1.  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.  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.  Analyze interactions btwn Nezha and MTs at cell-junctions  Look at Nezha signals at cell-cell bounderies

6.  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.  Knockdown KIFC3 (siRNA)

9.  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.  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?