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Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking.

Solis GP, Hülsbusch N, Radon Y, Katanaev VL, Plattner H, Stuermer CA - Mol. Biol. Cell (2013)

Bottom Line: Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane.Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies.Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, 78467 Konstanz, Germany. Claudia.Stuermer@uni-konstanz.de

ABSTRACT
The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

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Characterization of reggie-tubules in HeLa cells. (A–C) Confocal microscopy of HeLa cells shows reggie-decorated tubules after expression of either reggie-1-EGFP (R1-EGFP; A) or reggie-2-EGFP (R2-EGFP; B) emanating from the perinuclear recycling compartment (arrowheads). Immunostaining of endogenous reggie-1 (R1; C) shows its localization at tubular structures and vesicles. Boxed area indicates adjacent enlargement. (D) Real-time images of HeLa cells expressing R1-EGFP were recorded for 2 min. In the first time-lapse recording (I; left, boxed region), a vesicle emerges from the end of a reggie-tubule and moves toward the PM (arrows). The second time-lapse recording (II; left, boxed region) shows the elongation of a reggie-tubule toward the PM (arrows). (E–H) R1-EGFP–decorated tubules in HeLa cells partially colocalize with the α-tubulin microtubules (α-tub; E) and the F-actin cytoskeleton (G). Exposure to nocodazole (F) or cytochalasin D (H) caused collapse and abnormal number and organization of reggie-tubules, respectively. Boxed areas are enlarged in insets. (I) Quantification of dimethyl sulfoxide (control), nocodazole (Noco), cytochalasin D (CytD), and filipin effects on reggie-tubule formation in HeLa cells (n = 3, **p < 0.01, ***p < 0.001, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.
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Figure 1: Characterization of reggie-tubules in HeLa cells. (A–C) Confocal microscopy of HeLa cells shows reggie-decorated tubules after expression of either reggie-1-EGFP (R1-EGFP; A) or reggie-2-EGFP (R2-EGFP; B) emanating from the perinuclear recycling compartment (arrowheads). Immunostaining of endogenous reggie-1 (R1; C) shows its localization at tubular structures and vesicles. Boxed area indicates adjacent enlargement. (D) Real-time images of HeLa cells expressing R1-EGFP were recorded for 2 min. In the first time-lapse recording (I; left, boxed region), a vesicle emerges from the end of a reggie-tubule and moves toward the PM (arrows). The second time-lapse recording (II; left, boxed region) shows the elongation of a reggie-tubule toward the PM (arrows). (E–H) R1-EGFP–decorated tubules in HeLa cells partially colocalize with the α-tubulin microtubules (α-tub; E) and the F-actin cytoskeleton (G). Exposure to nocodazole (F) or cytochalasin D (H) caused collapse and abnormal number and organization of reggie-tubules, respectively. Boxed areas are enlarged in insets. (I) Quantification of dimethyl sulfoxide (control), nocodazole (Noco), cytochalasin D (CytD), and filipin effects on reggie-tubule formation in HeLa cells (n = 3, **p < 0.01, ***p < 0.001, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.

Mentions: In our attempt to understand the contribution of reggie to the intracellular trafficking and recycling of membrane proteins (TfR in HeLa and E-cadherin in A431 cells), we transfected HeLa cells with reggie-1–enhanced green fluorescent protein (EGFP) or reggie-2–EGFP. Of interest, expression of either reggie-1 or -2 led to the formation of prominent reggie-positive tubular structures, which emerged from the perinuclear recycling compartment and reached to the vicinity of the PM (Figure 1, A and B). Such tubules were also apparent after immunostaining with a reggie-1–specific antibody (Ab) consisting of conspicuous rows of orderly aligned puncta and centered on the recycling compartment (Figure 1C). In addition, reggie-1–specific Abs labeled small and larger vesicles (the latter representing lysosomes; Stuermer et al., 2001). In further experiments aimed at characterizing of tubulovesicular system, we focused on reggie-1 and determined by immunostaining analyses that tubules decorated by reggie-1 (hereafter reggie-tubules) are not constituents of the endoplasmic reticulum, Golgi, and mitochondrial endomembrane systems (Supplemental Figure S1, A–C). Reggie-tubules appeared highly dynamic, with vesicles deriving from and merging with tubules and moving toward and away from the PM (Figure 1D and Supplemental Movie S1). Although less prominent, dynamic reggie-tubules were also observed in A431 cells (Supplemental Figure S1D and Supplemental Movie S2).


Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking.

