1 / 11

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?.

mayten
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related