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. 2011 Mar;1(2):57-68.
doi: 10.4161/cl.1.2.15289.

Rab11-FIP2 influences multiple components of the endosomal system in polarized MDCK cells

Nicole A Ducharme  1 , Amy-Joan L Ham, Lynne A Lapierre, James R Goldenring
Affiliations

Rab11-FIP2 influences multiple components of the endosomal system in polarized MDCK cells

Nicole A Ducharme et al. Cell Logist. 2011 Mar.

Abstract

The Rab11 Family Interacting Proteins (Rab11-FIPs) are hypothesized to regulate sequential steps in the apical recycling and transcytotic pathways of polarized epithelial cells. Previous studies have suggested that Rab11-FIP proteins assemble into multi-protein complexes regulating plasma membrane recycling. Rab11-FIP2 interacts with both myosin Vb and Rab11. Recent investigations have noted that that Rab11-FIP2 mutants [Rab11-FIP2(129-512), also designated Rab11-FIP2(ΔC2) and Rab11-FIP2(S229A, R413G), also designated Rab11-FIP2(SARG)], are potent inhibitors of transcytosis in polarized MDCK cells. Interestingly, Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), also altered the morphology of the EEA-1 positive early endosomal compartment. These findings suggested that Rab11-FIP2 mutants could differentiate different points along the recycling pathway. We therefore sought to investigate whether Rab11-FIP2 is a general regulator of the early endosomal system. Both Rab11-FIP2 mutants altered the localization and co-localized with dynein heavy chain. In contrast, both clathrin heavy chain and AP-1 accumulated with membranes containing Rab11-FIP2(SARG), but not with Rab11-FIP2(ΔC2). Expression of Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), caused clustering of early endosomal markers Rab5b, Epsin 4 and IQGAP1, around a collapsed Rab11-FIP2 containing membranous cisternum. Interestingly, neither Rab11-FIP2 mutant had any effect on the distribution of Rab5a, a classical early endosome marker. The results support the view that Rab11-FIP2 may influence microtubule-dependent centripetal movement of subsets of early endosomes as well as processing through the common and recycling endosomal systems.

