Cell adhesion and ECM PowerPoint Presentation

Cell adhesion and ECM

Lecture 10:

Cell Biology interactive  media  ”video” or ”animation”

The eukaryotic kingdom

1

Cell biology 2014

(revised

11/2-14

)

The four major tissues in the human body

- Epithelial

- Muscle

- Connective

- Nerve

Cells

Cells +

ECM

Metazoan cells form organs with specialized tissues:

2

3

Stratum Corneum

Basal layer

ECM /residual cells*(”fibroblasts”)

Epidermis

Dermis

Basal

lamina

1.

2.

3.

1.

2.

3.

*

Different types of cell adhesion

Homophilic binding

Heterophilic binding

4

Cell-cell contacts in

columnar epithelia

Gap junction

Tight junction

Adherens junction

Desmosome

Connection allowing local communication

Cell-cell adhesion

5

hemidesmosome

Cell-ECM adhesion

Basal lamina

Restricting movement of

extra-cellular fluids

Tight junctions seal epithelial sheets to block passage of fluids in between cells

I. Tight junctions: function

Intestine

Blood

vessels

Glucose

Glucose

Active and selective transport

through the cytosol

of cells by e.g., the Na

+

driven glucose symport

6

II. Tight junctions: Architecture

The appearance of tight junctions

resemble stitches across the plasma

membranes of the two cells

Tight junctions are made up by occludin and claudin. These are

transmembrane proteins, which form tight connections across

the extracellular space

Linking protein attaches occludin and

claudin to the cortical actin cytoskeleton

7

P.M.

a

-catenin

a

-catenin

Cadherin

(calcium-dependent adhesion

)

Cell #1

Cell #2

P.M.

b

-catenin

b

-catenin

Actin

filament

Many cadherins are known:

E

-cadherin in

E

pithelia

N

-cadherin in

N

eural cells

Linkers of cadherins

to the actin cytoskeleton

Adherence junctions from stable cell-cell

adhesion points between adjacent cells

video 19.1-

adhesion_junctions

animation

20.2

-

contact_inhibition

8

Ca

2+

I. Cadherins:

adherence junctions

Wnt

Dishevelled

LRP

Frizzled

Target genes

TCF

b

-catenin

GSK-3

b

b

-catenin

P

Ub

Ub

Ub

Target

genes

TCF

Groucho

Axin

APC

GSK-3

b

b

-catenin

Axin

APC

Dishevelled

ZZZ

9

Cell cycle entry

+

etc

G1

Cytosolic

b

-catenin is stabilized by

Wnt

signaling

II. Cadherins:

growth arrest at cell-cell contact

P.M.

G1

Wnt

G1 cyclin

gene

..but is stabilized

by Wnt signaling

a

-catenin

b

-catenin

b

-catenin

TCF

b

-catenin

Cytosolic

b

-catenin is

by default unstable

.....

(

Ubiq

. dep. degradation

)

1.

2.

3.

b

-catenin enters the nucleus:

 G1 cyclin

transcription

 cell proliferation

4.

Sequestering

of cytosolic

b

-catenin at the adherence junctions

formed after cell proliferation

(at ”density

arrest”)

10

Ca

2+

III. Cadherins: organization of cells into organs

Cells expressing

different cadherins

Cells expressing

different amounts of

the same cadherin

Cadherins are important for organ formation during development

11

+Ca

2+

+Ca

2+

Desmosomes hold cells togetherlike rivets. Through linkage toIFs, they distribute shear forcesevenly within the cell

P.M.

Cell #1

Cell #2

P.M.

Linkers

Linkers

Intermediate

filament (IF)

Cadherin

family protein

Structure and function of the desmosome

animation 16.4- intermediate_filament

12

Ca

2+

Structure and regulation of gap junctions

Connexon

= ~1.5 nm

Free passage of: Amino acids

Nucleotides

Sugars

Ions

”2nd messengers”

Connexin

P

P

P

P

P

P

Different connexins –

different pore size

Cell #1

Cell #2

Regulation of pore size

13

I. Integrins: Structure and ligand specificity

Hetero-dimeric proteins consisting of

a- and b- chains ii) At least 21 cell-type specific isoforms of a/b-chain pairsiii) Integrin ligands include ECM components (collagen, fibronectin, laminin) and structures on neighboring cells

14

ECM: Basal lamina

ECM: connective tissue



x



x

(ligand: laminin)



y



y

(ligand: fibronectin)

I

ntegrins

linked to

IF

(

hemidesmosomes

: epithelia)

Integrins linked to

actin

(focal

adhesions: fibroblasts)

II. Integrins:

Anchorage to ECM

ECM:

connective tissue (contains residual migratory cells)

15

ECM: Basal lamina

Inactive

integrin

I

ntegrins linked to

IF

(hemidesmosomes)



Static

c

ell-ECM interactions , e.g

.

epithelial sheets

Integrins linked to

actin

(focal adhesions)



Dynamic

c

ell-ECM

interactions, e.g., during migration of fibroblasts or leukocytes

Basal lamina: barrier towards connective tissue

III. Integrins:

Architecture of the focal adhesion

ECM

P. M.

Active integrin

Focal adhesions exist only in motile cells (i.e., not in epithelia)

The dynamic nature of focal adhesion is dependent on both “Inside-out” and “Outside-in” signaling

FAK: Focal adhesion kinase



integrin dependant signaling

16



recruitment of SH2-domain signaling

proteins

(

Clustering of FAK

 trans-phosphorylation, i.e. the

same principle as for tyrosine kinase receptors, which are dimerized by ligand binding

)

Talin

Linker

FAK

Tyr-

P

Talin

FAK

Tyr-

P

Tyr-

P

IV. Integrins: Regulation of ligand-affinity

17

Outside-in activation of ECM-binding

Inside-out activation of ECM-binding

1.

