes → joins intermediates to ECM
→ INTEGRINS as TM adhesion proteins
What are all the proteins involved in Adherens junctions (and their properties)?
- Intracellular anchor proteins (adaptor proteins): catenins (alpha, beta), vinculin
- 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
What is the organization and importance of actin filaments in adherens junctions
*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
What allows folding of the epithelial sheet in embryogenesis (What junctions/organization)?
- 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 - Epithelial tube piches off from overlying sheet of cell → neural tube formation for example (1st structure of CNS)
What system provide contractility to non-skeletal muscle cells (ex: epithelial cells)?
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
What are the intermediate filament proteins involved in formation of desmosomes?
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
What proteins of the cadherin family are involved in desmosomes anchoring junctions?
*Cadherin family → extracellular, TM, intracellular domains, dimerize and interact with cadherins of neighbouring cells
- Desmoglein
- Desmocollin
What is the structure of desmosomes anchoring junctions (different poteins and their functions)?
- At the surface of the cell → formation of a dense cytoplasmic plaque made of intracellular anchor protein → Desmoplakin + Plakoglobin + Plakophilin
- Keratin filaments (IF) are anchored to cytoplasmic plaque (laterally) → bind to desmoplakin
- Cadherins: Desmoglein + desmocollin → dimerize and interact with neighbouring cells
Cytoplasmic tails of cadherins bind plakoglobin (g-catenin) → binds desmoplakin → binds intermediate filaments
What is Pemphigous vulgaris?
*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
What is the definition of a homophillic interaction?
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)
What are the MW and characteristics of plakoglobulins and desmoplakins?
*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
What diseases can be caused by mutations in plakoglobin and desmoplakin (in humans)?
Cardiomyopathy
Skin diseases
What are focal adhesions? (proteins involved)
Achoring junctions attaching actin filaments in a Cell-ECM fashion
- Integrins interact with ECM as heterodimers:
- 18 alpha subunits + 8 beta subunit → 24 integrin heterodimers (alpha and beta are TM proteins) - alpha-actinin, talin or filamin binds the intracellular tail of the beta-subunit + binds vinculin
- Actin filament are attached to integrins via a-actinin, filamin, talin, vinculin
How does immunofluorescence imaging differ when visualizing Actin and Vinculin between WT and integral alpha subunit KO?
*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
What proteins are involved in hemidesmosomes?
*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)
What is Epidermolysis bullosa?
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
What are the TM adhesion proteins responsible for the different anchoring junctions?
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
What molecules can and cannot pass freely through Gap junctions?
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
What is the structure and the composition of Gap junctions?
- 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
What is the structure/characteristics of connexins?
- 4 pass TM proteins
- 21 different connexins → different genes, different tissue distribution
- 20-60 kDa (humans)
*involved in Gap junctions
How does Gap junction regulation occur?
Why is it important?
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+
What are intracellular and extracellular physiological concentrations of calcium?
Intracellular = 0.1 uM
Extracellular = 2 mM (ex: blood, ECF)
What are the functions of Gap junctions?
- Signals spread rapidly (nerve, muscles)
- Electric coupling invertebrates → synchronization of contractions of heart muscle cells and smooth muscles (intestine)
- Non excitable cells → sharing of the small metabolites and ions
Ex: in the liver, noradrenaline/glucose/glycogen shared between cells
What are large and small Gap junctions?
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
What is the structure of classical cadherins? By which technique can it be visualized?
- 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
What is the importance of calcium in cadherins?
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
What characterizes the binding of cadherins with each other in cell-cell adhesions?
- 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)
What are the definitions of avidity and affinity?
Affinity → interaction of 2 components (Kd) → low for cadherins
Avidity → accumulated strength of multile affinities → high for cadherins
When do the cells in the mouse embryo start expressing E-cadherin? What is the effect?
~ 8 cell stage → cells stick together much more strongly, close adherence between cells
How does expression of different classical cadherins vary in the embryonic mouse brain?
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
What is the importance and specific characteristics of protocadherins (non-classical)?
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)
What is the effect of a mutation in Cldn-14 and tricellulin
non-syndromic deaffness (DFNB29)
You have cells on a plate bound to a support. How would you treat these cells to detach from the plate, to resuspend them?
- EDTA chelates Calcium
- Add trypsin (protease) → denatures cadherins?
What is the process of transport of glucose from the blood the the lumen of the gut?
*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
What are the anchoring proteins for each anchoring junction?
Adherens junctions: p120, a-, b-catenins, vinculin
Desmosomes: Plakoglobin, desmoplakin, plakophilin
Focal adhesions: a-actinin, filamen, talin, vinculin
Hemidesmosomes: plectin
