Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: During recovery from low calcium, Eps15 arrived at the lateral membrane before pS227-FIP2. MDCK cells were grown on Transwell supports for 5 d postconfluence, incubated overnight in low calcium, and then returned to regular media and allowed to recovery for the hours indicated on the left. Cells were fixed in methanol and stained for pS227-FIP2 (green merge), Eps15 (red in merge), and E-cadherin (blue in merge). White arrowheads indicate areas of colocalization of pS227-FIP2, Eps15, and p120. Black arrowheads indicate where the xy - and xz -slices were taken. Bar, 10 μm.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Membrane, Incubation, Staining

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: Eps15 accumulated in GFP-FIP2(WT) and GFP-FIP2(S227E)–containing compartments but not the GFP-FIP2(S227A)–containing membranes. (A) The GFP-FIP2–expressing MDCK cell lines were grown to subconfluence on coverglass slips, fixed in 4% paraformaldehyde, and stained for Eps15 (red in merge) and Rab5 (blue in merge). Bar, 5 μm. (B) Manders quantification of colocalization in A; a minimum of 60 cells were counted per cell line. Eps15 colocalization with GFP-FIP2(S227A) exhibited a significant decrease compared with Eps15 with GFP-FIP2(WT) or GFP-FIP2(S227E). * p < 0.05 by Dunn’s test.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: Mutation of all three NPF domains or any individual NPF domain of FIP2 released Eps15 from the GFP-FIP2(S227E) compartment. (A) The GFP-FIP2(S227E) MDCK cell lines with or without the NPF domain muta­tions were plated onto glass coverslips, fixed with 4% paraformaldehyde, and stained for Eps15 (red in merge) and F-actin (phalloidin; blue in merge). Bar, 5 μm. (B) Manders quantification of the colocalization shown in A; minimum of 30 cells per cell line were counted. The GFP-FIP2(S227E) containing any of the NPF domain mutations (SEdNPF1, SEdNPF2, or SEdNPF3) exhibited a significant loss of colo­cali­zation compared with GFP-FIP2(S227E). Mutation of all three NPF domains (SEdNPF123) elicited a further significant decrease in colocalization. * p < 0.05 by Dunn’s test vs. SE. ** p < 0.05 vs. all other groups. (C) Results of yeast two-hybrid assay. The amount of β-gala­ctosidase activity was calculated by compassion to a standard curve of known β-gala­ctosidase concentrations. The assay was performed three separate times. NEG, negative control. The GFP-FIP2(S227E) containing any of the NPF domain mutations exhibited a signi­ficant loss of colocalization compared with GFP-FIP2(S227E). * p < 0.05 by Dunn’s test. (D) GFP-FIP2(S227E) and GFP-FIP2(S227EΔNFP123) MDCK cells were transfected mCherry-Eps15, fixed, and stained for p120 (blue in merge). mCherry-Eps15 was localized with the GFP-FIP2(S227E) but not with coexpressed GFP-FIP2(S227ΔNPF123). (E) Western blot of mCherry-Eps15 precipitated from GFP-FIP2(S227) or GFP-FIP2(S227ΔNPF123)–expressing cells. The blot was probed simultaneously for GFP (top) and Eps15 (bottom) and imaged on a LiCor Odyssey FC imager. Size makers are shown on the left.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Mutagenesis, Staining, Y2H Assay, Activity Assay, Negative Control, Transfection, Western Blot, Expressing

