Figure 7.

Overexpression of GFP-Rab11-FIP1C(S234A) causes reorganization of recycling system markers after 8-hour calcium switch. GFP-FIP1C(S234A) expressing cells, either non-switched (NS) or fixed at 8 hours following calcium re-addition were examined for localization of Rab11a (A-C) or Rab11b (D-F) along with phalloidin staining or for Syntaxin 3 with p120 staining (G-I). A. Endogenous Rab11a colocalized with GFP-Rab11-FIP1C(S234A) in non-switched cells and at 8 hours after calcium switch. A still image taken from a 3-dimensional reconstruction shows Rab11a co-localizing with GFP-FIP1C(S234A) beneath the surface of a lateral lumen (B, see Movie S1). C. Colocalization analysis confirmed an increase in colocalization between Rab11a and GFP-FIP1C(S234A) following calcium switch (*p = 0.0001). D. Endogenous staining of Rab11b also colocalized with GFP-Rab11-FIP1C (S234A), however, to a lesser extent than Rab11a. The colocalization decreased significantly in cells after calcium switch (see F; *p = 0.003). E. A still image taken from a 3-dimensional reconstruction of Rab11b co-localizing with GFP-FIP1C(S234A) beneath a lateral lumen (Movie not shown). G. While Syntaxin 3 localized to the apical membrane region in non-switched cells, Syntaxin 3 relocalized following calcium switch in GFP-FIP1C(S234A) cells to the lateral lumen. P120 was used to label lateral membranes and was excluded from the lateral lumen surface. H. A still image taken from a 3-dimensional reconstruction of Syntaxin 3 at the surface of a lateral lumen (see Movie S2). P120 is absent from the surface of the lateral lumen (see Movie S3). I. Colocalization analysis confirms an increase in colocalization between GFP-FIP1C(S234A) and Syntaxin3 (*p = 0.000005). J. Little colocalization was present between GFP-FIP1C(S234A) and p120. X/Z images are shown below X/Y images, < indicates where images were taken for X/Y and X/Z planes. All scale bars = 10 µm. (C. F. I. J.) Graphs plot mean Pearson's Coefficient and error bars represent standard deviation.