Solis GP, Hülsbusch N, Radon Y, Katanaev VL, Plattner H, Stuermer CA - Mol. Biol. Cell (2013)

Characterization of reggie-tubules in HeLa cells. (A–C) Confocal microscopy of HeLa cells shows reggie-decorated tubules after expression of either reggie-1-EGFP (R1-EGFP; A) or reggie-2-EGFP (R2-EGFP; B) emanating from the perinuclear recycling compartment (arrowheads). Immunostaining of endogenous reggie-1 (R1; C) shows its localization at tubular structures and vesicles. Boxed area indicates adjacent enlargement. (D) Real-time images of HeLa cells expressing R1-EGFP were recorded for 2 min. In the first time-lapse recording (I; left, boxed region), a vesicle emerges from the end of a reggie-tubule and moves toward the PM (arrows). The second time-lapse recording (II; left, boxed region) shows the elongation of a reggie-tubule toward the PM (arrows). (E–H) R1-EGFP–decorated tubules in HeLa cells partially colocalize with the α-tubulin microtubules (α-tub; E) and the F-actin cytoskeleton (G). Exposure to nocodazole (F) or cytochalasin D (H) caused collapse and abnormal number and organization of reggie-tubules, respectively. Boxed areas are enlarged in insets. (I) Quantification of dimethyl sulfoxide (control), nocodazole (Noco), cytochalasin D (CytD), and filipin effects on reggie-tubule formation in HeLa cells (n = 3, **p < 0.01, ***p < 0.001, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.
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Related In: Results  -  Collection

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Figure 1: Characterization of reggie-tubules in HeLa cells. (A–C) Confocal microscopy of HeLa cells shows reggie-decorated tubules after expression of either reggie-1-EGFP (R1-EGFP; A) or reggie-2-EGFP (R2-EGFP; B) emanating from the perinuclear recycling compartment (arrowheads). Immunostaining of endogenous reggie-1 (R1; C) shows its localization at tubular structures and vesicles. Boxed area indicates adjacent enlargement. (D) Real-time images of HeLa cells expressing R1-EGFP were recorded for 2 min. In the first time-lapse recording (I; left, boxed region), a vesicle emerges from the end of a reggie-tubule and moves toward the PM (arrows). The second time-lapse recording (II; left, boxed region) shows the elongation of a reggie-tubule toward the PM (arrows). (E–H) R1-EGFP–decorated tubules in HeLa cells partially colocalize with the α-tubulin microtubules (α-tub; E) and the F-actin cytoskeleton (G). Exposure to nocodazole (F) or cytochalasin D (H) caused collapse and abnormal number and organization of reggie-tubules, respectively. Boxed areas are enlarged in insets. (I) Quantification of dimethyl sulfoxide (control), nocodazole (Noco), cytochalasin D (CytD), and filipin effects on reggie-tubule formation in HeLa cells (n = 3, **p < 0.01, ***p < 0.001, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.
Mentions: In our attempt to understand the contribution of reggie to the intracellular trafficking and recycling of membrane proteins (TfR in HeLa and E-cadherin in A431 cells), we transfected HeLa cells with reggie-1–enhanced green fluorescent protein (EGFP) or reggie-2–EGFP. Of interest, expression of either reggie-1 or -2 led to the formation of prominent reggie-positive tubular structures, which emerged from the perinuclear recycling compartment and reached to the vicinity of the PM (Figure 1, A and B). Such tubules were also apparent after immunostaining with a reggie-1–specific antibody (Ab) consisting of conspicuous rows of orderly aligned puncta and centered on the recycling compartment (Figure 1C). In addition, reggie-1–specific Abs labeled small and larger vesicles (the latter representing lysosomes; Stuermer et al., 2001). In further experiments aimed at characterizing of tubulovesicular system, we focused on reggie-1 and determined by immunostaining analyses that tubules decorated by reggie-1 (hereafter reggie-tubules) are not constituents of the endoplasmic reticulum, Golgi, and mitochondrial endomembrane systems (Supplemental Figure S1, A–C). Reggie-tubules appeared highly dynamic, with vesicles deriving from and merging with tubules and moving toward and away from the PM (Figure 1D and Supplemental Movie S1). Although less prominent, dynamic reggie-tubules were also observed in A431 cells (Supplemental Figure S1D and Supplemental Movie S2).

Bottom Line: Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane.Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies.Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, 78467 Konstanz, Germany. Claudia.Stuermer@uni-konstanz.de

ABSTRACT
The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

Show MeSH
Related in: MedlinePlus