Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

Supplemental Material

This article contains the following supporting material:

• Supplemental Materials
• Supplementary Figure 1 - (A) Tannic acid (0.5%) does not affect the trans- epithelial electrical resistance (TER) of fully polarized MDCK cells over a 2 hr time course. Tannic acid was added into the basolateral compartment of MDCK cells that had been cultured on Transwell filter for 4 days at which time the TER exceeded 200 ohms/cm². (B) ¹⁴C-inulin flux was determined for MDCK cells cultured on Transwell filters from 1-5 days. ¹⁴C-inulin flux was measured 15 and 30 min after addition of tannic acid to the basolateral medium. ¹⁴C-inulin flux was < 0.1% for MDCK cells cultured on Transwell filters for 4 and 5 days.
• Supplementary Figure 2 - (A) Naked2-EGFP retained its normal basolateral localization after addition of tannic acid to the apical medium. MDCK cells stably transfected with Naked2-EGFP cDNA were cultured on Transwell filters for 4 days and then were treated apically with 0.5% tannic acid for 2 hrs. F-actin was stained with rhodamine-phalloidin (red) to outline the cell borders. Bar, 10 μm.
  (B) MDCK cells stably infected with p120-EGFP and CD147 cDNA were cultured on Transwell filters for 4 days and then placed at 40°C for 4 hrs. In contrast to Naked2-EGFP, this "heat bleaching" did not affect p120-EGFP. After 40°C "heat bleaching," cells were then placed at 10°C for 10 min with 0.5% tannic acid added selectively to the basolateral medium, and then cells were restored to 37°C in the continued presence of tannic acid. Basolateral localization of p120 and CD147 were not affected by 60 min exposure to tannic acid.
• Supplemental video 1 - Locomotion of Naked2-associated vesicles in MDCK cells stably transfected with GFP-tagged Naked2. Time-lapse images were taken at 5 sec intervals for 10 min using a Zeiss LSM 510 confocal microscope. MDCK cells were seeded onto MatTek dishes and maintained at 37°C and 5% CO₂ with a controlled stage (Bioptechs Delta T System). The movie shows representative vesicles at the corner of the cell that move upwards along the plasma membrane with dimming of fluorescence over time.