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Involvement of class II phosphoinositide 3-kinase α-isoform in antigen-induced degranulation in RBL-2H3 cells.

Nigorikawa K, Hazeki K, Guo Y, Hazeki O - PLoS ONE (2014)

Bottom Line: The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells.The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α.These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT
In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca2+ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P2 was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

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Existence of CD63 and PtdIns(3,4)P2 on large vesicles in FcεRI-stimulated RBL-2H3 cells.(A) Existence of CD63 on large vesicles. RBL-2H3 cells were stimulated, fixed and prepared for staining with anti-CD63 and Alexa 488-labeled anti-mouse IgG antibodies. Scale bar = 5 µm. (B) Numbers of cells containing CD63-positive large vesicles (>2.5-µm diameter). For each experimental condition, 75 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (C) Existence of CD63 on large vesicles in control and PI3K-C2α-knockdown cells. Scale bar = 5 µm. (D) Numbers of CD63-positive large vesicles (>2.5-µm diameter) per cell. For each experimental condition, 161 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (E) Absence of PtdIns(3,4)P2 in CD63-positive large vesicles in PI3K-C2α-knockdown cells. The cells were transfected with EGFP-Akt-PH. Before or after FcεRI stimulation, the cells were fixed. Scale bar = 5 µm. (F) Presence of PtdIns(3)P on CD63-positive large vesicles in control and PI3K-C2α-knockdown cells. The cells were transfected with EGFP-3×FYVEEEA1. Scale bar = 5 µm. (G) Numbers of large vesicles containing PtdIns(3,4)P2 or PtdIns(3)P. The cells were transfected with EGFP-Akt-PH or EGFP-3×FYVEEEA1. The number of vesicles displaying EGFP fluorescence was counted. For each experimental condition, 210 cells were analyzed. The data are shown as the means ± s.e.m. from three separate experiments.
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pone-0111698-g005: Existence of CD63 and PtdIns(3,4)P2 on large vesicles in FcεRI-stimulated RBL-2H3 cells.(A) Existence of CD63 on large vesicles. RBL-2H3 cells were stimulated, fixed and prepared for staining with anti-CD63 and Alexa 488-labeled anti-mouse IgG antibodies. Scale bar = 5 µm. (B) Numbers of cells containing CD63-positive large vesicles (>2.5-µm diameter). For each experimental condition, 75 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (C) Existence of CD63 on large vesicles in control and PI3K-C2α-knockdown cells. Scale bar = 5 µm. (D) Numbers of CD63-positive large vesicles (>2.5-µm diameter) per cell. For each experimental condition, 161 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (E) Absence of PtdIns(3,4)P2 in CD63-positive large vesicles in PI3K-C2α-knockdown cells. The cells were transfected with EGFP-Akt-PH. Before or after FcεRI stimulation, the cells were fixed. Scale bar = 5 µm. (F) Presence of PtdIns(3)P on CD63-positive large vesicles in control and PI3K-C2α-knockdown cells. The cells were transfected with EGFP-3×FYVEEEA1. Scale bar = 5 µm. (G) Numbers of large vesicles containing PtdIns(3,4)P2 or PtdIns(3)P. The cells were transfected with EGFP-Akt-PH or EGFP-3×FYVEEEA1. The number of vesicles displaying EGFP fluorescence was counted. For each experimental condition, 210 cells were analyzed. The data are shown as the means ± s.e.m. from three separate experiments.

Mentions: We then examined the effect of PI3K-C2α knockdown on the granule dynamics following FcεRI stimulation. In the experiment shown in Figure 5A, RBL-2H3 cells were stimulated with antigen, fixed, and then stained with a specific antibody against CD63, a marker protein of secretory granules (also known as lysosome-associated membrane protein 3, LAMP3). In the resting state, most of the CD63-positive granules had a diameter smaller than 1 µm, as has been reported in a previous study [19]. Upon antigen stimulation, CD63-positive large vesicles (>2.5-µm diameter) appeared in more than a half of the cell population (Figure 5A, B) likely due to the granule-granule fusion and the granule swelling that occur during the process of exocytosis [20]–[23].


