Animal Connective Tissues

• ECM is a matrix of proteins and proteoglycans that surrounds tissues and contains multiple structures.
• Basal lamina underlies epithelial tissue, with connective tissue beneath it.

Extracellular Matrix of Animal Connective Tissues

• Basal lamina serves as an instance of extracellular matrix common to multicellular animals and an essential aspect of epithelial tissues
• Extracellular matrix in connective tissues exhibits varied and bulky forms
• The diversity of tissues arises from variations in relative amounts and organization of the macromolecules constituting the ECM
• ECM can calcify into bone or teeth, become the transparent substance of the cornea, or form the rope-like organization of tendons
• Cells within the ECM produce and orient it, aiding in matrix organization through cytoskeleton orientation
• Matrix molecules get secreted by fibroblasts.
• Fibroblasts differentiate into chondrocytes in cartilage and osteoblasts in bone

Key Components Building Connective Tissue

• The matrix is constructed from two main classes of macromolecules, as in basal lamina.
• Glycosaminoglycan (GAG) polysaccharide chains are covalently linked to proteins, forming proteoglycans.
• Fibrous proteins such as collagen exist.
• Polysaccharides resist compressive forces while allowing rapid diffusion of nutrients and hormones.
• Collagen fibers strengthen and organize the matrix, while elastin provides resilience

Collagens

• All multi-cellular animals contain this major protein of the extracellular matrix
• Mammals contain ~25% collagen
• Connective tissue cells secrete in large quantities, other cells secrete in smaller quantities
• 42 genes code for collagens, but less than 40 collagen molecule types have been found
• Type I collagen, the primary collagen in skin and bone, features rope-like fibrillar structures
• Collagen molecules assemble into higher-order polymers called collagen fibrils after secretion

Collagen Structure

• The primary feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure.
• Three collagen polypeptide chains called α chains wind around one another in a rope-like superhelix.
• Collagens contain extremely high levels of glycine (every 3rd amino acid)
• Collagen coils tightly due to Glycine's simple structure, lacking steric hindrance from side chains.
• Collagen polypeptide chains can form a triple-stranded, helical procollagen after being trimmed, hydroxylated, and glycosylated in the rough ER from membrane-bound ribosomes
• Hydroxylysines and hydroxyprolines are infrequently found in other animal proteins, the location helps stabilize the triple-stranded helix.
• Hydroxyl groups facilitate hydrogen bonding, stabilizing the triple-stranded helix

Elastin

• Elastin gives elasticity to tissue
• Many vertebrate tissues, including skin, blood vessels, and lungs, require both strength and elasticity
• Elastic fibers network in the ECM, providing resilience for recoil after stretch which can be 5x more extensible than rubber
• Non-elastic collagen fibrils intertwine with elastic fibers to mitigate stretching and tearing
• Elastin comprises 50% of the aorta by dry weight
• Elastin, like collagen, is rich in proline and glycine, yet it isn't glycosylated, although it does have hydroxyproline but not hydroxylysine.
• Soluble tropoelastin gets secreted into the extracellular space before assembling at the plasma membrane to form elastic fibers.
• After secretion, tropoelastin gets crosslinked to one another between lysines.
• Elastin contains hydrophobic and alanine- and lysine-rich segments, the prior being key in elastic properties, the latter for cross-links

Fibronectin

• Fibronectin gets located outside of the cell to allow the cell to attach to the matrix.
• The ECM contains non-collagen proteins to perform the function of cell-surface receptors and macromolecules
• A glycoprotein found in all vertebrates, vital for cell-matrix interactions is fibronectin
• Mutant mice lacking fibronectin cannot form proper blood vessels die early in embryogenesis.
• Fibronectin dimers are composed of 2 large subunits, with 50 exons each
• Subunits of fibronectin are composed of 50 exons and join by disulfide bonds at one end
• Alternative splicing produces various isoforms from a gene, which each fold into functionally distinct domains.
• The domains of fibronectin monomers consist of self-association, collagen binding, cell binding, and heparin.
• Fibronectin exists in a soluble circulating form or as insoluble fibrils cross-linked by disulfide bonds in the ECM.
• Fibronectin assembles into fibrils on cell surfaces with appropriate binding partners through integrins
• Integrins provide a linkage from fibronectin outside a cell to the actin cytoskeleton inside.

