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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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Down-regulation of reggie-1 impairs Tf recycling in HeLa cells. (A) Wild-type and shRNA stably transfected HeLa cells were pulsed with Tf-rhod for 5 min and then chased for 10 and 20 min. Reggie-depleted (shR1) cells showed no defects in Tf-rhod uptake (5-min pulse) and transport from early endosomes to the recycling compartment (10-min chase) compared with control transfected (shLuc) and untransfected HeLa cells. After a 20-min chase, however, the accumulation of Tf-rhod was retained at the perinuclear compartment in the majority of shR1 cells but reduced in shLuc and HeLa cells. (B, C) Quantification of the effect of reggie-1 down-regulation on Tf-rhod uptake (B) and recycling (C) in HeLa cells (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). (D) Western blot (WB) analysis of pulse-chase experiments in shLuc and shR1 cells using biotinylated Tf (Tf-biotin) confirmed that Tf recycling was delayed in reggie-depleted cells after a 20-min chase. No significant difference was observed in Tf-biotin uptake upon reggie down-regulation (n = 4, **p < 0.01, paired t test, mean ± SEM). α-Tubulin (α-tub) was used as loading control. (E) Expression of a shRNA-resistant reggie-1 construct (R1-EGFP rescue) rescued the Tf-rhod recycling defects observed after a 20-min chase in transfected (arrowheads) but not in untransfected shR1 cells. (F, G) Pulse-chase experiments were performed in shR1 (F) and control shLuc (G) cells expressing a Rab11a constitutively active (EGFP-Rab11a-CA) and dominant-negative (EGFP-Rab11a-DN) mutant, respectively. Whereas the Rab11a-CA construct was able to rescue the Tf-rhod recycling defects in shR1 cells without affecting its uptake (arrowheads; F), in shLuc cells the Rab11a-DN mutant impaired both Tf-rhod uptake and recycling (arrowheads; G). (H, I) Quantification of Tf-rhod perinuclear accumulation from pulse-chase experiments in E–G. A constitutively active mutant of Rab8a (EGFP-Rab8a-CA) was not able to rescue or mimic the effects of reggie depletion in shR1 or shLuc cells, respectively (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.
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Figure 5: Down-regulation of reggie-1 impairs Tf recycling in HeLa cells. (A) Wild-type and shRNA stably transfected HeLa cells were pulsed with Tf-rhod for 5 min and then chased for 10 and 20 min. Reggie-depleted (shR1) cells showed no defects in Tf-rhod uptake (5-min pulse) and transport from early endosomes to the recycling compartment (10-min chase) compared with control transfected (shLuc) and untransfected HeLa cells. After a 20-min chase, however, the accumulation of Tf-rhod was retained at the perinuclear compartment in the majority of shR1 cells but reduced in shLuc and HeLa cells. (B, C) Quantification of the effect of reggie-1 down-regulation on Tf-rhod uptake (B) and recycling (C) in HeLa cells (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). (D) Western blot (WB) analysis of pulse-chase experiments in shLuc and shR1 cells using biotinylated Tf (Tf-biotin) confirmed that Tf recycling was delayed in reggie-depleted cells after a 20-min chase. No significant difference was observed in Tf-biotin uptake upon reggie down-regulation (n = 4, **p < 0.01, paired t test, mean ± SEM). α-Tubulin (α-tub) was used as loading control. (E) Expression of a shRNA-resistant reggie-1 construct (R1-EGFP rescue) rescued the Tf-rhod recycling defects observed after a 20-min chase in transfected (arrowheads) but not in untransfected shR1 cells. (F, G) Pulse-chase experiments were performed in shR1 (F) and control shLuc (G) cells expressing a Rab11a constitutively active (EGFP-Rab11a-CA) and dominant-negative (EGFP-Rab11a-DN) mutant, respectively. Whereas the Rab11a-CA construct was able to rescue the Tf-rhod recycling defects in shR1 cells without affecting its uptake (arrowheads; F), in shLuc cells the Rab11a-DN mutant impaired both Tf-rhod uptake and recycling (arrowheads; G). (H, I) Quantification of Tf-rhod perinuclear accumulation from pulse-chase experiments in E–G. A constitutively active mutant of Rab8a (EGFP-Rab8a-CA) was not able to rescue or mimic the effects of reggie depletion in shR1 or shLuc cells, respectively (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.

