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. 2015 Jan 5;25(1):61–68. doi: 10.1016/j.cub.2014.10.052

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© 2015 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

PMC Copyright notice

Aurora Kinases Phosphorylate Lgl to Relocalize It to the Cytoplasm during Mitosis in the Wing Epithelium

(A–C) Live imaging of third instar wing imaginal discs. Fluorescently tagged Crumbs (Crb-GFP; A) and Par-6 (Par-6-GFP; B) remain apically localized during mitosis (apical section; diagrammed on the left). Baz-mCherry is partially downregulated but remains apical (C).

(D and E) Fluorescently tagged Discs-large (Dlg-GFP; D) and Scribble (Scrib-GFP; E) remain basolaterally localized during mitosis (basal section; diagrammed on the left).

(F–H) Fluorescently tagged Lethal giant larvae (Lgl-GFP) becomes cytoplasmic at mitosis (basal section; diagrammed on the left): note the complete relocalization at mitosis with no membrane staining (G).

(I) Lgl-GFP relocalizes to the cytoplasm at mitosis normally in an aPKC⁴¹⁷ kinase-dead MARCM clone in wing disc epithelia, indicating that aPKC phosphorylation is not responsible for regulating Lgl at mitosis. Other kinase-dead alleles of aPKC gave similar results (not shown).

(J) Phosphomutant Lgl3A-GFP fails to relocalize to the cytoplasm during mitosis in wing disc epithelia.

(K) Relocalization of Lgl-GFP to the cytoplasm in mitosis is strongly delayed in aurA^87Ac-3/Df mutants. Similar results were obtained with other aurora A mutant alleles or with AurA RNAi (not shown).

(L) Relocalization of Lgl-GFP to the cytoplasm in mitosis is blocked upon treatment of the epithelium with the Aurora A/B inhibitor VX-680.

(M) Quantification of control and VX-680 treated wing discs expressing Lgl-GFP.

(N) Western blotting analysis of actin5c.Gal4 UAS-Lgl-GFP wing discs showing absence of a strong phosphorylated Lgl band upon treatment of samples with λ-phosphatase or treatment of the epithelium with VX-680 prior to sample preparation.

(O) Aurora A and B directly phosphorylate the Lgl phosphosite motif in an in vitro kinase assay. CENP-A is a positive control substrate that is known to be phosphorylated by both Aurora A and B.

Error bars indicate 1 SD from the mean. See also Figures S1 and S3 and Movies S1, S2, and S3.