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Nezha Binds with MTs at the Minus Ends

Nezha Binds with MTs at the Minus Ends. Use GFP-tagged Nahza deletions Stain for EGFP and tubulin Add carrier tubulins and Taxol to Caco2 extracts Collect MT pellets Mix Nehza-Flag w/ purified tubulins See if they CO-IP. Nezha Binds with MTs at the Minus Ends.

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Nezha Binds with MTs at the Minus Ends

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  1. Nezha Binds with MTs at the Minus Ends • Use GFP-tagged Nahza deletions • Stain for EGFP and tubulin • Add carrier tubulins and Taxol to Caco2 extracts • Collect MT pellets • Mix Nehza-Flag w/ purified tubulins • See if they CO-IP

  2. Nezha Binds with MTs at the Minus Ends • Look at localization of Nezha and MTs • Characterize by including EB1 • In vitro MT-capping • Nezha-CC1F, Alexa labeled, His tagged • Add rhodamine-labeled and unlabeled tubulins sequentially

  3. Dynamics of MTs Anchored at Nezha Transfect Caco2 w/ Nezha-KOR and GFP-α-tubulin Record behavior (time lapse)

  4. Dynamics of MTs Anchored at Nezha Transfect Caco2 w/ Nezha-KOR and EB1-GFP Record behavior (time lapse)

  5. Dynamics of MTs Anchored at Nezha • Analyze interactions btwn Nezha and MTs at cell-junctions • Look at Nezha signals at cell-cell bounderies

  6. KIFC3 ZA recruitment is PLEKHA7/Nezha Dependant • Tested to see if MTs tethering to PLEKHA7-Nezha complex is to provide a track for dynein or another motor • Found KIFC3 • Knockdown Nezha or PLEKHA7 by siRNA • See how KIFC3 is affected • Overexpress Nezha • See how KIFC3 is affected

  7. KIFC3 ZA recruitment is PLEKHA7/Nezha Dependant Transfect Caco2 w/ KIFC3-GFP and E-cadherin-KOR Record behavior (time lapse)

  8. KIFC3 ZA recruitment is PLEKHA7/Nezha Dependant • Knockdown KIFC3 (siRNA)

  9. MTs Are Essential in the Actions of the Nezha-KIFC3 System • Treat cells w/ nocodazole • Observe • MTs are required for ZA assembly • Sensitivity of these proteins to MT depolymerization differs • Use FRAP to test whether KIFC3 uses Nezha-anchored MTs to acess cell junction

  10. Organizing Junctions at the Cell-Cell Interface A Complex of Nezha and PLEKHA7 Links Microtubules with Cadherins at Junctions between Epithelial Cells

  11. Future Directions • Does Nezha have nucleation activity? • Does it cooperate with ninein, which also captures MTs at their minus ends? • Is there a role for Nezha in the cytoplasm? • How did it localize there? • Is there a cooperative interaction between Nezha and MTs to regulate the binding of PLEKHA7? • What is the mechanism that makes this complex ZA specific? • Is there a role for these proteins at the centrosome? • What is the detailed role of KIFC3? • How is tissue morphogenesis affected?

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