Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila - PubMed (original) (raw)

Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila

Sang-Chul Nam et al. Dev Biol. 2007.

Abstract

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, we studied the effects of Par-1 on Baz localization in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. We also show that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, we propose that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis.

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Figures

Fig. 1. Requirement of Baz for localization of Par-6 and aPKC

(A) Schematic view of longitudinal (left) and tangential (right) sections of a photoreceptor cluster in mid pupal stage (40h pd) prior to rhabdomere formation. At this stage, the apical side (green) of the cells and the region of AJ (red) is oriented toward the center of the ommaidial cluster, as photoreceptor cells have rotated 90° inward during earlier pupal stage. In a tangential section indicated by the dashed arrow, Crb, Sdt, Dpatj, Par-6 and aPKC colocalize to the apical domain whereas Arm and Baz are localized at AJs. Later, the green apical region becomes the rhabdomere stalk as the rhabdomeres develop from the apical surface of photoreceptors. (B, C) In baz mutant photoreceptor cells marked by the absence of GFP (blue in B” and C”), Par-6 (B’), aPKC (C’) and Dpatj (C) are absent or strongly reduced (arrows). In contrast, Arm (B) is mislocalized basolaterally. (D) apkc mutant clones show the presence of Baz but its mislocalization (arrows). Dpatj is mispositioned basal to Baz. (E) par-6 mutant clones also display misplaced Dpatj basal to Baz (arrows). Clone boundaries are marked by white lines.

Fig. 2. Localization of Par-1 and loss of function phenotype of par-1 in photoreceptors

(A) Par-1 expression was examined in par-1 mosaic eye at 40% pd. The white line indicates the clone boundary. In par-1+ wild-type ommatidia marked by the expression of GFP clone marker (A’), Par-1 is strongly expressed in the interommatidial cells (arrowhead). In photoreceptor cells, Par-1 is weakly localized along the basolateral cell membranes but is enriched in the central (apical) region of photoreceptor clusters (arrow). _par-1_- mutant cells without GFP expression show no Par-1 expression (A). (B) Pattern of Arm (red) and Dpatj (blue) in par-1 mutant clones marked by GFP (green, B’) and lines. Note that although Arm is often expanded basolaterally (arrows) some clusters look relatively normal (arrowheads). (C) A series of cross-sections of a mosaic eye from the distal to the proximal shows that Baz (red) and Crb (blue) are gradually lost in the proximal sections of a par-1 mutant clone as marked by the absence of GFP (green). (D) 40h pd retina with par-1 mutant clones stained for Arm and Dlg. This image is a confocal section at the middle region along the proximal-distal axis of the retina. Some mutant photoreceptors show significant loss of Arm (arrowheads) or ectopic Arm at basolateral positions (arrows) (D and D”). (D’) Basolateral membrane marked by Dlg staining. Dlg is also detected in the apical region. (E and E’) High magnification of the box region in (D). Dlg staining is not affected by the presence of ectopic Arm localization in the basolateral membrane (arrows).

Fig. 3. Par-1 overexpression causes severe disruption of Arm and Dpatj localization

(A) Par-1 overexpression by GMR-Gal4 causes diffusion and expansion of Arm (A, red) and Dpatj (A’, green). (B) A magnified view of the square region of (A). Often, Dpatj is displaced basal to Arm, suggesting the loss of cell polarity (A”, arrows). (C) GMR>Par-1 stained for Arm and Baz shows significant overlap of these markers. (D) Par-1(KN), a kinase-inactive mutant of Par-1, shows a near normal pattern of Arm and Dpatj.

Fig. 4. Localization of GFP-Baz and GFP-BazSA

(A) Overexpression of wild-type GFP-Baz by GMR-Gal4 causes an expansion of Arm (A, red) and Dpatj (A’, green), as well as cell polarity defects (A”). (B) The unphosphorylated form of GFP-Baz (GFP-BazSA) shows relatively normal localization of GFP-Baz (B”, green), although weak GFP-BazSA staining is occasionally found at ectopic positions (arrows). Most Dpatj (B’, blue) staining is also normally localized to the apical region, (C) Localization of Arm (C, red) and Dpatj (C’, blue) in the heterozygote of GMR-Gal4/+ as control. The table in the panel (D) shows the frequency of severely affected ommatidia in each genotype.

Fig. 5. Effects of PP2A inhibition by dominant-negative Mts

(A) dnMts overexpression by GMR-Gal4 causes the expansion of Arm (A, red) and Dpatj (A’, green), as well as cell polarity defects (A”, arrows). (B) High magnification of the rectangle area in (A”). Note that Arm and Dpatj are diffused and abnormally positioned. (C) GMR>dnMts stained for Arm and Baz showing their overlap. (D) Localization of Arm (A, red) and Dpatj (A’, green) in GMR-Gal4/+ control.

Fig. 6. Genetic interaction of Par-1 and Mts

(A-C) Scanning electron microscope images of GMR>Par-1(KN) (A), GMR>Par-1 (B) and GMR>Par-1; mts/+ (C). (D) Localization of Arm (A, red) and Dpatj (A’, green) in a kinase-inactive mutant form of Par-1 (Par-1(KN)) overexpressed by GMR-Gal4. (E) Par-1 overexpression by GMR-Gal4 causes severe mislocalization of Arm (E, red) and Dpatj (E’, green), resulting in cell polarity defects (E”, arrows). The same images shown in Fig. 3B are provided here for phenotype comparison purposes. (F) Par-1 overexpression by GMR-Gal4 in the mts/+ heterozygous background (GMR>Par-1; mts/+) shows further reduction or loss of Arm (F, red) and Dpatj (F’, green), compared to GMR>Par-1 (E).

Fig. 7. A model for the function of Par-1 and PP2A in Baz localization

During early-mid pupal stage, Par-6 and Crb complex proteins are targeted to the apical region of photoreceptor cells, except that Baz is localized to AJ between the apical and the basolateral domains. Baz protein phosphorylated by Par-1 is displaced from the AJ whereas dephosphorylation of Baz at the S151/S1085 Par-1 sites allows Baz localization to AJ. Mts, the catalytic subunit of PP2A, antagonizes Par-1 function by dephosphorylating Baz and/or by inactivating Par-1.

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Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila - PubMed (original) (raw)