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PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.

Matsui T, Watanabe T, Matsuzawa K, Kakeno M, Okumura N, Sugiyama I, Itoh N, Kaibuchi K - Mol. Biol. Cell (2014)

Bottom Line: CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185.This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2.Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

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CLASP2 phosphorylation by aPKC is essential for Golgi ribbon organization. (A) Rescue experiments for Golgi ribbon organization. RPE-1 cells were transfected with siRNA for CLASPs along with the indicated plasmids. These cells were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-GFP (gray and green), anti-GCC185 (magenta), and anti-GM130 (cyan) antibodies. The expression of siRNA-resistant CLASP2γ-WT restored Golgi ribbon organization, but CLASP2-3A failed to do so. Right, magnifications of left insets. Bars, 10 μm. (B) Circularity index of the Golgi morphology in indicated cells. n > 40. ***p < 0.001. All results are representative of three independent experiments.
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Figure 6: CLASP2 phosphorylation by aPKC is essential for Golgi ribbon organization. (A) Rescue experiments for Golgi ribbon organization. RPE-1 cells were transfected with siRNA for CLASPs along with the indicated plasmids. These cells were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-GFP (gray and green), anti-GCC185 (magenta), and anti-GM130 (cyan) antibodies. The expression of siRNA-resistant CLASP2γ-WT restored Golgi ribbon organization, but CLASP2-3A failed to do so. Right, magnifications of left insets. Bars, 10 μm. (B) Circularity index of the Golgi morphology in indicated cells. n > 40. ***p < 0.001. All results are representative of three independent experiments.

Mentions: Finally, we performed rescue experiments in CLASP-depleted cells with GFP–CLASP2γ-3A. The expression of the siRNA-resistant wild-type CLASP2γ restored the organization of the Golgi ribbon to that observed in control cells. However, GFP–CLASP2γ-3A failed to restore the organization of the Golgi ribbon (Figure 6, A and B). In addition, compared with the wild type, GFP–CLASP2γ-3A strongly colocalized with GCC185, which was similar to the behavior of endogenous CLASP2 in PAR3- and aPKC-depleted cells (Figures 3B and 6A). These results suggest that PAR3 and aPKC regulate the organization of the Golgi ribbon through the phosphorylation of CLASP2.


PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.

Matsui T, Watanabe T, Matsuzawa K, Kakeno M, Okumura N, Sugiyama I, Itoh N, Kaibuchi K - Mol. Biol. Cell (2014)

CLASP2 phosphorylation by aPKC is essential for Golgi ribbon organization. (A) Rescue experiments for Golgi ribbon organization. RPE-1 cells were transfected with siRNA for CLASPs along with the indicated plasmids. These cells were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-GFP (gray and green), anti-GCC185 (magenta), and anti-GM130 (cyan) antibodies. The expression of siRNA-resistant CLASP2γ-WT restored Golgi ribbon organization, but CLASP2-3A failed to do so. Right, magnifications of left insets. Bars, 10 μm. (B) Circularity index of the Golgi morphology in indicated cells. n > 40. ***p < 0.001. All results are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 6: CLASP2 phosphorylation by aPKC is essential for Golgi ribbon organization. (A) Rescue experiments for Golgi ribbon organization. RPE-1 cells were transfected with siRNA for CLASPs along with the indicated plasmids. These cells were fixed with methanol at −30°C for 20 min, followed by immunostaining with anti-GFP (gray and green), anti-GCC185 (magenta), and anti-GM130 (cyan) antibodies. The expression of siRNA-resistant CLASP2γ-WT restored Golgi ribbon organization, but CLASP2-3A failed to do so. Right, magnifications of left insets. Bars, 10 μm. (B) Circularity index of the Golgi morphology in indicated cells. n > 40. ***p < 0.001. All results are representative of three independent experiments.
Mentions: Finally, we performed rescue experiments in CLASP-depleted cells with GFP–CLASP2γ-3A. The expression of the siRNA-resistant wild-type CLASP2γ restored the organization of the Golgi ribbon to that observed in control cells. However, GFP–CLASP2γ-3A failed to restore the organization of the Golgi ribbon (Figure 6, A and B). In addition, compared with the wild type, GFP–CLASP2γ-3A strongly colocalized with GCC185, which was similar to the behavior of endogenous CLASP2 in PAR3- and aPKC-depleted cells (Figures 3B and 6A). These results suggest that PAR3 and aPKC regulate the organization of the Golgi ribbon through the phosphorylation of CLASP2.

Bottom Line: CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185.This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2.Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

Show MeSH
Related in: MedlinePlus