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Anchorage of Microtubule Minus Ends to Adherens Junctions Regulates Epithelial Cell-Cell Contacts

Wenxiang Meng, Yoshimi Mushika, Tetsuo Ichii and Masatoshi Takeichi. Anchorage of Microtubule Minus Ends to Adherens Junctions Regulates Epithelial Cell-Cell Contacts. Presented By Vanessa Mondol BGGN 222. What is the zonula adherens?.

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Anchorage of Microtubule Minus Ends to Adherens Junctions Regulates Epithelial Cell-Cell Contacts

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  1. Wenxiang Meng, Yoshimi Mushika,Tetsuo Ichii and Masatoshi Takeichi Anchorage of Microtubule Minus Ends to Adherens Junctions Regulates Epithelial Cell-Cell Contacts Presented By Vanessa Mondol BGGN 222

  2. What is the zonula adherens? • 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)

  3. Hypothesis: • p120-catenin (p120) coupled with cadherin might interact with microtubules (MT) at endogenous levels • This interaction is probably mediated by other proteins

  4. Searched for proteins interacting with the p120 N-terminal region • 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

  5. Identification of PLEKHA7 as a p120-Catenin Partner • 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)

  6. Characterization of PLEKHA7-p120 binding Further analysis shows region between aa 538 – 696 in PLEKHA7 is required to bind to p120

  7. Identification of PLEKHA7 as a p120-Catenin Partner • 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)

  8. PLEKHA7 Regulates Zonula Adherens Integrity • overexpress PLEKHA7-GFP, see what happens • siRNA of PLEKHA-7 • PLEKHA7 is important for cadherin-catenin accumulation at the ZA

  9. PLEKHA7 Binds Nezha • 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

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

  11. PLEKHA7 Binds Nezha • 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

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