Lecture 10:

Cell Biology interactive media ”video” or ”animation”

The eukaryotic kingdom

1

Cell biology 2014 (revised 12/2-13)

The four major tissues in the human body

- Epithelial

- Muscle

- Connective

- Nerve

Cells

Cells + ECM

Metazoan cells form organs with specialized tissues:

2

Different types of cell adhesion

Homophilic binding Heterophilic binding

3

Cell-cell contacts in columnar epithelia

Gap junction

Tight junction

Adherens junction

Desmosome

Connection allowing local communication

Cell-cell adhesion

4

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

GlucoseActive and selective transport through the cytosol of cells by e.g., the Na+ driven glucose symport

5

II. Tight junctions: Architecture

The appearance of tight junctionsresemble 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

6

P.M.

a-catenin

a-catenin

Cadherin (calcium-dependent adhesion)

Cell #1

Cell #2

P.M.

b-catenin

b-catenin

Actinfilament

Many cadherins are known: E-cadherin in EpitheliaN-cadherin in Neural cells

Linkers of cadherinsto the actin cytoskeleton

Adherence junctions from stable cell-cell adhesion points between adjacent cells

video 19.1- adhesion_junctions7

Ca2+

I. Cadherins: adherence junctions

II. Cadherins: growth arrest at cell-cell contact

P.M.

G1

Wnt

G1 cyclin gene

..but is stabilized by Wnt signaling

a-cateninb-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 (i.e., at ”density arrest”)

8

Ca2+

III. Cadherins: organization of cells into organs

Cells expressing different cadherins

Cells expressing different amounts ofthe same cadherin

Cadherins are important for organ formation during development9

+Ca2+

+Ca2+

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

Intermediatefilament (IF)

Cadherin family protein

Structure and function of the desmosome

animation 16.4- intermediate_filament 10

Ca2+

Structure and regulation of gap junctionsConnexon

= ~1.5 nmFree passage of: Amino acids Nucleotides Sugars Ions ”2nd messengers”

Connexin

P PP PP P

Different connexins –different pore size

Cell #1

Cell #2Regulation of pore size

11

I. Integrins: Structure and ligand specificityi) 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

12

ECM: Basal laminaECM: connective tissue

bx ax (ligand: laminin)by ay (ligand: fibronectin)

Integrins linked to IF (hemidesmosomes: epithelia)

Integrins linked to actin (focal adhesions: fibroblasts)

II. Integrins: Anchorage to ECM

ECM: connective tissue (contains residual migratory cells)

13

ECM: Basal lamina

Inactiveintegrin

Integrins linked to IF (hemidesmosomes) Static cell-ECM interactions , e.g. epithelial sheets

Integrins linked to actin (focal adhesions) Dynamic cell-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

14

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)

TalinLinkerFAK

Tyr- P

TalinFAK

Tyr- P

Tyr- P

IV. Integrins: Regulation of ligand-affinity

15

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:i) Pushes a- and b-chains apartii) Clusters the cytosolic parts of integrin b-chains iii) Links b-chains with actin filamentsiv) 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 avidity

Albert et al. Fig 19-49

4.

16

Inactive talin

VI. Integrins: Outside-in activationBinding by (very) high affinity ECM ligands……..breaks the interaction between the a- and b-chainsThe exposed b-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.

inactivetalin

ECM

4.

17

VII. Integrins: survival and cell proliferation signals

Plasma membrane

MAPK P

P

PPKB/Akt

P

PI-3 K

P

myc

Cell cycle entry

FAK

BadP

14-3-3

18

RasGTP

Motile cell types requires ECM for both growth and survival

Survival

PP3

-Tyr

- P

-Tyr

- P-T

yr- P

- 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)

19

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 networksof 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 nutrients20

III. ECM: general structure of proteoglycan

H2O

Na+

--

--

-

Ca2+

Protein core

Polysaccharide sidechain

Negatively charged saccharides attract counter ionsand water, giving the ECM the property to resist compression and bounce back to its original shape

Osmosis

Linking saccharidesGlucosamino-glycans (GAGs)linear polymers of repeating disaccharides

O-linked sugar

21

IV. ECM: Proteoglycan aggregates

Hyaluronan, up to 50 000repeating disaccharides

Linker protein

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

These aggregates have a very high shock absorbing capacity and are highly enriched in cartilage 22

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 andflexible fibers. Many types (at least 15)

23

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

24

VII. ECM: different types of connective tissue

”Normal” connective tissue Cartilage

Fibroblast Chondrocyte

Ca10(PO4)10(OH)2

Ca10(PO4)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 25

Summary: ECM – a sticky business!

26

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. 27

Differential means to achieve mechanical strength

Epithelial cells

Basal lamina(dense ECM)

Connective tissue(ECM + cells)

Cells resistant to mechanical stress

ECM (but not cells) resistent to mechanical stress

“Recommended reading”

Alberts et al5th edition

Chapter 191131-11451150-11621164-1194

Focus on the general principlesand topics highlighted inthe lecture synopsis

28