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The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics.

Garbett D, Sauvanet C, Viswanatha R, Bretscher A - Mol. Biol. Cell (2013)

Bottom Line: Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53).Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively.Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853.

ABSTRACT
The closely related apical scaffolding proteins ERM-binding phosphoprotein of 50 kDa (EBP50) and NHE3 kinase A regulatory protein (E3KARP) both consist of two postsynaptic density 95/disks large/zona occludens-1 (PDZ) domains and a tail ending in an ezrin-binding domain. Scaffolding proteins are thought to provide stable linkages between components of multiprotein complexes, yet in several types of epithelial cells, EBP50, but not E3KARP, shows rapid exchange from microvilli compared with its binding partners. The difference in dynamics is determined by the proteins' tail regions. Exchange rates of EBP50 and E3KARP correlated strongly with their abilities to precipitate ezrin in vivo. The EBP50 tail alone is highly dynamic, but in the context of the full-length protein, the dynamics is lost when the PDZ domains are unable to bind ligand. Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53). Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively. Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

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The tail of E3KARP causes preferential localization to the base of microvilli. (A) Representative maximum projection images of JEG-3 cells expressing GFP-tagged EBP50, E3KARP, or the chimeras EBP50-E3tail and E3KARP-50tail. GFP signal is shown in green; ezrin staining in red; actin in blue. Scale bars: 10 μm. (B) Top, highly magnified regions of microvilli containing GFP-tagged EBP50, E3KARP, or the tail chimeras, and stained for ezrin (red). Scale bars: 2 μm. Bottom, graphs of normalized intensity of ezrin (red) and GFP (green) of multiple microvilli (n ≥ 5) were plotted (open circles) as a function of the percent of the total microvillar length with 0 and 100% representing the tip and base respectively. The data were fitted to a LOWESS function (shown as a solid line).
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Figure 7: The tail of E3KARP causes preferential localization to the base of microvilli. (A) Representative maximum projection images of JEG-3 cells expressing GFP-tagged EBP50, E3KARP, or the chimeras EBP50-E3tail and E3KARP-50tail. GFP signal is shown in green; ezrin staining in red; actin in blue. Scale bars: 10 μm. (B) Top, highly magnified regions of microvilli containing GFP-tagged EBP50, E3KARP, or the tail chimeras, and stained for ezrin (red). Scale bars: 2 μm. Bottom, graphs of normalized intensity of ezrin (red) and GFP (green) of multiple microvilli (n ≥ 5) were plotted (open circles) as a function of the percent of the total microvillar length with 0 and 100% representing the tip and base respectively. The data were fitted to a LOWESS function (shown as a solid line).

Mentions: It has been suggested that E3KARP localizes toward the terminal web region below microvilli in renal proximal tubules (Wade et al., 2003). Using high-resolution confocal microscopy of JEG-3 cells, we have shown that we are able to localize microvillar proteins to different subdomains within a single microvillus (Hanono et al., 2006; Viswanatha et al., 2012). To further explore the localization differences between EBP50 and E3KARP on the submicrovillar scale, we expressed GFP-EBP50, GFP-E3KARP, or the chimeras GFP-EBP50-E3tail and GFP-E3KARP-50tail in JEG-3 cells and visualized the GFP signal in relation to endogenous ezrin (Figure 7A). The fluorescence intensity of GFP and ezrin was then measured within multiple microvilli and fitted to a locally weighted scatterplot-smoothing (LOWESS) function. In cells with both high and moderate levels of GFP fluorescence, EBP50 and E3KARP-50tail both localized along the full-length of microvilli as determined by ezrin staining (Figure 7B). In contrast, in cells with moderate levels of GFP fluorescence, E3KARP and EBP50-E3tail both showed a strong localization toward the base of microvilli that also persisted slightly into the terminal web area, where ezrin staining disappears (Figure 7B). Deletion of EBP50 residues 263–268, 271–279, 281–289, or 313–320 did not have any effect on its localization within microvilli (Supplemental Figure S1). Additionally, the tails of EBP50 (residues 242–358) and E3KARP (residues 239–337) alone show similar localizations to full-length EBP50 and E3KARP, respectively (Figure S2), although the base localization of the E3KARP tail alone was less prevalent than with full-length E3KARP. Therefore E3KARP localizes toward the base of microvilli, and this is a characteristic of its tail, which can shift EBP50 to the microvillar base, as seen with the EBP50-E3tail chimera.


The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics.

