Wenxiang Meng, Yoshimi Mushika, Tetsuo Ichii and Masatoshi Takeichi Presented By Vanessa Mondol BGGN 222 Presented By Vanessa Mondol BGGN 222.

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Wenxiang Meng, Yoshimi Mushika, Tetsuo Ichii and Masatoshi Takeichi Presented By Vanessa Mondol BGGN 222 Presented By Vanessa Mondol BGGN 222

 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)

Characterization of PLEKHA7-p120 binding 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

Characterization of PLEKHA7-Nezha binding 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