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Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

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NHE5 is potent acidifier of recycling endosomes. (A) Endosomal pH in control PC12 cells and shNHE5 cells in the presence or absence of bafilomycin treatment. A mixture of fluorescein-conjugated (pH-sensitive) and Alexa Fluor 568–conjugated (pH-insensitive) transferrin was used as a ratiometric recycling endosomal pH reporter. N = 100–200 cells per condition. Error bars represent SEM, **p < 0.01 (Student's t test). (B) A set of representative live-cell images used for pH measurement experiments. Fluorescence signals for transferrin associated with perinuclear structures (arrowheads) were captured under live-cell confocal imaging and analyzed for pH determination (see Materials and Methods). Scale bar, 10 μm. (C) Double immunofluorescence labeling of transferrin and endosomal markers. PC12 cells were treated with Alexa Fluor 568–conjugated transferrin and subjected to chase incubation under the same condition as in A and B. Rab11 antibody, heterologously expressed mCherry-tagged Rab5, and mCherry-tagged Rab7 were used to visualize recycling and early and late endosomes, respectively. Scale bars, 10 μm.
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Figure 2: NHE5 is potent acidifier of recycling endosomes. (A) Endosomal pH in control PC12 cells and shNHE5 cells in the presence or absence of bafilomycin treatment. A mixture of fluorescein-conjugated (pH-sensitive) and Alexa Fluor 568–conjugated (pH-insensitive) transferrin was used as a ratiometric recycling endosomal pH reporter. N = 100–200 cells per condition. Error bars represent SEM, **p < 0.01 (Student's t test). (B) A set of representative live-cell images used for pH measurement experiments. Fluorescence signals for transferrin associated with perinuclear structures (arrowheads) were captured under live-cell confocal imaging and analyzed for pH determination (see Materials and Methods). Scale bar, 10 μm. (C) Double immunofluorescence labeling of transferrin and endosomal markers. PC12 cells were treated with Alexa Fluor 568–conjugated transferrin and subjected to chase incubation under the same condition as in A and B. Rab11 antibody, heterologously expressed mCherry-tagged Rab5, and mCherry-tagged Rab7 were used to visualize recycling and early and late endosomes, respectively. Scale bars, 10 μm.

Mentions: Previous studies showed that NHE5 is largely associated with recycling endosomes under steady-state conditions (Szaszi et al., 2002, Szabo et al., 2005; Diering et al., 2009). We next tested the possible involvement of NHE5 in recycling endosomal pH regulation. Control PC12 cells or PC12 cells stably expressing NHE5-shRNAs (described in Diering et al., 2011) were incubated with a mixture of two types of transferrin (Tfn), one conjugated with the pH-sensitive fluorescent dye fluorescein and the other conjugated with the pH-insensitive dye Alexa Fluor 568, to generate a ratiometric reporter of recycling endosomal pH. Cells were rinsed three times with prewarmed serum-free media and transferred to the preheated (37°C) stage of a confocal microscope, and 15 min later, image capture began for fluorescein/Alexa Fluor 568 emission intensity ratio measurements. Under these conditions, Tfn was observed mainly in perinuclear structures (Figure 2B), a distribution typical of recycling endosomes (Figure 2C). The fluorescence ratios collected from live-cell imaging were calibrated to endosomal pH using the high–[K+]/nigericin technique (Presley et al., 1997; D'Souza et al., 1998; Sonawane et al., 2002; Brett et al., 2005; Ohgaki et al., 2010). In control PC12 cells, recycling endosomal pH was 6.20 ± 0.02 (SEM), whereas in shNHE5 cells recycling endosomal pH was 6.48 ± 0.02, a value significantly more alkaline than in parental PC12 cells (Figure 2A). After 5-min treatment with the V-ATPase inhibitor bafilomycin, we observed endosomal alkalinization to pH 6.52 ± 0.02 in control PC12 cells and 6.75 ± 0.03 in shNHE5 cells (Figure 2A). These results suggest that NHE5 is a potent recycling endosomal acidifier, playing an equally important role as the V-ATPase in PC12 cells. We next addressed the effect of NHE5 knockdown on early endosomal pH using a previously described protocol with some modifications (Gagescu et al., 2000). As shown in Supplemental Figure S2, there was no significant difference in early endosomal pH between the two cell lines (pH = 6.06 ± 0.02 in PC12 cells vs. 6.01 ± 0.02 in shNHE5 cells).


Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

NHE5 is potent acidifier of recycling endosomes. (A) Endosomal pH in control PC12 cells and shNHE5 cells in the presence or absence of bafilomycin treatment. A mixture of fluorescein-conjugated (pH-sensitive) and Alexa Fluor 568–conjugated (pH-insensitive) transferrin was used as a ratiometric recycling endosomal pH reporter. N = 100–200 cells per condition. Error bars represent SEM, **p < 0.01 (Student's t test). (B) A set of representative live-cell images used for pH measurement experiments. Fluorescence signals for transferrin associated with perinuclear structures (arrowheads) were captured under live-cell confocal imaging and analyzed for pH determination (see Materials and Methods). Scale bar, 10 μm. (C) Double immunofluorescence labeling of transferrin and endosomal markers. PC12 cells were treated with Alexa Fluor 568–conjugated transferrin and subjected to chase incubation under the same condition as in A and B. Rab11 antibody, heterologously expressed mCherry-tagged Rab5, and mCherry-tagged Rab7 were used to visualize recycling and early and late endosomes, respectively. Scale bars, 10 μm.
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Related In: Results  -  Collection

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Figure 2: NHE5 is potent acidifier of recycling endosomes. (A) Endosomal pH in control PC12 cells and shNHE5 cells in the presence or absence of bafilomycin treatment. A mixture of fluorescein-conjugated (pH-sensitive) and Alexa Fluor 568–conjugated (pH-insensitive) transferrin was used as a ratiometric recycling endosomal pH reporter. N = 100–200 cells per condition. Error bars represent SEM, **p < 0.01 (Student's t test). (B) A set of representative live-cell images used for pH measurement experiments. Fluorescence signals for transferrin associated with perinuclear structures (arrowheads) were captured under live-cell confocal imaging and analyzed for pH determination (see Materials and Methods). Scale bar, 10 μm. (C) Double immunofluorescence labeling of transferrin and endosomal markers. PC12 cells were treated with Alexa Fluor 568–conjugated transferrin and subjected to chase incubation under the same condition as in A and B. Rab11 antibody, heterologously expressed mCherry-tagged Rab5, and mCherry-tagged Rab7 were used to visualize recycling and early and late endosomes, respectively. Scale bars, 10 μm.
Mentions: Previous studies showed that NHE5 is largely associated with recycling endosomes under steady-state conditions (Szaszi et al., 2002, Szabo et al., 2005; Diering et al., 2009). We next tested the possible involvement of NHE5 in recycling endosomal pH regulation. Control PC12 cells or PC12 cells stably expressing NHE5-shRNAs (described in Diering et al., 2011) were incubated with a mixture of two types of transferrin (Tfn), one conjugated with the pH-sensitive fluorescent dye fluorescein and the other conjugated with the pH-insensitive dye Alexa Fluor 568, to generate a ratiometric reporter of recycling endosomal pH. Cells were rinsed three times with prewarmed serum-free media and transferred to the preheated (37°C) stage of a confocal microscope, and 15 min later, image capture began for fluorescein/Alexa Fluor 568 emission intensity ratio measurements. Under these conditions, Tfn was observed mainly in perinuclear structures (Figure 2B), a distribution typical of recycling endosomes (Figure 2C). The fluorescence ratios collected from live-cell imaging were calibrated to endosomal pH using the high–[K+]/nigericin technique (Presley et al., 1997; D'Souza et al., 1998; Sonawane et al., 2002; Brett et al., 2005; Ohgaki et al., 2010). In control PC12 cells, recycling endosomal pH was 6.20 ± 0.02 (SEM), whereas in shNHE5 cells recycling endosomal pH was 6.48 ± 0.02, a value significantly more alkaline than in parental PC12 cells (Figure 2A). After 5-min treatment with the V-ATPase inhibitor bafilomycin, we observed endosomal alkalinization to pH 6.52 ± 0.02 in control PC12 cells and 6.75 ± 0.03 in shNHE5 cells (Figure 2A). These results suggest that NHE5 is a potent recycling endosomal acidifier, playing an equally important role as the V-ATPase in PC12 cells. We next addressed the effect of NHE5 knockdown on early endosomal pH using a previously described protocol with some modifications (Gagescu et al., 2000). As shown in Supplemental Figure S2, there was no significant difference in early endosomal pH between the two cell lines (pH = 6.06 ± 0.02 in PC12 cells vs. 6.01 ± 0.02 in shNHE5 cells).

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

Show MeSH
Related in: MedlinePlus