•Last Class: Cytoskeleton

•1. Cytoskeleton components: –Microtubules–Actin Filaments–Intermediate Filaments

•2. The regulation of microtubules and Actin • filaments•3. Molecular Motors•4. Cell behaviors related to cytoskeleton

• Cell Adhesion on Extracellular Matrix (ECM)

Cells surrounded by ECM (macromolecules consisting of proteins and polysacchrides)

Embryonic chick limb bud

Connective Tissue underlying an epitheliumFibroblast, primary cell secreting ECM

Fibroblasts in Connective TissueWith Collagen fibers

(SEM image of rat cornea, no elastic fiber existing, hyaluronan, proteoglycans and glycoproteins have been removed by enzymes

and acids)

ECM

• Glycosaminoglycans (GAGs) covalently linked to protein, proteoglycans forming gels

• Fibrous proteins: collagen, elastin, fibronectin, Laminin.

The repeating disaccharide sequence of a GAGSulfate, carboxyl groups, negative charges

GAGs

• Hyaluronan• Chondroitin sulfate and dermatan sulfate• Heparan sulfate• Keratan sulfate

The relative dimensions and volumes of different

moleculesVery stiff and hydrophilic,

can’t be packed

Negative charge->cation, Na+ ->water molecules->swelling

pressure->compression resistent

The repeating sequence of hyoluronanSingle chain of 25,000 sugars; No sulfate

The linkage of GAG to protein, the assembly of proteoglycanStart from a serine, 4 saccharide and GAG

Except hyalurona

Diversity of ProteoglycansMembrane-bound ribosome-> endoplasmic reticulum ->golgi apparatus

Aggrecan aggregatesNon-covalently bound to hyaluronan chainsJust like decorin decorates collagen fibrils

Proteoglycan functions

• Constrain the range of embedded protein actions• Affect embedded protein activities• Control embedded protein release rate• Prevent embedded protein degradation• Control the local concentration of embedded

proteins• Function as co-receptors

Fibrous Proteins:

Collagen, elastin, fibronectin, laminin

Collagen MoleculesGlycine, proline, hydroxyproline

Triple helix (three fibers interwined together)

Embryonic chick skinFibroblasts surrounded by collagen fibrils (high order polymers)

Degraded fragment of XVII, endostatin, inhibits angiogenesis and hence tumor growth

Crosslinking of collagen fibrilsDeaminated by lysyl oxidase to yield aldehyde groups, spontaneous

covelent bond between aldehyde groups (about every 67 nm)If crosslink is broken, collagen is easy to tear

The Life of Collagen fibril

Collagen fibrils form collagen fibers

Fibril-associated collagen helps the organization of fibrils to resist tensile force.

Collagen fibrils in the tadpole skin. Tendon and bone are different

Cells help the organization of collagen fibrilsTwo embryonic chick hearts at the ends and collagen gel in the center

Hearts full of fibroblasts and heart muscle cells

Elastic Fibers consisting of Elastin (for tissue elasticity)Vessel Walls: EM image of dog aorta

Stretching of a network of elastin moleculesElastin is highly hydrophobic

Mainly two features: hydrophobic and cross-linked segmentsHydrophobic segments provide elasticity

Cross-linking provides stability

Fibronectin (crucial for angiogenesis)A glycoprotein dimer connected by disulfite bonds at one end

Can exist in soluble or fibrillar forms

Co-alignment of extracellular fibronectin fibrils and intracellular actin filament bundlesRed: fibronectin fibrils

Green: actin filament bundlesNot only important for adhesion, but also for migration

Laminin(consisting of three polypeptides , , )

3 kinds of Basal LaminaeA thin, flexible mats underlying all epithelial cells and tubes

Not only for structural support and filtering, but also determine cellular functions

Basal Laminae in the cornea of a chick embryoUsually synthesized by the cells seeded on it

Most laminar consists of type IV collagen, laminin and nidogen (enactin)

A molecular structure Model of basal Laminae

Function of Basal Laminae in neuromuscular junctions (besides the supporting or filtering functions)

Muscle cells: laminin; neuronal cells: agrin (heparan sulfate proteoglycan)

Cells communicate through ECM to affect Cell shape

ECM affects Cell survival and proliferation

Cancer Cells and ECM DegradationMatrix metalloproteinase, serine proteaseProteases bound on cell surface receptor

Controlling of protease activity: 1. local activation; 2. surface receptor binding; 3. inhibitor secretion

tPA: tissue-type plasminogen activator; uPA: urokinase-type plasminogen activator

ECM Receptors

IntegrinsTransmembrane heterodimers

20 nm above cell surface, dependent on Ca2+ and Mg2+

Integrins couple ECM to cytoskeleton through cytosolic

proteins, talin, -atinin, paxillin, filamin, vinculin

Inside-out Signaling for Integrins

Outside-in Signaling of integrinsFAK, a key molecule for integrin functions

FAK can bind to Talin which associates with integrin subunit, to paxillin which associates with integrin subunit

Matrix-dependent cell survival in the formation of proamniotic cavityDuring embryonic development

•Summary•1. ECM components: glycosaminoglycans (GAGs) and fibrous proteins

GAGs: Hyaluronan; Chondroitin sulfate and dermatan sulfate; Heparan sulfate; Keratan sulfate

Fibrous proteins: collagen, elastin, fibronectin, laminin•2. basal laminae•3. Cell-ECM interaction, ECM degration•4. ECM receptor, integrins