Lecture 18

Question 1

structural composition of ECM

Answer 1

• fibrillar proteins: collagen, elastin
• specialized (adhesion) proteins: fibronectin, laminin
• ground substance: proteoglycans

Question 2

collagens

Answer 2

• multiple types with multiple functions
• mostly structure and tension
• alignment leads to tensile strength (think Ehlers-Danlos)
• highly interactive with other ECM molecules -> organize connective tissues
• very stable (some forms have been reported to remain after 10 years)

Question 3

elastin function

Answer 3

• elasticity -> ability (and force) to return to original shape
• a folded protein is in its lower free energy state
• crosslinks and anchors force the elastin network to extend (change conformation)
• extended proteins want to return to low energy position (folded) -> elastic force
• more elastin fibers -> the more the tissue wants to go back to its shape (example - arteries and other BV)
• elastin proteins do NOT need chaperones -> natural folding gives the force to return to original position

Question 4

α1-antitripsin deficiency

Answer 4

• α1-antitripsin not blocking the elastase (released by neutrophils) -> elastase breaks down the elastin fibers in the tissues
• loss of elastic tissue decreases the recoil in the lungs and the force to push the air out is lost -> lung tissues break down leading to a type of emphysema
• lungs need elasticity -> emphysema develops and patients experience shortness of breath and decreased oxygenation that worsens

Question 5

shock amortization by GAGs

Answer 5

• highly negative charges attract cations (mainly Na²⁺) and water
• generates a balance between chemical forces pulling water in and physical forces pushing water out (competition)
• physical compression displaces the water, allowing it to function as a hydraulic cushion
• once force is removed, the joint rehydrates and recovers
• NO covalent or irreversible change -> simply hydration forces regulating cushioning

Question 6

osteoarthritis

Answer 6

• degenerative condition of joint cartilage (most often knees and hips, but any joint may be affected)
• friction/damage leads to inflammation which leads to more damage
• leads to removal of cartilage and friction between bones -> painful
• new therapies using glucosamine sulphate and chondroitin sulfate (oral GAGs) have promising results in reducing pain and advancement of disease
• pro-water -> increase cushioning in joint and reduce the inflammation cycle

Question 7

classes of cell adhesion molecules (CAMs)

Answer 7

cell-matrix and cell-cell adhesion
1. IgCAMs -> intracellular domains folded LIKE an immunoglobulin domain (similar folding structure, NOT related)
2. CAD (cadherin) domains -> bind the same class of cadherin on a neighboring cell; Ca²⁺ stabilizes interaction; cytoplasmic tails bind cytoskeletal adapter proteins
3. integrins -> heterodimers of one α and one β subunit; bind a wide range of matrix (ECM) molecules
4. selectins -> Ca2+ dependent lectin (carb-binding) domain, an epidermal growth factor (EGF)-like domain, and a variable number of complement regulatory domains
5. mucins -> use multiple carbs for interactions with other cells
a. hydrations of side chain carbs allow them to stretch very far and reach other components
- anchored by a single transmembrane helix anchor
- adapter proteins link the cytoplasmic tails of most adhesion proteins to the actin cytoskeleton or to intermediate filaments

Question 8

principles of cell adhesion

Answer 8

1. Cell adhesion is determined by the selective expression of adhesion molecules
2. Many adhesion proteins bind ONE ligand, and many ligands bind only ONE type of receptor
   a. although some do not follow this
3. Adhesion can be regulated by surface density, state of aggregation, and state of activation of adhesion receptors.
   a. those with high levels of adhesion molecules bind with each other strongly and move to the center of the cluster (i.e. lipid rafts)
4. Ligand affinity is low, leading to fast dissociation and rapidly reversible interactions.
   a. molecules could not break interactions if affinity is high -> need to move along the surface
5. Many additional receptors connect with the cytoskeleton and transduce force and tension from the environment.
   a. provides signals to allow the cell to function properly based on where it is (like movement)
6. Ligand binding activates intracellular signaling pathways.

Question 9

immunoglobulin family

Answer 9

• extracellular immunoglobulin domains anchored to plasma membrane by a single transmembrane helix
• C-terminus is cytoplasm and interacts with intracellular ligands, including tyrosine kinases, PDK domains (in cytoskeleton), and adaptive proteins (with nectin to actin filaments)
• IgCAMs are used in neural development for synapse (guide synapse orientation) and myelin sheet formation
• ICAM-1 interaction with blood cell integrin is needed for adhesion and extravasation in response to inflammation
• tyrosine kinases in CD lymphocytes involved in immune activation

