Subapical distribution of EGFP-Rab11-FIP2 in stably expressing cell lines. Localization of the apical maker GP135 with EGFP-Rab11-FIP2 and EGFP-Rab11-FIP2(S227A) was compared in stable cell lines. Confocal immunofluorescence imaging of the EGFP-Rab11-FIP2 constructs with GP135 showed that EGFP-Rab11-FIP2 partially colocalized and interspersed with the apical marker GP135 (red in merge). EGFP-Rab11-FIP2(S227A) was also partially colocalized with and partially beneath the apical marker GP135 (red in merge). The x-y planes are optical sections taken 2.5 μm apart.

Phosphorylation of Rab11-FIP2 is necessary for the proper reestablishment of polarity in a replating assay. Cell lines expressing EGFP-Rab11-FIP2 or EGFP-Rab11-FIP2(S227A) were trypsinized and replated on laminin-coated filters. The cells were fixed at the indicated time points and stained with p120 (pseudocolored red) and ZO-1 (pseudocolored blue). These overlays show x-y planes after replating. Although ZO-1 was reestablished at tight junctions in both lines, EGFP-Rab11-FIP2(S227A)–expressing cells demonstrated a deficit in p120 localization 4 h after replating.

Dominant-negative Rab11a recycling system trafficking mutants do not colocalize with junctional proteins p120 or ZO-1. (A) X-Y confocal sections showing dual labeling of p120 catenin (Cy 3; pseudocolored red) and ZO-1 (Cy5; pseudocolored blue) in EGFP-myosin Vb tail cells. No colocalization was found between the junctional markers and EGFP-myosin Vb tail. (B) Projections showing dual labeling of p120 catenin (Cy 3; pseudocolored red) and ZO-1 (Cy5; pseudocolored blue) in EGFP-Rab11-FIP2(129-512) and EGFP-myosin Vb tail cells after calcium switch at t = 0. Junctional markers do not colocalize with the dominant-negative inhibitors of the Rab11a apical recycling system. No localization with the collapsed recycling system was seen at any time point after reestablishment of extracellular calcium.

EGFP-Rab11-FIP2 is phosphorylated in situ. Top blot, Phosphorimage of phosphorylation of immunoprecipitated EGFP-Rab11-FIP2 from MDCK cell lines stably expressing either EGFP-Rab11-FIP2 or EGFP-Rab11-FIP2(S227A). Cells were loaded with [³²P]orthophosphate, lysed, and immunoprecipitated with an anti-GFP antibody, and proteins were resolved on SDS-PAGE gels. Bottom blot, Immunoblot probing for GFP of immunoprecipitated lysates. Full-length EGFP-Rab11-FIP2 is 75 kDa (denoted with a solid square), but it is consistently broken down to a lower molecular mass species (indicated with a star) by an as yet unknown mechanism.

Distribution of EGFP-Rab11-FIP2 during calcium switch. Cell lines expressing EGFP-Rab11-FIP2 or its phosphorylation mutant were subjected to the calcium switch protocol and fixed at the indicated time points after readdition of extracellular calcium. The EGFP-Rab11-FIP2 initially moved to a lateral membrane before accumulating in a tight spot internally. As the junctions reformed, the construct localized to the apical membrane as seen for those cells not subject to the switch. EGFP-Rab11-FIP2(S227A) initially localized in subapical rings. As the junctions reformed, the construct was less compact and became diffusely apical. The x-y planes are compiled stacks of optical sections taken 2.5 μm apart.

Phosphorylation of Rab11-FIP2 is necessary for the proper reestablishment of p120 at adherens junctions but not ZO-1 at tight junctions. Cell lines expressing EGFP-Rab11-FIP2 or its phosphorylation mutant were subjected to the calcium switch protocol and fixed at the indicated time points. (A) Z sections from cells in the calcium switch experiment stained for p120 catenin. Note the delayed establishment of p120 localization in Rab11-FIP2(S227A) compared with wild type. p120 staining showed a normal reestablishment at the adherens junction in EGFP-Rab11-FIP2 cells grown in the presence of doxycycline (uninduced). (B) Projections of z-stacks from ZO-1 staining during the calcium switch experiment demonstrate the normal reestablishment of ZO-1 at tight junctions in all cell lines.

Rab11-FIP2 kinase activities. (A) A kinase activity associated with rabbit gastric microsomes phosphorylates Rab11-FIP2 on serine 227. Top, Coomassie stain for protein levels. Bottom, phosphorimage. (B) Phospho-amino acid analysis demonstrates phosphorylation of Rab11-FIP2 is on serine residues. Positions of phospho-amino acid standards are shown by dotted lines. PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine. (C) Rab11-FIP2 is phosphorylated by recombinant activated MARK2 in vitro on serine 227 of Rab11-FIP2 wild type by not Rab11-FIP2(S227A). Top blot, Coomassie stain for protein levels. Bottom blot, phosphorimage. (D) Amino acid alignment of the putative Rab11-FIP2 sequences. The conserved phosphorylation site is boxed and in bold. The phosphorylated serine is marked by a star.

Distribution of EGFP-Rab11-FIP2 in stably overexpressing cell lines. (A) T23 cells stably expressing EGFP-Rab11-FIP2 exhibit two different morphologies. White arrowhead indicates a cell with a more diffuse distribution of EGFP-Rab11-FIP2. (B) Transiently transfected MDCK II cells show only a punctuate subapical distribution of EGFP-Rab11-FIP2. (C) T23 cells expressing EGFP-Rab11-FIP2 in the presence or absence of pIgA (red in merge). On induction of trafficking, the EGFP-Rab11-FIP2 exhibits dynamic relocalization to the more traditional recycling system. EGFP images are from two different z-positions in the same confocal stack, as indicated in the x-y orthogonal view, to enhance the visualization of both morphologies.

Phosphorylation of Rab11-FIP2 does not alter interaction with myosin Vb tail or pIgA trafficking. (A) The phosphorylation state of Rab11-FIP2 does not affect known interactions with myosin Vb. Stable EGFP-RAB11-FIP2 cells lines were transiently transfected with Ds-Red2 myosin Vb tail. Rab11-FIP2 wild type and Rab11-FIP2(S227A) as well as Rab11a were pulled into the myosin Vb tail collapsed recycling system as expected. (B) Recycling functions of Rab11-FIP2 are not affected by phosphorylation on serine 227. Apical recycling and transcytosis of ¹²⁵I-IgA were assessed in stable transfected cell lines in the presence or absence of doxycycline. Neither apical recycling nor transcytosis were affected by the phosphorylation state of Rab11-FIP2. Top graphs, EGFP-Rab11-FIP2 wild type. Bottom graphs, EGFP-Rab11-FIP2(S227A). Left-hand side shows data from transcytosis assays, whereas the right-hand side shows results from apical recycling assays. Dark blue and pink lines indicate that doxycycline was present in the media (inhibiting expression of the EGFP-chimera). Yellow and light blue lines indicate that the cells were incubated in doxycycline-free media allowing for expression of EGFP-chimera proteins. Dark blue and yellow lines indicate media was collected from the apical side of the transwell. Pink and light blue lines indicate media was collected from the basal side of the transwell. The results show no effects of EGFP-Rab11-FIP2 expression on either transcytosis or apical recycling.