Mentions: To determine whether reggie-1 is indeed involved in cargo recycling, we analyzed the trafficking of rhodamine-labeled transferrin (Tf-rhod) in HeLa cells expressing reggie-1–EGFP. No apparent colocalization between Tf-rhod and reggie-positive perinuclear structures was observed after 5 min of incubation (Figure 4E), suggesting that reggie-1 is not involved in Tf endocytosis. In a pulse-chase experiment, HeLa cells were incubated for 5 min with Tf-rhod (pulse), washed, and left for 10 min (chase) to allow Tf trafficking to the recycling compartment. As expected, Tf-rhod accumulated at the reggie-positive perinuclear recycling compartment (Figure 4F). Accordingly, quantification of the Pearson's r revealed a twofold increase in the colocalization of Tf-rhod and reggie-1-EGFP after a 10-min chase (0.21 ± 0.02 for a 5-min pulse and 0.42 ± 0.04 for a 5-min pulse/10-min chase; p < 0.001). Moreover, the TfR also accumulated at the perinuclear compartment in a similar pulse-chase experiment and colocalized with endogenous reggie-1 (Supplemental Figure S5E), suggesting that reggies may be involved in TfR recycling. How reggies affect Tf trafficking was examined using the pulse-chase method in shR1 cells. The amount and distribution of incorporated Tf-rhod did not differ between shR1, shLuc, and untransfected HeLa cells after a 5-min pulse (Figure 5, A and B). After a 10-min chase, cells showed similar accumulation of Tf-rhod at the perinuclear compartment (Figure 5A), excluding a major role of reggies in the endocytosis of Tf-rhod and its transport from early endosomes to the recycling compartment. Of importance, however, the perinuclear accumulation of Tf-rhod increased ∼40% in shR1 cells after a 20-min chase compared with shLuc and untransfected HeLa cells (Figure 5, A and C). Immunostainings also revealed increased accumulation of TfR at the perinuclear compartment in shR1 cells after a 20-min chase (Supplemental Figure S5, F and G). Therefore the absence of reggies seems to impair TfR recycling. Biochemical analysis of pulse-chase experiments using biotinylated Tf confirmed that down-regulation of reggies did not affect Tf endocytosis but significantly delayed its recycling after a 20-min chase (Figure 5D). The specificity of this phenotype was supported by a rescue experiment in which the shR1 cells were transfected with a shRNA-resistant reggie-1 construct (Solis et al., 2007). After a 20-min chase, the Tf-rhod accumulation at the perinuclear region was reduced to the normal level in cells in which reggie-1 was reintroduced (Figure 5, E and H) but not in untransfected shR1 cells.


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)

Down-regulation of reggie-1 impairs Tf recycling in HeLa cells. (A) Wild-type and shRNA stably transfected HeLa cells were pulsed with Tf-rhod for 5 min and then chased for 10 and 20 min. Reggie-depleted (shR1) cells showed no defects in Tf-rhod uptake (5-min pulse) and transport from early endosomes to the recycling compartment (10-min chase) compared with control transfected (shLuc) and untransfected HeLa cells. After a 20-min chase, however, the accumulation of Tf-rhod was retained at the perinuclear compartment in the majority of shR1 cells but reduced in shLuc and HeLa cells. (B, C) Quantification of the effect of reggie-1 down-regulation on Tf-rhod uptake (B) and recycling (C) in HeLa cells (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). (D) Western blot (WB) analysis of pulse-chase experiments in shLuc and shR1 cells using biotinylated Tf (Tf-biotin) confirmed that Tf recycling was delayed in reggie-depleted cells after a 20-min chase. No significant difference was observed in Tf-biotin uptake upon reggie down-regulation (n = 4, **p < 0.01, paired t test, mean ± SEM). α-Tubulin (α-tub) was used as loading control. (E) Expression of a shRNA-resistant reggie-1 construct (R1-EGFP rescue) rescued the Tf-rhod recycling defects observed after a 20-min chase in transfected (arrowheads) but not in untransfected shR1 cells. (F, G) Pulse-chase experiments were performed in shR1 (F) and control shLuc (G) cells expressing a Rab11a constitutively active (EGFP-Rab11a-CA) and dominant-negative (EGFP-Rab11a-DN) mutant, respectively. Whereas the Rab11a-CA construct was able to rescue the Tf-rhod recycling defects in shR1 cells without affecting its uptake (arrowheads; F), in shLuc cells the Rab11a-DN mutant impaired both Tf-rhod uptake and recycling (arrowheads; G). (H, I) Quantification of Tf-rhod perinuclear accumulation from pulse-chase experiments in E–G. A constitutively active mutant of Rab8a (EGFP-Rab8a-CA) was not able to rescue or mimic the effects of reggie depletion in shR1 or shLuc cells, respectively (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.