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Figures

Figure 1
Figure 1
Rab11-FIP2 mutants alter the localization of the microtubule motor protein, dynein heavy chain. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). (A) Parental T23 MDCK cells were stained with Alexa-488-phalloidin (pseudo-colored green) and antibodies against dynein (pseudo-colored red) and imaged by confocal microscopy. Dynein was observed in association in vesicles at the periphery of the cells, especially at the lateral borders. (B) T23 MDCK cells stably expressing either EGFP-Rab11-FIP2(SARG) or EGFP-Rab11-FIP2(ΔC2) were stained with antibodies against dynein and imaged by confocal microscopy. Expression of both Rab11-FIP2 mutants caused accumulation of dynein within the collapsed membrane cisternae. Images were taken with a 100x lens. Scale bars represent 5 µm.
Figure 2
Figure 2
Rab11-FIP2 mutants alter the distribution of clathrin heavy chain. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). (A) Parental T23 MDCK cells were stained with Alexa-488-phalloidin (pseudo-colored green) to show the cell boundaries and clathrin heavy chain (pseudo-colored red) and imaged by confocal microscopy. Punctate clathrin staining was observed on vesicles throughout the cytoplasm. (B) T23 MDCK cells stably expressing EGFP-Rab11-FIP2(SARG) and EGFP-Rab11-FIP2(ΔC2) were stained for endogenous clathrin heavy chain (pseudo-colored red) and imaged by confocal microscopy. Clathrin heavy chain staining was accumulated with the EGFP-Rab11-FIP2(SARG) mutant in its collapsed cisternum. In EGFP-FIP2(ΔC2)-expressing cells, clathrin staining was observed predominantly as vesicles surrounding the Rab11-FIP2 containing membranes. Images were taken with a 100x lens with a 3x zoom. Scale bars represent 5 µm.
Figure 3
Figure 3
Rab11-FIP2 mutants alter AP-1 distribution. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). (A) Parental T23 MDCK cells were stained with Alexa-488-phalloidin (pseudo-colored green) to show the cell boundaries and antibodies against AP-1 (pseudo-colored red) and imaged by confocal microscopy. AP-1 staining vesicles were observed diffusely within the subapical cytoplasm. (B) T23 MDCK cells stably expressing EGFP-Rab11-FIP2(SARG) or EGFP-Rab11-FIP2(ΔC2) were stained for endogenous AP-1 (pseudo-colored red) and imaged by confocal microscopy. AP-1 showed a prominent accumulation with EGFP-Rab11-FIP2(SARG) containing cisternae. In Rab11-FIP2(ΔC2)-expressing cells, AP-1-containing vesicles were clustered around the Rab11-FIP2-containing cisternum. Images were taken with a 100x lens with a 3x zoom. Scale bars represent 5 µm.
Figure 4
Figure 4
Rab11-FIP2 does not alter the localization of Rab5a. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). Parent T23 MDCK cells and T23 MDCK cells stably expressing EGFP-Rab11-FIP2(ΔC2) were transiently transfected with mRFP-Rab5a and imaged by confocal microscopy. Upper parts: Parent T23 MDCK cells were co-stained with Alexa-488-phalloidin (pseudo-colored green) and showed normal diffuse localization of mRFP-Rab5a. Lower parts: The mRFP-Rab5a did not show a relocalization towards the Rab11-FIP2 containing cisternae in cells overexpressing Rab11-FIP2(ΔC2). Images were taken with a 100x lens with a 3x zoom. Scale bars represent 5 µm.
Figure 5
Figure 5
Differentiable influences Rab11-FIP2 mutants on mRFP-Rab5b. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). Parental MDCK-T23 cells and cells overexpressing EGFP-Rab11-FIP2 constructs were transiently transfected with mRFP-Rab5b. Top part: In parental T23 cells, mRFP-Rab5b was distributed in fine punctate structures throughout the cells. Lower parts: T23 MDCK cells stably expressing wild-type EGFP-FIP2 and each of the mutants (EGFP-FIP2(SARG) and EGFP-FIP2(ΔC2)) were transiently transfected with mRFP-Rab5b and imaged by confocal microscopy. Overexpression of either wild-type Rab11-FIP2 or Rab11-FIP2(SARG) had no effect on the distribution of Rab5b. However, overexpression of EGFP-FIP2(ΔC2) caused a redistribution of mRFP-Rab5b-containing vesicles towards the Rab11-FIP2 containing membrane cisternae. Nevertheless, little direct co-localization was observed. Images were taken with a 100x lens with a 3x zoom. Scale bars represent 5 µm.
Figure 6
Figure 6
Differentiable influences of Rab11-FIP2 mutants on Epsin 4 localization. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). (A) Parental T23 MDCK cells were stained with Alexa-488-phalloidin (pseudo-colored green) and Epsin 4 (pseudo-colored red) antibodies and imaged by confocal microscopy to show endogenous ubiquitous localization. Epsin 4 was diffusely distributed throughout the cytoplasm. (B) Similarly, in T23 cells stably expressing EGFP-FIP2(SARG), Epsin 4 was distributed throughout the cytoplasm. However, overexpression of Rab11-FIP2(ΔC2) caused accumulation of Epsin 4-containing vesicles around the collapsed Rab11-FIP2-containing cisternae. All images are derived from 100x images. Scale bars are 10 µm.
Figure 7
Figure 7
Rab11-FIP2(ΔC2) alters the distribution of both Epsin 4 and EEA-1. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). T23 cells stably expressing either (upper parts) EGFP-FIP2(SARG) or (lower parts) EGFP-FIP2(ΔC2) were stained with antibodies against Epsin 4 (Cy3 secondary, pseudo-colored red) and EEA-1 (Cy5 secondary, pseudo-colored blue) antibodies and imaged by confocal microscopy. EGFP-FIP2(ΔC2) expression caused accumulation of both EEA-1 and Epsin 4 around the collapsed membrane cisternum. Images were taken with a 100x lens with a 3x zoom. Scale bars represent 5 µm.
Figure 8
Figure 8
Rab11-FIP2(ΔC2) alters the distribution of IQGAP1. Confocal fluorescence microscopic images of polarized MDCK cells. X-Y plane images are shown flanked by X-Z projections (horizontal) and Y-Z projections (vertical). (A) Parental T23 MDCK cells were stained with Alexa-488-phalloidin (pseudo-colored green) and antibodies against IQGAP1 (pseudo-colored red) and imaged by confocal microscopy. IQGAP was distributed throughout the cytosol with some concentration near lateral membranes. (B) In T23 cells stably expressing wild-type EGFP-FIP2, IQGAP1 was distributed diffusely within the cytoplasm. However, overexpression of Rab11-FIP2(ΔC2) caused the accumulation of vesicles containing IQGAP1 around the collapsed membrane cisternae containing EGFP-Rab11-FIP2. Images were taken with a 100x lens with a 3x zoom. Scale bars represent 5 µm.
Figure 9
Figure 9
Scheme for the influence of Rab11-FIP2 mutants on trafficking in polarized MDCK cells. While both Rab11-FIP2(ΔC2) and Rab11-FIP2(SARG) cause collapse of the apical recycling endosome (ARE) in a compressed membranous nexus, the morphologies are dissimilar. Rab11-FIP2(ΔC2) causes the formation of a doughnut shaped tubular cisternum that is located next to the centrosome (black bars depict the centrioles) (red arrow). In contrast, Rab11-FIP2(SARG) causes the formation of a tighter and more peripheral membrane nexus (green arrow). Additionally, it appears that the influence of Rab11-FIP2(ΔC2) is felt on earlier steps and also impacts on a range of early endosomal trafficking pathways. In contrast, Rab11-FIP2(SARG) seems to divert trafficking out of the ARE at a later stage and does not perturb early endosomal systems. Apical early endosomal (AEE) trafficking leading to apical recycling appears to avoid the influence of Rab11-FIP2 mutants, while the pathway initiated by basolateral early endosomes (BEE) for basolateral to apical transcytosis is strongly inhibited by both Rab11-FIP2 mutants. These findings support a hypothesis that Rab5a may regulate AEE function and apical recycling, while Rab5b and EEA-1 mediate BEE function and transcytosis.
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