Three types of molecules are abundant in the extracellular matrix of all tissues:

為何ECM (GAG)可以保水??????????

GAG (---)→attaract cations (+)→

more concentrate → osmotic pressure

↑↑↑→ water income → keep in ECM

The extracellular matix (ECM)

Three types of molecules are abundant in the extracellular matrix of all tissues:

1. proteoglycan: a glycoproteins, high viscosity, it can bound variety of ECMs

2. Collagen fibers: provide mechanical strength and resilience.

3. Soluble multiadhesive matrix proteins: bind to and cross-link cell-

surface adhesion receptors and other ECM components

Fibronectins connect many cells to fibrous collagens and other matrix components

Fibronectin = example of “adhesive” ECM protein

Is a protein with multiple domains with number of specific binding sites for macromolecules and for receptors on cell surface.

Plasma Fibronectin; soluble, circulates in the blood and other body fluids → enhances blood clotting, wound healing and phagocytosis

Fibronectin Fibrils; insoluble → assemble on surface of cells & deposited in ECM

Fibrils assemble on surface of cells via fibronectin-binding integrins.

Usually aligned with adjacent intracellular actin fibres.

Fibronectin is a dimer, two chains linked via disulfide bonding at the carboxyl terminus

Fibrin, heparan sulfate proteoglycan, and collagen:

– bind to distinct regions in fibronectin

– integrate fibronectin fibers into the extracellular matrix network

Some cells express integrin receptors that bind to the Arg-Gly-Asp (RGD) sequence of fibronectin.

At least 20 different forms of fibronectin have been identified.

– All of them arise from alternative splicing of a single fibronectin gene.

The soluble forms of fibronectin are found in tissue fluids.

The insoluble forms are organized into fibers in the extracellular matrix.

Fibronectin fibers consist of crosslinked polymers of fibronectin homodimers.

Fibronectin proteins contain six structural regions.

– Each has a series of repeating units.

cell

RGD is the letter

designation for the amino acids Arginine, Glycine and Aspartic Acid

協同區

FIBRONECTINS: attach cells to collagen matrices Regulates cell shape/cytoskeleton

Dimers of two similar polypeptides linked by disulfide bonds Forms fibrils when exposed to cells expressing intergrins

Circulating fibronectin can bind to fibrin, with the result that platelets associate at the site of blood clots via integrin binding

‹纖連蛋白是高分子量糖蛋白（220-250KD）

‹纖連蛋白模型

‹纖連蛋白的主要功能︰

—介導細胞黏著，進而調節細胞的形狀和細胞骨 架的組織，促進細胞鋪展；

—在胚胎發生過程中，纖連蛋白對于許多類型細 胞的遷移和分化是必需的；

—在創傷修復中，纖連蛋白促進巨噬細胞和其它 免疫細胞遷移到受損部位；

—在血凝塊形成中，纖連蛋白促進血小板附著于 血管受損部位。

Specific tripeptide sequence RGD is important role of the cell-bind region of fibronectin is require adhesion of cells

mutation

•Arg-Gly-Asp

•Cell binding region

•Type III repeat/binds integrins

Integrins mediate linkage between fibronectin in the extracellular matrix and the cytoskeleton

Green: integrin Red: actin

Stress fiber are long bundles of actin microfilamenht that radiate inward from points where the cell contacts a substratum

Adhesive interactions in motile and nonmotile cell

Integrins relay signals between cells and their 3D environment Integrins interact with ECM: regulation of integrin-mediated

adhesion and signaling controls cell movement

Human fibroblast Cell grown directly on the flat surface of culture dish (a)

On 3D ECM components (b)

Integrins cluster into adhesive structures with various morphologies in nonepithelial cell

Integrins

At least 12 distinct α- subunits and 9 β- subunits identified;

single subunit can associate with more than one partner

Difficult to crystallize and therefore 3D structures not readily available Cloning experiments

indicate that integrins have short cytoplasmic tail (~ 50 amino acids) and 4 Ca²⁺ binding sites identified in the a subunit

Numerous disulfide bonds- difficult identify all of them

Muscular dystrophy: connections between the ECM and cytoskeleton are defective

About 1/3300 boys, heat or lung failure

Mutation of dystrophin (a cytoslic protein), bind to dystroglycan

Dystroglycan: α-subunit (peripheral protein) plus β-subunit

(transmembrane protein); the a- subunit also has O-linked

oligosaccharides to bind various basal lamina components

DGC (dystrophin glycoprotein

complex) links extracellular matrix to the cytoskeleton and singling pathways enzyme for muscle’s function

Mutations in components of this pathway (e.g. muscular dystrophy) results in mechanical instability of muscle cells.