Cell Matrix Degradation

• Cells need degradation for matrix division and travel
• White blood cells require the matrix to cross the basal lamina
• Degradation is exploited by cancer cells to spread through the body
• Cells use extracellular proteolytic enzymes (proteases) that act close to the cells to degrade matrix components

Proteases

• The two main classes of proteases involved in matrix degradation include matrix metalloproteases for Cat2+ and Znt2+, and serine protease
• Some proteases are highly specific, cleaving particular proteins at a few sites such as collagenases, while others are less specific but act only where needed through plasma membrane anchoring
• Controlling Breakdown of ECM components happens via: specific protease that identifies ECM and location through anchored protease

Control Mechanisms for Proteases That Degrade Matrix

• Local activation happens through precursor activation of proteases when needed
• Plasminogens activate plasmin breaking blood clots with plasminogen activators
• Many cell-surface receptors confine enzymes where needed, urokinase plasminogen activator is a confined one for migrating cells.
• Secretion of inhibitors occur by tissue inhibitors of metalloproteases and serine protease inhibitors (serpins)

Signalling from Cell-Extracellular Matrix (ECM)

• Cells make, organize, and degrade the extracellular matrix
• The matrix significantly impacts cells through transmembrane proteins that act as matrix receptors
• Components of the ECM can affect cellular behavior through transmembrane cell adhesion protein receptors

Integrins

• Principal animal receptors of ECM proteins
• Integrins bind laminins and fibronectins.
• Transmembrane adhesion molecules possess the ability to transmit signals in both directions across the cell membrane.
• Transmembrane heterodimers composed of integrins bind to the cytoskeleton
• The ECM is attached to integrins, which are associated with the cytoskeleton
• Anchorage is required for cellular migration
• An integrin molecule consists of two non-covalently associated glycoproteins called α and β.
• The extracellular portion binds to ECM proteins such as laminin or fibronectin, or to ligands on the surface of other cells.
• The intracellular portion binds to actin, via the protein talin and a set of anchorage proteins.
• Integrins can switch between an active and an inactive conformation to make and break attachments, especially when travelling
• Assembly and disassembly of cytoskeletal attachments mediates coupling inside the cell
• Allosteric regulation underlies these phenomena

Integrins in Action

• Integrins undergo conformational changes affecting both intracellular and extracellular ends when binding
• Signals for integrin signalling: outside-in and inside - out
• Outside-in signalling occurs through the binding to RGD which reveals the talin, so the integrins can bind to the actin cytoskeleton
• Binding to the fibronectin outside of the cell transduces the signal to the cell itself
• For inside-out activation to occur, a signal from an RTK activates PIP2 to transduce and open Talin by changing its structure such that it binds to an integrin
• A conformational change in the integrin allows it to activate the outside and bind the ECM.

Additional Integrin Information

• Integrins in white blood cells and platelets regulate activity through inside signaling
• Rapid signaling stems from pre-existing integrins
• Integrins exhibit lower affinity but greater abundance (10-100 fold), clustering to form adhesions
• Integrins activate signalling pathways to allow behaviour according to matrix.
• Anchorage-dependence is exhibited in cultures where cells require attachment to ECM
• ECM attachment through an integrin pathway mediates cell proliferation and survival, initiating apoptosis when contact is lost

Cell Extracellular Matrix Experiment

• Cells forced to spread out survive better
• In an experiment, the placement of the growth affects anchoring which leads to more or less growth

Integrins and Signalling Proteins

• Integrins recruit intracellular signalling to cells: focal adhesion kinase (FAK),
• FAKs are recruited by talin and clustered. Cross-phosphorylation creates docking sites for Src family which help anchor tyrosine kinases
• Outside-in signalling travels through FAK and anchors, where phophorylation regulates adhesion
• Mice lacking FAK adhere through fibronectin however, too many focal adhesions are formed, preventing the cell from migrating
• FAK helps disassemble focal adhesions

Animal connective tissues are surrounded by a matrix of proteins and proteoglycans. Basal lamina underlies epithelial tissue. The diversity of tissues arises from variations in relative amounts and organization of the macromolecules constituting the ECM.