A two-tiered mechanism for stabilization and immobilization of

E-cadherin

Matthiue Cavey, Matteo Rauzi, Pierre-François Lenne & Thomas Lecuit

Presented by Shig3/5/10

Background: E-cadherinCadherin – calcium dependent adhesion molecules that mediate cell-cell interactions

E-cadherin – epithelial cadherin

Homo-E-cad – E-cad homodimer that localizes at SAJs

SAJ – spot adherens junction

Role in development: – spatio-temporal cadherin levels change

Role in adult – ie: intestine and bloodstream barrier

Homophilic E-cadherin clusters in stable microdomains

FRAP

E-cad-GFP fusion

Figure 1: FRAP of E-cad-GFP

Bright region Low-intensity region

Also demonstrated with E-cad-EosFPPhotoconvert -> no exchange w/neighbors Bright SAJ

Two dynamically distinct actin populations

Figure 2: Didn’t observe depolymerization of all F-actin with latrunculin-A (Lat-A)

Actin patches underlie SAJ stability: SAJs dimmer in Lat-A treated embryos.

α-Cat is not essential for homo-E-cad stability

β-catenin

α-catenin

E-cad

F-actinTraditional View

Knockdown α-Cat by RNAi

No detectible α-Cat in SAJs

E-cad-β-Cat spots still colocalize with F-actin

Figure 2: α-Cat not required to retain Lat-A resistant F-actin patches at SAJs

A tensile actin network controls mobility of SAJs

Lat-A: prevents actin polymerization

RNAi

Figure 3

Bitesize: organizes actin filaments at cell junctions through Moesin (F-actin binding protein)

Reorganize actin network:increase mobility of SAJs.

A tensile actin network controls mobility of SAJs

DiaCA: Diaphanous – promotes unbranched actin elongation

myrWasp: promotes cortical actin branching

Rok inhibitor: removes myosin-II motors within the cortical actin network

Figure 3

Fortify actin network:decrease mobility of SAJs.

α-Cat mediates tethering ofhomo-E-cad by cortical actin

Figure 3: α-Cat RNAi increases SAJ mobility

α-Cat mediates tethering ofhomo-E-cad by cortical actin

Figure 4: Nano-joule ablation to sever actin networkUncaged fluorescein

Model: two-tiered stabilization and immobilization of E-cadherin

Stability:

Mobility:

homo-E-cad at SAJs is stable

independent of α-catenin

E-Cad-β-Cat-F-actin structural units

linker between E-Cad-β-Cat and actin?

α-catenin links E-cad to actin

actin rearrangement delocalizes E-cad

factors acting on actin effect SAJ mobility

Future directions

• Calcium necessary for E-cad rigidity and adhesion– calcium regulation during development

• More linkers between SAJs and actin?– genetic screen

• Role of microtubules and associated proteins• Better characterization of nano-scissors