Question 10

cadherin family

Answer 10

calcium dependent adhesion proteins
* most are homophilic -> bind to same cadherin on neighboring cell (bind the same type -> like-cells)
* other are heterophilic -> bind to other types of cadherin (bind to different cell types -> like in epithelium binding cells below)
* specialized cell-cell junctions like desmosomes and adherens junctions in epithelial and muscle cells -> RICH in homophilic cadherins

Question 11

cadherin structure

Answer 11

• characterized by CAD domains that require Ca²⁺ to form rigid rods (Ca²⁺ allows chain to be stable and stretch)
• some have ICS (intracellular cadherin segments) that link with catenins to interact with actin filaments or with intermediate filaments

Question 12

cadherin signaling

Answer 12

• contact inhibition: normal cells sense they are close to other cells and shift out of cell cycle (stop dividing)/stop migrating
• E-cadherin mutations can contribute to formation of malignancy -> cancer forms b/c cells keep dividing
• B-catenin regulates cell proliferation and is inhibited by the Wnt pathway -> leads to digestion of B-catenin (which binds to cytoplasmic tail of cadherins)
• cadherin RET is a TKR; over-activation by mutations that cause dimerization causes cancer and deficiency causes Hirschsprung disease in which autonomic nerves in the wall of the intestines fail to develop
• balance is important!

Question 13

cadherin functions in embryo development

Answer 13

when embryo starts differentiating, cells with same type of cadherins clump together -> remain together and prevent foreign cells (those for other tissues) from growing (those destined to the same tissue type/organ clump together based on cadherins)

Question 14

integrin family

Answer 14

• main cellular receptors for ECM molecules, but some bind adhesion molecules on other cells
• bind epithelial and muscle cells to laminin in the basal lamina (membrane), providing the physical attachments necessary to transmit internal forces to the matrix and to resist external forces
• interactions of ligands with integrins and mechanical forces also generate signals that control cell growth and structure
• ex: allow endothelial cells to stay on surface of BV despite shear forces of blood flow

Question 15

integrin structure

Answer 15

• one ligand (intracellular and extracellular) increases affinity for the other site
• intracellular binding primes for extracellular ligands and vice versa
• α and β domain interact with each other, but are NOT covalently bound
• when there is no ligand inside OR outside, it decreases their affinity on both sides
• when they bind something on the inside, they stretch to want to bind something on the outside (AND VICE VERSA)

Question 16

integrin extracellular ligands

Answer 16

• approximately 1/3 of matrix ligands for integrins involve the sequence motif RGD or other simple sequences in otherwise unrelated proteins -> can bind multiple ligands
• for example, fibronectin binds to at least 9 different integrins and laminin, Von Willebrand factor bind at least 5 differnt
• affinity for ligands is low, allowing for fast dissociation and cell movement

Question 17

integrin intracellular ligands

Answer 17

• integrins cluster together in focal contacts
• adapter proteins talin and vinulin link conserved binding sites on the cytoplasmic domains of B-integrins directly to actin filaments at the ends of stress fibers
• activation of talin by PIP2 induces extension of integrins to facilitate extracellular binding
• paxilin is an adaptor for signaling proteins, such as SRC protein kinases and focal adhesion kinases

Question 18

outside-in signaling from integrins

Answer 18

• in seconds -> tyrosine kinases phosphorylate several focal adhesion proteins which has a central role in transducing these signals (ACTIVATION)
• in a minute -> cells raise Ca2+ conc high enough to initiate calcium-dependent processes
• in a few minutes -> ligand binding activates Rho-family GTPases that stimulate actin assembly and spreading of the cell on the ligand-coated surfaces (cell starts migrating)
• integrins cluster in “focal complexes” at the leading edge and anchor actin filament stress fibers to the cell membrane and the membrane to the substrate (allows for migration of the cell)
• contraction of stress fibers applies tension to the focal contacts, which remain stationary causing the cell to advance past them
• after hours -> activation of RAS/mitogen-activated protein kinase pathway turns on expression of selected genes, leading to differentiation during development

smooth muscle contraction from integrins

Question 19

inside-out signaling to integrins

Answer 19

• cells fine-tune their interactions with matrix molecules by regulating the activity of cell-surface integrins
• example: integrins on WBCs and platelets require “inside-out” activation before they can bind their extracellular ligands
• activation also regulates cellular interactions during development
• cytoplasmic proteins, talin and kindlins, activate integrins by binding the cytoplasmic tail of the B-integrin and separating the two transmembrane domains
• cells can mobilize integrins stored in cytoplasmic vesicles within minutes in response to chemo-attractants

Question 20

selectin family

Answer 20

• bind to glycoproteins and mucins
• particularly important in immune cell migration and embryo implantation
• allow activation of cells for integrins to lead transendothelial migration

Question 21

mucin family

Answer 21

• primarily bind to selectins
• abundant negative charges in their glycan groups allow them to extend far from the cell surface