D103 ECM (ALS 7, Videos 12 and 13)

function of multi-domain proteins

organize the ECM

• binds to multiple other proteins via specialized binding domains

how do you predict a FRAP curve for fibronectin to look

there is a lot of crosslinking, meaning they want to stay together

proteins are not very mobile

what is #1 

collagen

what is #2

fibronectin

what is #5

integrin

outside-in integrin activation

ecm ligand binding activates integrin and intracellular events

inside-out integrin activation

actin-associated protein binding activates integrin and extracellular events 

bidirectional regulation

binding of ligand/adaptor on one side of the membrane induces conformational change on the other side 

collagen 

central component of the ECM 

• long sturdy, rope structure

• many variations of collagen

• assembled in ER in a trimeric lattice structure

fibroblasts were cultured on dishes coated with different ECM proteins. aftr 2 hours, the average cell spreading area was measured using fluorescence microscopy and image analysis. which of the following conclusions is best supported by these data?

cell spreading is greater on ECM proteins

• ECM proteins include collagen, fibronectin, and laminin

epithelial cells

adhere to their neighbors and the basal membrane

tight juntion

seals gap between epithelial cells 

adherens junction

connects actin filament bundle in one cell with that in the next cell

desmosome

connects intermediate filaments in one cell to those in the next cell

gap junction

allows the passage of small water-soluble molecules from cell to cell 

hemidesmosomes

anchor intermediate filaments in a cell to ECM

actin-linked cell-matrix adhesion

anchors actin filaments in cell to ECM

integrins 

connect cell to the ECM 

• ex: actin-linked cell-matrix adhesion; hemidesmosomes

• cell-matrix anchoring junctions 

cadherins

connect adjacent cells

• ex: tight junction, adherens, desmosome, gap junction

ex of occluding junction

tight junction

cell-cell anchoring junctions

adherens and desmosomes 

ex of channel-forming junctions

gap junctions

in which buffer would you carry out this cell sorting experiment

CaCl2

• Ca2+ binding induces conformational changes

• Ca2+ binding proteins: tryponin and calmodulin

e-cadherin and cancer: loss of function mutations of e-cadherin correlate with increased invasiveness and metastasis of tumors. which of the following describes the most direct consequence of this mutation 

neighboring cells can no longer adhere to each other 

• most direct consequence 

core matrisome

encoded by collagens, proteoglycans, and non-collagenous glycoproteins

genes encoding matrisome associated proteins: ECM-afiliated, ECM regulators, secreted factors

adaptors between actin and ecm

vinculin, talin, kindlin

assist in protrusion

cell migration involves the maturation of adhesion sites

how does a cell look in which the GAP of Rho is mutated so that it cannot function 

full of stress fibers 

which of the following protein-protein interactions are critical for attachment of this migrating cell to the substratum

integrin binding to fibronectin

which of the following factors does NOT contribute to cell migration

cadherin

ecm

a collection of extracellular molecules secreted by fibroblasts within ECM including many insoluble fibers

• varies between different tissues

• provides structural support for the surrounding cells

ecm formation

synthesized and secreted by fibroblasts (within ecm)

remodeled and degraded by proteases in the ecm

reason for specialized properites of the ecm 

differential enrichment of ecm components 

main components of ecm

1. collagen (most abundant)

2. elastin

3. gags (glycosaminoglycans)

4. proteoglycans

5. multi-adhesive proteins (fibronectin, laminin)

osteogenesis imperfecta

brittle bone disease due to a mutation in Gly in COL1A

why is collagen processing necessary 

necessary for its assembly and function 

• intracellular: vitamin C (ascorbic acid) as a cofactor for lysine and proline hydroxylation → scurvy 