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Figure 5: Down-regulation of reggie-1 impairs Tf recycling in HeLa cells. (A) Wild-type and shRNA stably transfected HeLa cells were pulsed with Tf-rhod for 5 min and then chased for 10 and 20 min. Reggie-depleted (shR1) cells showed no defects in Tf-rhod uptake (5-min pulse) and transport from early endosomes to the recycling compartment (10-min chase) compared with control transfected (shLuc) and untransfected HeLa cells. After a 20-min chase, however, the accumulation of Tf-rhod was retained at the perinuclear compartment in the majority of shR1 cells but reduced in shLuc and HeLa cells. (B, C) Quantification of the effect of reggie-1 down-regulation on Tf-rhod uptake (B) and recycling (C) in HeLa cells (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). (D) Western blot (WB) analysis of pulse-chase experiments in shLuc and shR1 cells using biotinylated Tf (Tf-biotin) confirmed that Tf recycling was delayed in reggie-depleted cells after a 20-min chase. No significant difference was observed in Tf-biotin uptake upon reggie down-regulation (n = 4, **p < 0.01, paired t test, mean ± SEM). α-Tubulin (α-tub) was used as loading control. (E) Expression of a shRNA-resistant reggie-1 construct (R1-EGFP rescue) rescued the Tf-rhod recycling defects observed after a 20-min chase in transfected (arrowheads) but not in untransfected shR1 cells. (F, G) Pulse-chase experiments were performed in shR1 (F) and control shLuc (G) cells expressing a Rab11a constitutively active (EGFP-Rab11a-CA) and dominant-negative (EGFP-Rab11a-DN) mutant, respectively. Whereas the Rab11a-CA construct was able to rescue the Tf-rhod recycling defects in shR1 cells without affecting its uptake (arrowheads; F), in shLuc cells the Rab11a-DN mutant impaired both Tf-rhod uptake and recycling (arrowheads; G). (H, I) Quantification of Tf-rhod perinuclear accumulation from pulse-chase experiments in E–G. A constitutively active mutant of Rab8a (EGFP-Rab8a-CA) was not able to rescue or mimic the effects of reggie depletion in shR1 or shLuc cells, respectively (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.
Mentions: To determine whether reggie-1 is indeed involved in cargo recycling, we analyzed the trafficking of rhodamine-labeled transferrin (Tf-rhod) in HeLa cells expressing reggie-1–EGFP. No apparent colocalization between Tf-rhod and reggie-positive perinuclear structures was observed after 5 min of incubation (Figure 4E), suggesting that reggie-1 is not involved in Tf endocytosis. In a pulse-chase experiment, HeLa cells were incubated for 5 min with Tf-rhod (pulse), washed, and left for 10 min (chase) to allow Tf trafficking to the recycling compartment. As expected, Tf-rhod accumulated at the reggie-positive perinuclear recycling compartment (Figure 4F). Accordingly, quantification of the Pearson's r revealed a twofold increase in the colocalization of Tf-rhod and reggie-1-EGFP after a 10-min chase (0.21 ± 0.02 for a 5-min pulse and 0.42 ± 0.04 for a 5-min pulse/10-min chase; p < 0.001). Moreover, the TfR also accumulated at the perinuclear compartment in a similar pulse-chase experiment and colocalized with endogenous reggie-1 (Supplemental Figure S5E), suggesting that reggies may be involved in TfR recycling. How reggies affect Tf trafficking was examined using the pulse-chase method in shR1 cells. The amount and distribution of incorporated Tf-rhod did not differ between shR1, shLuc, and untransfected HeLa cells after a 5-min pulse (Figure 5, A and B). After a 10-min chase, cells showed similar accumulation of Tf-rhod at the perinuclear compartment (Figure 5A), excluding a major role of reggies in the endocytosis of Tf-rhod and its transport from early endosomes to the recycling compartment. Of importance, however, the perinuclear accumulation of Tf-rhod increased ∼40% in shR1 cells after a 20-min chase compared with shLuc and untransfected HeLa cells (Figure 5, A and C). Immunostainings also revealed increased accumulation of TfR at the perinuclear compartment in shR1 cells after a 20-min chase (Supplemental Figure S5, F and G). Therefore the absence of reggies seems to impair TfR recycling. Biochemical analysis of pulse-chase experiments using biotinylated Tf confirmed that down-regulation of reggies did not affect Tf endocytosis but significantly delayed its recycling after a 20-min chase (Figure 5D). The specificity of this phenotype was supported by a rescue experiment in which the shR1 cells were transfected with a shRNA-resistant reggie-1 construct (Solis et al., 2007). After a 20-min chase, the Tf-rhod accumulation at the perinuclear region was reduced to the normal level in cells in which reggie-1 was reintroduced (Figure 5, E and H) but not in untransfected shR1 cells.

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