Duchenne muscular dystrophy (DMD) 裘馨氏肌肉萎縮症

X染色體

P21基因缺損(DMD)。

DMD基因會製造一種重要的肌肉萎縮蛋白

(Dystrophin)，此症患者的細胞內完全缺少肌肉 萎縮蛋白，肌纖維膜變得無力脆弱，經年累月伸 展後終於撕裂，肌細胞易死亡。

患者的肌肉隨著年齡的增長會逐漸退化消失。

Affects structure (DGC) involved in linking muscle cell to basal lamina This structure provides strength to cell membrane

disruption leads to weakening of mechanical stability of cell Affects cytosolic adapter protein-Dystrophin

Interacts with dystroglycan (glycoprotein

Model for integrin activation

X-ray crysal structure

(inactive form)

RGD

Ligand bind and induced conformational change

Diversity of ligand-integrin interaction contributes to number biological processes

Cell-matrix adhesion is modulated by changes in the binding

activity and neumber of integrins

Integrin superfamily 的黏著分子主要參與細胞與細胞外基質的黏附，使細胞 得以附著而形成integration。此外，這些分子還參與白血球與血管內皮細 胞的黏附。

這些分子都是由α 、β兩條鏈以非共價鍵連接組成的heterodimer。

α鏈的分子量為120～180kD, β鏈的分子量為90～110kD,不同的α鏈或β鏈胺 基酸組成和序列有不同程度的同源性，在架構上有其共同的特點。

α鏈和β鏈均由胞漿區、穿膜區、胞膜外區三部分組成。胞漿區一般較短，可 能與細胞骨架相聯。穿膜區富含疏水胺基酸。

α subunits和β subunits組合構成並不是隨機的，多數α subunits只能與一 種β subunits結合成heterodimer，而大部分β subunits則可以結合數種不 同α subunits。目前依β subunits的不同將與動脈粥狀硬化相關的integrin superfamily分為3個不同的組。

Integrin在與ligand結合時所識別的只是ligand分子中由數個胺基酸組成的序 列，例如Arg-Gly-Asp ( RGD )序列。不同的integrin可以識別相同的序列或 同一個ligand中不同的序列。

Integrins在體內分佈很廣泛，多數integrins可以表現於多種組織、細胞，如

VLA組的integrins在體內廣泛分佈于各種組織、細胞，而多數細胞可同時表 達數種不同的integrins。

vinculin Actin

(Fibronectin, laminin)

Focal adhesions

Laminin and fibronectin provide an adhesive

substrate for cells

The Binding of Cytoskeleton to the Extracellular Matrix Through

the Integrin Molecule

Many cell-matrix and some cell-cell interactions are mediated

by integrins

Immunoglobulin superfamily

Immunoglobulin Superfamily CAMs (ICAMs)

Ca2+ independent cell-cell adhesion in neuronal and other tissue is mediated by CAMs (IgCAM) in the immunoglobulin

superfamily

Movement of leukocytes into tissue depends on a precise sequence of combinatorially diverse set of adhesive interactions.

Movement across an endothelial cell layer of:

• Monocytes (macrophage precursors - cells that ingest foreign particles)

• Neutrophils (release antibacterial)

• T and B Lymphocytes (antigen-specific)

Movement is mediated by selectins, a class of CAMs specific for leukocyte/vascular cell interactions

• Activate integrin binds ICAM-1 and ICAM-2

• Cells move from the blood into infected or inflamed tissue

Immunoglobulin superfamily的家族成員眾多，且均與immunoglobulin 有一定的同 源性，其分子架構中含有多個90-100個胺基酸的Ig-like domains。廣泛分佈於 淋巴細胞、單核細胞、內皮細胞等多種細胞的表面。

重要的成員有intercellular adhesion molecule-1、2、3 ( ICAM-1、2、3）、

vascular cell adhesion molecule-1 ( VCAM-1 ) 和platelet-endothelial cell adhesion molecule ( PECAM )。