• extracellular: collagent fibril and cross-striations 

elastin

essential for tissue elasticity

• critical component of tissues that experience mechanical stress (ex: blood vessels, skin, and lungs)

elastin monomer

proline and glycine-rich hydrophobic domain → able to extend and recoil

• monomers are crosslinked via Ala and Lys-rich alpha-helical domains

cutis laxa 

genetic disorder where there is little skin elasticity bc no elastin 

composition of glycosaminogycans (GAGs) and proteoglycans

repeating diassacharides

• one sugar: GlcNAc or GalNAc

• other sugar: D-glucoronic acid, L-iduronic acid or D-galactose

what can GAGs be linked to

proteoglycan as the core protein

• unlike glycoproteins, where there is more protein than sugar bound in the complex

purpose of multi-domain proteins 

organize the ECM 

• binds to multiple other proteins via specialized binding domains (ex: RGD)

• fibronectin and laminin

fibronectin

forms homodimer that is joined by disulfide bonds

used in lab to coat tissue culture dishes

laminin

heterotrimer (has 3 different chains)

laminin a2 deficiency 

congential muscular dystrophy 

collagen overview

triple helix made up fo 3 collagen alpha-chains, large protein with complex processing during its secretion

elastin overview

prominent ECM component, makes up elastic fibers that can recoil after a stretch

• 2 repeating domaints (Ala and Lys rich alpha helices → crosslinking; hydrophobic domains which provide the elastic properties)

GAGs

large repeated disaccharide chains with highly extended conformation

• the presence of charged sugars leads to hydrophilic character

• can withstand compressive forces

proteoglycans

95% of weight of those porteins are sugar (GAGs)

• several classes that differ in the comp of disaccharide portion (GAGs)

• stiff, hydrophilic molecule → attracts water → gel like appearance

multi-domain proteins (fibronectin and laminin)

contain specific bindings sites for multiple other proteins; provides binding sites for cells during migration

• all components are secreted by fibroblasts in the ECM

when do cells adhere to each other

during early embryonic development

result of loss of e-cadherin function

cells did not adhere and resulted in early embryonic death

• suggests a role for e-cadherin in cell adhesion and embryonic development

cadherin-mediated sorting out of cells

layers separate as development progresses 

cells are segregated from each other because they: 

• express different cadherins 

• express different levels of the same cadherin (sort based on the amount of cadherin present) 

calcium and cadherin

cadherin undergoes a Ca2+ induced conformational change 

• each cadherin (located extracellularly) has a Ca2+ binding site 

desmosomes vs adherens junctions

both are cadherins that link the cytoskeleton of neighboring cells

• homotypic binding to cadherins of the neighboring cell

desmosomes: structural support of great tensile strength; link via intermediate filaments

adherens junctions: adhesion/ contractile belt; link via f-actin

pemphigus

autoimmune disease that affects ability to generate antibodies against desmosomal cadherins

• causes skin blistering and leakage of body fluids into epithelium

• affects cadherin function

purpose of integrins

link the cytoskeleton to the ecm 

• a family of transmembrane proteins that form heterodimers (a and B subunit) 

• at least 24 different family members 

• connect cytoskeletal elements (f-actin/ intermediate fil) to the ECM indirectly via adaptor proteins

• individual integrins cluster, which leads to high affinity binding

in outside-in signaling, what induces a cell to adhere to the substratum

ecm components (fibronectin) associates with integrin via RGD domain

• enhancement of the interaction thru integrin clusterin (leads to high affinity binding)

• cells become attached thru association of integrins with f-actin (formation of focal adhesions)

in inside-out signaling, what induces integrin activation and focal adhesion formation

actin polymerization activates integrins via actin-accessory proteins

• focal adhesion: protein complex that forms

adhesion proteins overview

TM proteins that participate in adhesion via an extracellular domain and that associate with the cytoskeleton via their intracellular domain 

cadherins overview

ca2+-dependnet conformational change, which promotes homotypic/homophilic interactions with neighboring cells

• adherens junctions (linkage of actin cytoskeleton → adhesion belt)

• desmosomes (linkage of intermediate filament → provides strength to cell layer)

integrins overview

form a heterodimer

• help motile cells to adhere to ECM (link between actin cytoskeleton and the ECM)

• tightly regulated conformational changes that are induced across the membrane

• inside-out and outside-in signaling