PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.
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.
Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.Show MeSH
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Mentions: CLASPs accumulate at the Golgi apparatus and the plus ends of MTs, depending on GCC185 and EBs (Mimori-Kiyosue et al., 2005; Efimov et al., 2007). We used RNA interference to examine whether PAR3 and aPKC participate in the localization of CLASP2 in RPE-1 cells. Because RPE-1 cells are supposed to express CLASP2α and its N-terminal truncated alternative isoform CLASP2γ, we used small interfering RNA (siRNA) targeting their common sequence to deplete both isoforms (Akhmanova et al., 2001; Mimori-Kiyosue et al., 2005; Miller et al., 2009; Watanabe et al., 2009). Depletion of PAR3, aPKC, or CLASPs in RPE-1 cells with siRNA was confirmed by immunoblot analysis (Figure 3A and Supplemental Figure S2). Depletion of CLASPs also reduced the level of GCC185 to ∼25.3% of that in control cells. CLASP2 accumulated at the Golgi apparatus and partially overlapped with GCC185 in scramble-transfected cells (Figure 3B), as previously described (Efimov et al., 2007). Those cells displayed tightly packed and elongated intact Golgi morphology—Golgi ribbons—judging by the fluorescence of the cis-Golgi protein GM130. However, depletion of CLASPs diminished the signal of CLASP2 and changed the Golgi ribbon morphology into a circular appearance (Miller et al., 2009). Under the same conditions, depletion of PAR3 and aPKC increased the accumulation of CLASP2 at the TGN and enhanced the colocalization of CLASP2 and GCC185 concomitantly with disruption of the Golgi ribbon (Figure 3B). On the basis of the method described in Miller et al. (2009), we quantified the disruption of the Golgi ribbon by measuring the circularity index of the Golgi. Depletion of PAR3 and aPKC increased the circularity of the Golgi ribbon (Figure 3D). Of note, depletion of PAR3 and aPKC did not noticeably affect the localization of CLASP2 at the plus ends of microtubules under this condition (unpublished data).
Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.