Garbett D, Sauvanet C, Viswanatha R, Bretscher A - Mol. Biol. Cell (2013)

The tail of E3KARP causes preferential localization to the base of microvilli. (A) Representative maximum projection images of JEG-3 cells expressing GFP-tagged EBP50, E3KARP, or the chimeras EBP50-E3tail and E3KARP-50tail. GFP signal is shown in green; ezrin staining in red; actin in blue. Scale bars: 10 μm. (B) Top, highly magnified regions of microvilli containing GFP-tagged EBP50, E3KARP, or the tail chimeras, and stained for ezrin (red). Scale bars: 2 μm. Bottom, graphs of normalized intensity of ezrin (red) and GFP (green) of multiple microvilli (n ≥ 5) were plotted (open circles) as a function of the percent of the total microvillar length with 0 and 100% representing the tip and base respectively. The data were fitted to a LOWESS function (shown as a solid line).
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Figure 7: The tail of E3KARP causes preferential localization to the base of microvilli. (A) Representative maximum projection images of JEG-3 cells expressing GFP-tagged EBP50, E3KARP, or the chimeras EBP50-E3tail and E3KARP-50tail. GFP signal is shown in green; ezrin staining in red; actin in blue. Scale bars: 10 μm. (B) Top, highly magnified regions of microvilli containing GFP-tagged EBP50, E3KARP, or the tail chimeras, and stained for ezrin (red). Scale bars: 2 μm. Bottom, graphs of normalized intensity of ezrin (red) and GFP (green) of multiple microvilli (n ≥ 5) were plotted (open circles) as a function of the percent of the total microvillar length with 0 and 100% representing the tip and base respectively. The data were fitted to a LOWESS function (shown as a solid line).
Mentions: It has been suggested that E3KARP localizes toward the terminal web region below microvilli in renal proximal tubules (Wade et al., 2003). Using high-resolution confocal microscopy of JEG-3 cells, we have shown that we are able to localize microvillar proteins to different subdomains within a single microvillus (Hanono et al., 2006; Viswanatha et al., 2012). To further explore the localization differences between EBP50 and E3KARP on the submicrovillar scale, we expressed GFP-EBP50, GFP-E3KARP, or the chimeras GFP-EBP50-E3tail and GFP-E3KARP-50tail in JEG-3 cells and visualized the GFP signal in relation to endogenous ezrin (Figure 7A). The fluorescence intensity of GFP and ezrin was then measured within multiple microvilli and fitted to a locally weighted scatterplot-smoothing (LOWESS) function. In cells with both high and moderate levels of GFP fluorescence, EBP50 and E3KARP-50tail both localized along the full-length of microvilli as determined by ezrin staining (Figure 7B). In contrast, in cells with moderate levels of GFP fluorescence, E3KARP and EBP50-E3tail both showed a strong localization toward the base of microvilli that also persisted slightly into the terminal web area, where ezrin staining disappears (Figure 7B). Deletion of EBP50 residues 263–268, 271–279, 281–289, or 313–320 did not have any effect on its localization within microvilli (Supplemental Figure S1). Additionally, the tails of EBP50 (residues 242–358) and E3KARP (residues 239–337) alone show similar localizations to full-length EBP50 and E3KARP, respectively (Figure S2), although the base localization of the E3KARP tail alone was less prevalent than with full-length E3KARP. Therefore E3KARP localizes toward the base of microvilli, and this is a characteristic of its tail, which can shift EBP50 to the microvillar base, as seen with the EBP50-E3tail chimera.

Bottom Line: Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53).Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively.Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853.

ABSTRACT
The closely related apical scaffolding proteins ERM-binding phosphoprotein of 50 kDa (EBP50) and NHE3 kinase A regulatory protein (E3KARP) both consist of two postsynaptic density 95/disks large/zona occludens-1 (PDZ) domains and a tail ending in an ezrin-binding domain. Scaffolding proteins are thought to provide stable linkages between components of multiprotein complexes, yet in several types of epithelial cells, EBP50, but not E3KARP, shows rapid exchange from microvilli compared with its binding partners. The difference in dynamics is determined by the proteins' tail regions. Exchange rates of EBP50 and E3KARP correlated strongly with their abilities to precipitate ezrin in vivo. The EBP50 tail alone is highly dynamic, but in the context of the full-length protein, the dynamics is lost when the PDZ domains are unable to bind ligand. Proteomic analysis of the effects of EBP50 dynamics on binding-partner preferences identified a novel PDZ1 binding partner, the I-BAR protein insulin receptor substrate p53 (IRSp53). Additionally, the tails promote different microvillar localizations for EBP50 and E3KARP, which localized along the full length and to the base of microvilli, respectively. Thus the tails define the localization and dynamics of these scaffolding proteins, and the high dynamics of EBP50 is regulated by the occupancy of its PDZ domains.

Show MeSH
Related in: MedlinePlus