該家族的主要功能是參與不同細胞間的識別與黏附，在與免疫、發炎有關的 細胞黏附中發揮重要作用。

ICAM-1是最早發現的immunoglobulin superfamily 黏附分子之一，以後又相繼發現了ICAM-2和 ICAM-3，它們的胺基酸序列具有同源性，且都 可以結合LFA-1分子(一種integrin )。不同的

ICAM分子在體內的分佈範圍有較大差異，

ICAM-1分子分佈廣泛，如白血球、內皮細胞、

某些腫瘤細胞、上皮細胞、肝細胞、平滑肌細 胞等，IL-1、TNF- α 、和LPS可促進ICAM-1分 子的表現；ICAM-2則分佈較局限，主要表現在 血管內皮細胞；而ICAM-3表現在T細胞、單核 細胞 。

IgCAMs comprise a diverse group of adhesion receptors, that are defined by the presence of one or several Ig folds; classical

examples are:

Neuronal CAM (NCAM) is implicated in neuronal guidance and establishment of new synapses

NCAM forms homotypic contacts

Intercellular CAM (ICAM) interacts heterotypically with integrins CAMs differ widely in their cytoplasmic binding partners

Selectins: mediate transient cell-cell adhesion in the bloodstream

White blood cells (WBCs) utilize the adhesive properties of selectins (and integrins) in order to move: blood ↔ tissue.

• Selectins are “lectins” – carbohydrate binding proteins. (Ca2+ dependent)

• TM protein with a highly conserved lectin

domain that binds to a specific oligosaccharide.

• Transient, calcium-dependent interactions.

• Selectin types:

9 L-selectin: WBCs

9 P-selectin: platelets and endothelial cells 9 E-selectin: activated endothelial cells

lectins” – carbohydrate binding proteins. (Ca2+

dependent

目前已發現selectin家族中有三個成員︰L-selectin、P-selectin和E-selectin，

L、P和E分別表leukocyte , platelet和endothelium , 是最初發現相應selectin 分子的三種細胞，故得名。

P-selectin，分子量 140KD，由單核巨細胞跟內皮細胞形成，貯存於血小板的 α顆粒及內皮細胞的Weibel-Palade小體。當受到thrombin跟histamine刺激 時，P-selectin會迅速到達血小板或內皮細胞表面，介導白血球與內皮細胞 的起始黏附。

E-selesctin，分子量115KD，正常的內皮細胞表面並無E-selesctin存在，細 胞內也沒有儲存。當受到IL-1及TNF- α等細胞因子的刺激時，會活化內 皮細胞，刺激E-selesctin的合成。 E-selesctin會幫助白血球與內皮細胞的 黏附作用。

L-selesctin廣泛存在於各種白血球的表面，參與發炎部位白血球的出血管過 程。白血球表面L-selesctin分子上的sLeA與活化的內皮細胞表面的P- selesctin及E -selesctin之間的識別與結合，可召集血液中快速流動的白血 球在發炎部位的血管內皮上減速滾動（即透過黏附、分離、再黏

附……，如此循環往複），最後穿過血管進入發炎部位。

MACROMOLECULAR ORGANIZATION OF ECM

Movement of leukocytes into tissue depends on a precise sequence of combinatorially diverse set of adhesive intereaction

Bacterial, infection or inflammation → tissue dysfunction → blood

(leukocyte) → expressed special adhesion molecules at endothelial

surface → bind leukocyte → induced adhesion molecule activation →

extravasation → extracellular ……….

P-selectin, a lectin (protein that binds carbohydrates) on activated endothelial cells, binds a specific ligand (an oligosaccharide sequence) on T cells

PAF (Platelet Activating Factor - a phospholipid) activates integrin on the leukocyte surface

CAM directly bind to leukocyte

1. Endothelium activatioin → selectin or carbohydrate ligand →weak, reversible binding

2. Infection or inflammation signal → chemokines or PAF → expressed special molecules → attached leukocyte

3. Additional activation dependent CAM, integrins → strong adhesion

Multi

Multi - - step Model of Leukocyte step Model of Leukocyte

Adhesion and Extravasation

Adhesion and Extravasation

Leukocyte-adhesion deficiency 淋巴球黏力缺失症

Genetic disorder

Leukocyte did not extravasation

Plant tissue

Unlike animals, plants do not replace or repair old or damaged cells or tissues; only grow new organs