Involvement of class II phosphoinositide 3-kinase α-isoform in antigen-induced degranulation in RBL-2H3 cells.

Nigorikawa K, Hazeki K, Guo Y, Hazeki O - PLoS ONE (2014)

Existence of CD63 and PtdIns(3,4)P2 on large vesicles in FcεRI-stimulated RBL-2H3 cells.(A) Existence of CD63 on large vesicles. RBL-2H3 cells were stimulated, fixed and prepared for staining with anti-CD63 and Alexa 488-labeled anti-mouse IgG antibodies. Scale bar = 5 µm. (B) Numbers of cells containing CD63-positive large vesicles (>2.5-µm diameter). For each experimental condition, 75 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (C) Existence of CD63 on large vesicles in control and PI3K-C2α-knockdown cells. Scale bar = 5 µm. (D) Numbers of CD63-positive large vesicles (>2.5-µm diameter) per cell. For each experimental condition, 161 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (E) Absence of PtdIns(3,4)P2 in CD63-positive large vesicles in PI3K-C2α-knockdown cells. The cells were transfected with EGFP-Akt-PH. Before or after FcεRI stimulation, the cells were fixed. Scale bar = 5 µm. (F) Presence of PtdIns(3)P on CD63-positive large vesicles in control and PI3K-C2α-knockdown cells. The cells were transfected with EGFP-3×FYVEEEA1. Scale bar = 5 µm. (G) Numbers of large vesicles containing PtdIns(3,4)P2 or PtdIns(3)P. The cells were transfected with EGFP-Akt-PH or EGFP-3×FYVEEEA1. The number of vesicles displaying EGFP fluorescence was counted. For each experimental condition, 210 cells were analyzed. The data are shown as the means ± s.e.m. from three separate experiments.
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pone-0111698-g005: Existence of CD63 and PtdIns(3,4)P2 on large vesicles in FcεRI-stimulated RBL-2H3 cells.(A) Existence of CD63 on large vesicles. RBL-2H3 cells were stimulated, fixed and prepared for staining with anti-CD63 and Alexa 488-labeled anti-mouse IgG antibodies. Scale bar = 5 µm. (B) Numbers of cells containing CD63-positive large vesicles (>2.5-µm diameter). For each experimental condition, 75 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (C) Existence of CD63 on large vesicles in control and PI3K-C2α-knockdown cells. Scale bar = 5 µm. (D) Numbers of CD63-positive large vesicles (>2.5-µm diameter) per cell. For each experimental condition, 161 cells were analyzed. The data are shown as the means ± s.e.m. from four separate experiments. (E) Absence of PtdIns(3,4)P2 in CD63-positive large vesicles in PI3K-C2α-knockdown cells. The cells were transfected with EGFP-Akt-PH. Before or after FcεRI stimulation, the cells were fixed. Scale bar = 5 µm. (F) Presence of PtdIns(3)P on CD63-positive large vesicles in control and PI3K-C2α-knockdown cells. The cells were transfected with EGFP-3×FYVEEEA1. Scale bar = 5 µm. (G) Numbers of large vesicles containing PtdIns(3,4)P2 or PtdIns(3)P. The cells were transfected with EGFP-Akt-PH or EGFP-3×FYVEEEA1. The number of vesicles displaying EGFP fluorescence was counted. For each experimental condition, 210 cells were analyzed. The data are shown as the means ± s.e.m. from three separate experiments.
Mentions: We then examined the effect of PI3K-C2α knockdown on the granule dynamics following FcεRI stimulation. In the experiment shown in Figure 5A, RBL-2H3 cells were stimulated with antigen, fixed, and then stained with a specific antibody against CD63, a marker protein of secretory granules (also known as lysosome-associated membrane protein 3, LAMP3). In the resting state, most of the CD63-positive granules had a diameter smaller than 1 µm, as has been reported in a previous study [19]. Upon antigen stimulation, CD63-positive large vesicles (>2.5-µm diameter) appeared in more than a half of the cell population (Figure 5A, B) likely due to the granule-granule fusion and the granule swelling that occur during the process of exocytosis [20]–[23].

Bottom Line: The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells.The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α.These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT
In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca2+ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P2 was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

Show MeSH
Related in: MedlinePlus