Wenxiang Meng, Yoshimi Mushika,Tetsuo Ichii and Masatoshi Takeichi

Adheren junctions (AJ)are protein complexes that occur at cell-cell junctions in epithelial tissues

Beltlike adherens junction that encircles the apical end of an epithelial cell and attaches it to the adjoining cell.

Known to interact with actin, to anchor it to the cell wall. Little is known about it’s interaction it microtubules (MT)

p120-catenin (p120) coupled with cadherin might interact with microtubules (MT) at endogenous levels

This interaction is probably mediated by other proteins

PLEKHA7 (pleckstrin homology domain-containing, family A member 7)

Nehza, a binding partner for PLEKHA7 Associated with minus ends of MTs

KIFC3, kinesin-14 family member concentrated in the ZA Localization abolshed by RNAi of PLEKHA7 or

Nehza

Depletion of any of these proteins led to disorganization of the ZA

Generate GST fusions (Fig 1A)

Incubate with lysates of Caco2

Mass spec materials pulled down, found PLEKHA7

To confirm finding, FLAG tag PLEKHA7, transfect Caco2

Incubate transfected lysates with GST fusions and GST , probe for FLAG (Fig 1C)

IP with α-FLAG, p120 and E-cadherin CO-IP (Fig 1D)

Further analysis shows region between aa 538 – 696 in PLEKHA7 is required to bind to p120

Developed Abs for PLEKHA7

Immunofluorescence staining of Caco2

Examine the effect of p120 depletion on PLEKHA7 by siRNA

Look at PLEKHA7 recruitment in Neuro-2a cells (cadherin-deficient)

overexpress PLEKHA7-GFP, see what happens

siRNA of PLEKHA-7

PLEKHA7 is important for cadherin-catenin accumulation at the ZA

Use GST-PLEKHA7 fusion to pull down Nehza

Developed Abs for Nehza

Transfect Caco2 w/ PLEKHA-FLAG, and Nehza-GFP

Use transfected cell lysates to see if they

CO-IP

Deletion analysis of Nehza shows C-terminal region from aa 943 – 1225 is required for association with PLEKHA7

Look at fluorescence to see where it localizes (Fig2D) Compare to PLEKHA7-

siRNA knockdowns

Examine localization of PLEKHA7 when Nehza is knockdown by siRNA

Nehza, once associated with PLEKHA7, maintains the integrity of the ZA