Default state

:

The

a-

and

b-

chains are tightly associated

Activated state: a- and b-chains are pushedapart and clustered by talin High affinity/avidity ECM-association

1.

2.

2.

The

a-

and

b-

chains of integrins have affinity for both “each other” and ECM ligands

 the concept of competing affinities

V. Integrins:

Inside-out activation

FAK

P

2.

1.

3.

2.

3.

Activated talin:

Pushes

a-

and

b-

chains

apart

Clusters

the cytosolic parts of integrin

b-

chains

Links 

-chains with

actin filaments

Recruit focal adhesions proteins (

vinculin

, FAK etc)



generation of a focal adhesion point

2.

1.

3.

4.

Activation of talin by a

RTK ligand (e.g. EGF)

Separation

of

a-

and

b-

chains

High

affinity ECM-binding Integrin clustering increased avidityAlbert et al. Fig 19-49

4.

18

Inactive talin

VI. Integrins: Outside-in activation

Binding by (

very

) high affinity ECM ligands……..breaks the interaction between the - and -chainsThe exposed -chain talin-binding site……..….activates talin  Generation of a focal adhesion point

outside-in + inside-out = positive feedback

1.

2.

3.

4.

ECM

1.

ECM

2.

2.

3.

inactive

talin

ECM

4.

19

VII. Integrins:

survival and cell proliferation signals

Plasma membrane

MAPK

P

P

P

PKB/

Akt

P

PI-3 K

P

myc

Cell cycle entry

FAK

Bad

P

14-3-3

20

Ras

GTP

Motile cell types requires ECM for both growth and survival

Survival

P

P

3

-Tyr-

P

-Tyr-

P

-Tyr-

P

Talin

- Provides mechanical support to tissues Organizes cells into tissues ‘Instructs’ cells as to where they are and what they should do- Reservoir for extra-cellular signaling molecules

I. The extra-cellular matrix (ECM)

21

II. The extracellular matrix (ECM)

Proteoglycan molecules form highly hydrated gel-like “ground

substance” in which the fibrous proteins are embedded

Structural proteins, such as collagen and elastin, strengthen and organize the matrix

Composed of polymeric networks

of several types of macromolecules.

Secreted by connective tissue cells,

such as fibroblasts & chondrocytes.

1.

2.

3.

1.

2.

3.

Multi-adhesive proteins, such as fibronectin and laminin,

facilitate cell attachment to the ECM

The aqueous phase of the ECM permits diffusion of nutrients

22

III. ECM: general structure of proteoglycan

H

2

O

Na

+

-

-

-

-

-

Ca

2+

Protein core

Polysaccharide

sidechain

Negatively charged saccharides attract counter ions

and water, giving the ECM the property to resist

compression and bounce back to its original shape

Osmosis

Linking saccharides

Glucosamino-

glycans (GAGs)

linear polymers

of repeating

disaccharides

O-linked sugar

23

IV. ECM: Proteoglycan aggregates

Hyaluronan,

up to 50 000

repeating

disaccharides

Linker protein

Proteoglycans can form huge aggregates onto hyaluronan. These aggregates can be up to 4

m

m in length

These aggregates have a very high shock absorbing capacity

and are highly enriched in cartilage

24

V. ECM:

Collagen architecture

Collagen

a

-chain

(single helix)

Collagen molecule

(triple helix)

Collagen fibril

Collagen fiber

Assembled

outside

the cell

Assembled

in ER

Collagen is the most common protein in body, it forms

strong and

flexible fibers. Many types (at least 15)

25

VI. ECM: Elastic elastin networks

Single elastin

molecule

In cases there ECM is very flexible, e.g.,

in skin, lungs and blood

vessel walls, some of the collagen is replaced by elastin.

Cross-linked elastin behaves like a rubber band!

Stretching

Relaxation

Crosslinking

26

VII. ECM:

different types of connective tissue

”Normal” connective tissue

Cartilage

Fibroblast

Chondrocyte

Ca

10

(PO

4

)

10

(OH)

2

Ca

10

(PO

4

)

10

(OH)

2

Bone

Osteoblast

Physical properties of the tissue depend on the content of the

ECM, which is determined by the residual cell type

27

Summary: ECM – a sticky business!

28

Laminin

- Present in basal lamina of epithelia and the

ligand for hemidesmosomes

Fibronectin

- Present in all ECM and primary high-affinity

ligand for focal adhesions

Fig. 19-1: Epithelial tissue: The intermediate filaments of the cells themselves (linked from cell to cell by desmosomes) provides mechanical strength. Hemidesmosomes (integrin binding to laminin) are only found in the epithelial cells that connect to the basal lamina. These epithelial cells are normally essentially non-motile.Connective tissue: ECM provides the mechanical strength, the sole role of the residual cells (fibroblasts) is to produce the ECM components. These residual cells move around and may migrate to e.g. a site of tissue damage.

29

Differential means to achieve mechanical strength

Epithelial cells

Basal lamina

(dense ECM)

Connective tissue(ECM + cells)

Cells resistant to mechanical stressECM (but not cells) resistent to mechanical stress

“Recommended reading”

Alberts

et al

5th edition

Chapter 19

1131-11451150-11621164-1194

Focus on the general principlesand topics highlighted inthe lecture synopsis

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