Plant only four broad types of cells (form four basic classes of tissue) 1.dermal tissue: interact with environment

2.vasculat tissue: transport water and dissolved substances 3.ground tissue: space filling

4.sporogenous tissue: forms the reproductive organs

Contain polysaccharides: cellulose (tensile 張力 拉力strength), hemicelluose

Allow soluble factor to pass to cell membrane, but less permeable than animal cell matrix

Functions of cell walls:

For plants:

Mechanical strength--> plant heights

Glue cells together--> dictate the way in which plant develop Exoskeleton--> control cell shapes

Control balance between cell turgor pressure and cell volume Diffusion barrier for macro-molecules and pathogens

Food reserves: endosperm cell walls degrade during seed during germination

Signaling

CELLULOSE AND HEMICELLULOSE ARE PRESENT IN A MATRIX OF PECTIN POLYMERS

The plant cell wall is a laminate (薄板) of cellulose fibrils in a matrix of glycoprotein

Primary wall consists of the following basic features

– Cellulose (strength) – Hemicellulose

– Pectins (flexibility)

– Structural proteins (rigidity)

– Non-structural proteins

Cellulose micorfibrilsLiner chains of (1->4)-linked beta-D-glucose. Each microfibril may consists of6 to 30-50 chains. Each chain has 2000 to 25,000 glucose residues. Cellulose has a high tensile strength, equivalent to steel.

Insoluble, chemically stable. Excellent “bones”for building a strong cell wall.

a “glucan”: a polymer made up of glucose

…”xylan”……….…………..xylose Xyloglucan: a glucan backbone (a liner

chain of glucose) with xlylose attached as side chains

Cellulose molecules can be arranged together to form fibrils

that have great tensile strength.

These fibrils are the main structural element in the cell

walls of plants.

xyloglucan structure

Loosening of the cell wall permits elongation of plant Auxin → induced weakening of cell wall → water into cell

→expansion of intracellular vaculoe →elongation of cell

Cell Wall Structure

The middle lamella, primarily pectin, ‘glues’ neighbouring cells together.

Primary cell wall:

The first wall laid down during growth.

This is soft and flexible so that the cell can expand during growth.

It contains a mixture of biopolymers, with typically ~20-30% cellulose

Once growth has stopped, the secondary cell wall is laid down, which provides structural support for the cells.

Secondary Cell Wall Structure:

Some cells have very thick secondary cell walls, to provide maximum support.

In the case of flax it can be μm in thickness (compared with ~100nm for the primary wall), and this was why flax was used for this study.

Typical cellulose content is ~50%, though can reach ~100% for e.g. cotton.

Structure is complex, and thought to consist of aligned 排成直線layers of cellulose microfibrils in a general biopolymer matrix, with systematic misorientations between the layers.

PLASMODESMATA INTERCONNECT CYTOPLASMS OF ADJACENT PLANT CELLS

60 nM diameter allows passage of molecules of up to 1000 MW

ER extensions (desmotubule) can pass through, allowing transit of membrane bound molecules

Elevation of cytosolic calcium inhibits transports (similar to gap junction)

The extracellular matrix and the cell wall are the “outside” of the cell Extracellular matrix (ECM): consists of collagen fibers and

proteoglycan. Collagen are a group of insoluble glycoproteins that

contain large amount of glycine and the hydroxylated forms of lysine and proline. (examples, tendons, cartilage, and bone)

Cell wall: consists cellulose microfibrils embedded in a matrix of other polysaccharides and small amounts of proteins (extensins)

Primary cell wall: cullulose fibrils and gel like polysaccharides, thus flexible and extensible

Secondary cell wall: additional cell wall materials deposited on the inner surface of the primary cell wall, thus thicker and rigid. Second cell wall also contains high concentration of lignin, a major component of Communication between cells:wood

Plasmadesmata: cytoplasmic bridges between plant cells

Animal cells: gap junctions, right junctions and adhesive junctions

Prokaryotes: cell walls consist of peptidoglycans with GlcNAc-MurNAc units