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: Eps15 localized to the central GFP-FIP2(S2227E) compartment and away from the lateral membrane in low calcium. The MDCK cell line expressing GFP-FIP2(S227E) was grown on Transwells, switched into low-calcium medium, and allowed to recover for the hours listed on the left. Cells were fixed in 4% paraformaldehyde and stained for Eps15 (red in merge) and p120 (blue in merge). Black arrowheads indicate where xy - and xz -slices were taken. Bar, 10 μm.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Membrane, Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: Mutation of the second NPF domains in GFP-FIP2(S227E) released Eps15 from the GFP-FIP2(S227E) compartment, allowing Eps15 to localize at the lateral membrane. The MDCK cell line expressing GFP-FIP2(S227EΔNPF2) was grown on Transwells, switched into low-calcium medium, and then allowed to recover for the hours listed on the left. Cells were fixed in 4% paraformaldehyde and stained for Eps15 (red in merge) and p120 (blue in merge). Black arrowheads indicate where the xy - and xz -slices were taken. Bar, 10 μm.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Mutagenesis, Membrane, Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: pS227-FIP2 did not colocalize with two known Eps15-binding proteins. The parental MDCK cells (top) or the GFP-FIP2(S227E)–expressing MDCK cells (bottom) were grown on coverslips, fixed with paraformaldehyde, and stained for Eps15 (blue in merge) and either AP-2 (A; red in merge) or Numb (B; red in merge). Bar, 10 μm. (C) Quantitation of Eps15, AP-2, and Numb colocalization with GFP-FIP2(S227E). At least 30 cells were counted for each staining pattern. * p < 0.05 by Dunn’s test compared with Eps15 colocalization.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Binding Assay, Expressing, Staining, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: Release of Eps15 from the GFP-FIP2(S227E) compartment by mutation of any of the NPF domains rescued the expression and localization of both E-cadherin and occludin at the junctions. The GFP-FIP2(S227E)–expressing MDCK cell lines were grown on Transwells, fixed with methanol, and stained for (A) E-cadherin (red in merge) and p120 (blue in merge) or (D) occludin (red in merge) and ZO-1 (blue in merge). Black arrowheads indicate where the xy - and xz -slices were taken. Bars, 10 μm. The cell lines were also grown in 10-cm tissue culture dishes for Western blots (B, E). (B) Representative blot probed for E-cadherin (top) and VDAC (bottom). (C) The adjusted density to VDAC of three similar blots for E-cadherin. (E) A representative blot probed for occludin (top) and VDAC (bottom). (F) The adjusted density to VDAC of three similar blots for occludin. * p < 0.05 by Dunn’s test vs. parental MDCK cells.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Mutagenesis, Expressing, Staining, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: Release of Eps15 from the GFP-FIP2(S227E) compartment by mutation of the second NPF domain resolved the multilumen GFP-FIP2(S227E) morphology back to a single- lumen morphology. Parental MDCK cells (top), GFP-FIP2(S227)–expressing MDCK cells (middle, green in merge), and GFP-FIP2(S227ΔNPF2)–expressing MDCK cells (bottom, green in merge) were grown in Matrigel for 7 d, fixed, and stained for podocalyxin (red in merge), F-actin (cyan in merge), and DAPI (blue in merge). Bars, 5 μm. Numbers in the lower right corner of the merged images indicate the percentage of cysts exhibiting the illustrated morphology; 15 cysts from three separate experiments were counted.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Mutagenesis, Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: The temporal and spatial timing of pS227-FIP2 was altered when Eps15 protein expression was knocked down. An MDCK cell line expressing shRNA to Eps15 was grown on Transwells, switched into low-calcium medium, allowed to recover from low calcium for the indicted hours, and then fixed in methanol and stained for pS227-FIP2 (green in merge), Eps15 (red in merge), and p120 (blue in merge). pS227-FIP2 was observed in almost all of the time points, with the least seen at 1 h. White arrowheads indicate lateral “corners” of cells where pS227-FIP2 was observed. White arrows indicate central areas where pS227-FIP2 was observed. Black arrowheads indicate where the xy - and xz -slices were taken. Bar, 10 μm.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Expressing, shRNA, Staining

Journal: Molecular Biology of the Cell

Article Title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

doi: 10.1091/mbc.E16-04-0214

Figure Lengend Snippet: Growth of the Eps15-knockdown MDCK cell line in Matrigel resulted in multilumen cysts. The MDCK control knockdown cell line (top) and the MDCK Eps15 knockdown cell line (bottom) were grown in Matrigel for 2 d (top) or 7 d (bottom), fixed, and stained for Eps15 (green in merge), podocalyxin (red in merge), F-actin (cyan in merge), and DAPI (blue in merge). Bars, 5 μm. Numbers in the lower right corner of the merged images indicate the percentage of cysts exhibiting the illustrated morphology; 15 cysts from three separate experiments were counted.

Article Snippet: The GFP-Rab11-FIP2 and mutant tet-off MDCK cell lines (kind gift of Keth Mostov, University California, San Francisco, San Francisco, CA) were grown as previously described ( Ducharme et al. , 2006 ) in DMEM supplemented with l -glutamine, nonessential amino acids, and G418 (all from Cellgro) and 10% fetal bovine serum (FBS; GE Heathcare Life Sciences-Hyclone, Logan, UT).

Techniques: Knockdown, Control, Staining