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Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells.

Fields IC, King SM, Shteyn E, Kang RS, Fölsch H - Mol. Biol. Cell (2009)

Bottom Line: We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)].Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane.In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell-specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits micro1A or micro1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of micro1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

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PI(3,4,5)P3 localizes in recycling endosomes. MDCK cells seeded on coverslips were transiently transfected with cDNAs encoding PH-Akt-GFP (A and B), in addition to RFP-cellubrevin (A), or RFP-PH-Grp1 (B). Twenty-four hours after transfection, cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Cy5-labeled secondary antibodies (B). Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. Arrow in B indicates plasma membrane ruffles. (C) HeLa, HEK293, LLC-PK1::μ1A, LLC-PK1::μ1B, or MDCK cells seeded on coverslips were transfected with cDNA encoding PH-Akt-GFP and stained for TfnR as described in Figure 1. Specimens were analyzed by confocal microscopy, and in cells expressing PH-Akt-GFP the degree of overlap with TfnR in recycling endosomes was analyzed with Volocity software as described in Materials and Methods. Data represent mean values of 16 cells (LLC-PK1::μ1A), 21 cells (LLC-PK1::μ1B), 30 cells (HeLa), 40 cells (HEK293), and 35 cells (MDCK). Error bars, SD. Student's unpaired t test for all pairwise combinations; statistically significant with *p < 0.0001 or *p = 0.0002 (MDCK vs. HeLa), except for the pairs LLC- PK1::μ1B and MDCK (p = 0.0955) as well as LLC-PK1::μ1A and HEK293 (p = 0.3505), which showed no statistical significance.
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Figure 2: PI(3,4,5)P3 localizes in recycling endosomes. MDCK cells seeded on coverslips were transiently transfected with cDNAs encoding PH-Akt-GFP (A and B), in addition to RFP-cellubrevin (A), or RFP-PH-Grp1 (B). Twenty-four hours after transfection, cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Cy5-labeled secondary antibodies (B). Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. Arrow in B indicates plasma membrane ruffles. (C) HeLa, HEK293, LLC-PK1::μ1A, LLC-PK1::μ1B, or MDCK cells seeded on coverslips were transfected with cDNA encoding PH-Akt-GFP and stained for TfnR as described in Figure 1. Specimens were analyzed by confocal microscopy, and in cells expressing PH-Akt-GFP the degree of overlap with TfnR in recycling endosomes was analyzed with Volocity software as described in Materials and Methods. Data represent mean values of 16 cells (LLC-PK1::μ1A), 21 cells (LLC-PK1::μ1B), 30 cells (HeLa), 40 cells (HEK293), and 35 cells (MDCK). Error bars, SD. Student's unpaired t test for all pairwise combinations; statistically significant with *p < 0.0001 or *p = 0.0002 (MDCK vs. HeLa), except for the pairs LLC- PK1::μ1B and MDCK (p = 0.0955) as well as LLC-PK1::μ1A and HEK293 (p = 0.3505), which showed no statistical significance.

Mentions: Colocalization of PH-Akt-GFP and TfnR in recycling endosomes (see Figures 2C and 3B) or GFP-PIPKIγ-90 and TfnR (see Figure 4B) was analyzed with Volocity 4.4 software. Confocal raw data were imported into Volocity (Improvision, Lexington, MA), and the background threshold of the images was adjusted to the same degree in all images analyzed. We then circled the region of perinuclear TfnR staining as the region of interest for calculation of the overlap coefficient according to Manders (Manders et al., 1993). The data are expressed as % overlap. Statistical analysis was then performed as described in the previous paragraph.


Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells.

Fields IC, King SM, Shteyn E, Kang RS, Fölsch H - Mol. Biol. Cell (2009)

PI(3,4,5)P3 localizes in recycling endosomes. MDCK cells seeded on coverslips were transiently transfected with cDNAs encoding PH-Akt-GFP (A and B), in addition to RFP-cellubrevin (A), or RFP-PH-Grp1 (B). Twenty-four hours after transfection, cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Cy5-labeled secondary antibodies (B). Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. Arrow in B indicates plasma membrane ruffles. (C) HeLa, HEK293, LLC-PK1::μ1A, LLC-PK1::μ1B, or MDCK cells seeded on coverslips were transfected with cDNA encoding PH-Akt-GFP and stained for TfnR as described in Figure 1. Specimens were analyzed by confocal microscopy, and in cells expressing PH-Akt-GFP the degree of overlap with TfnR in recycling endosomes was analyzed with Volocity software as described in Materials and Methods. Data represent mean values of 16 cells (LLC-PK1::μ1A), 21 cells (LLC-PK1::μ1B), 30 cells (HeLa), 40 cells (HEK293), and 35 cells (MDCK). Error bars, SD. Student's unpaired t test for all pairwise combinations; statistically significant with *p < 0.0001 or *p = 0.0002 (MDCK vs. HeLa), except for the pairs LLC- PK1::μ1B and MDCK (p = 0.0955) as well as LLC-PK1::μ1A and HEK293 (p = 0.3505), which showed no statistical significance.
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Figure 2: PI(3,4,5)P3 localizes in recycling endosomes. MDCK cells seeded on coverslips were transiently transfected with cDNAs encoding PH-Akt-GFP (A and B), in addition to RFP-cellubrevin (A), or RFP-PH-Grp1 (B). Twenty-four hours after transfection, cells were fixed and incubated with anti-TfnR antibodies (H68.4) followed by incubation with Cy5-labeled secondary antibodies (B). Specimens were analyzed by confocal microscopy and representative images are shown. Scale bars, 10 μm. Arrow in B indicates plasma membrane ruffles. (C) HeLa, HEK293, LLC-PK1::μ1A, LLC-PK1::μ1B, or MDCK cells seeded on coverslips were transfected with cDNA encoding PH-Akt-GFP and stained for TfnR as described in Figure 1. Specimens were analyzed by confocal microscopy, and in cells expressing PH-Akt-GFP the degree of overlap with TfnR in recycling endosomes was analyzed with Volocity software as described in Materials and Methods. Data represent mean values of 16 cells (LLC-PK1::μ1A), 21 cells (LLC-PK1::μ1B), 30 cells (HeLa), 40 cells (HEK293), and 35 cells (MDCK). Error bars, SD. Student's unpaired t test for all pairwise combinations; statistically significant with *p < 0.0001 or *p = 0.0002 (MDCK vs. HeLa), except for the pairs LLC- PK1::μ1B and MDCK (p = 0.0955) as well as LLC-PK1::μ1A and HEK293 (p = 0.3505), which showed no statistical significance.
Mentions: Colocalization of PH-Akt-GFP and TfnR in recycling endosomes (see Figures 2C and 3B) or GFP-PIPKIγ-90 and TfnR (see Figure 4B) was analyzed with Volocity 4.4 software. Confocal raw data were imported into Volocity (Improvision, Lexington, MA), and the background threshold of the images was adjusted to the same degree in all images analyzed. We then circled the region of perinuclear TfnR staining as the region of interest for calculation of the overlap coefficient according to Manders (Manders et al., 1993). The data are expressed as % overlap. Statistical analysis was then performed as described in the previous paragraph.

Bottom Line: We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)].Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane.In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell-specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits micro1A or micro1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of micro1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

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