Dynein-mediated apical localization of crumbs transcripts is required for Crumbs activity in epithelial polarity

Abstract

Asymmetrical localization of transcripts coupled with localized translation constitutes an important mechanism widely deployed to regulate gene activity in a spatial manner. The conserved transmembrane protein Crumbs (Crb) is an important regulator of epithelial polarity. However, it remains unclear how Crb is targeted to the apical domain. Here, we show that the cytoplasmic dynein complex transports both Crb protein and transcripts to the apical domain of Drosophila melanogaster follicular cells (FCs). The crb 3' untranslated region (UTR) is necessary and sufficient for the apical localization of its transcript and this apical transcript localization is crucial for crb function. In crb mutant FCs, Crb protein derived from transgenes lacking the 3' UTR does not effectively localize to the apical domain and does not effectively restore normal epithelial polarity. We propose that dynein-mediated messenger RNA transport coupled with a localized translation mechanism is involved in localizing Crb to the apical domain to mediate epithelial apicobasal polarity and that this mechanism might be widely used to regulate cellular polarity.

Figure 1.

Dynein function is required for FC A/B polarity. GFPnls are shown as green, DNA is shown as blue, and mutant clones are marked by the absence of GFP and apical up unless otherwise stated. (A) wt FCs display regular cuboidal morphology by rhodamine phalloidin staining (red). Dhc64C⁹⁰² (B) and Gl^1L (C) mutant FCs show altered morphology. wt FCs are monolayered (D), whereas Dhc64C⁹⁰² (E) and Gl^1L (F) mutant FCs are multiple layered at the posterior end. Bars, 5 μm.

Figure 2.

Dynein is required for the apical localization of the Crb complex. Rhodamine phalloidin is shown in red and GFPnls is shown in green. Crb (blue) localizes to the apical domain of wt FCs (A) but is lost from the apical domain in the dynein mutant and colchicine-treated FCs (B, C, and M). Arm (blue) localizes to the adherens junctions in wt FCs (D) and this localization is largely normal in dynein mutant and colchicine-treated FCs (E, F, and N). aPKC (blue) localizes to the apical region of wt FCs (G) and is largely unaffected in dynein mutant and colchicine-treated FCs (H, I, and O). Scrib (blue) localizes along the lateral domain of wt FCs (J) and is only slightly apically expanded in the dynein mutant cells (K, L, and P). Bars, 5 μm.

Figure 3.

Apical crb transcript localization is required for Crb activity. Crb protein is shown in red, and crb mRNA (green) localizes apically in wt FCs (A) but delocalizes in the dynein mutant (B). Injected crb^intra-myc transcripts (red) localize apically in wt (C) and anti-Myc 9E10 antibody–treated (F) but not in P1H4 (anti-Dhc64C) antibody–treated or colchicine-treated blastoderm embryos (D and E). crb^intra-myc-wo transcripts (red) fail to localize apically in wt blastoderm embryos (G). lacZ-crb 3′ UTR (H, red) but not lacZ-SV40 3′ UTR transcripts (I, red) localize apically in blastoderm embryos. crb^intra-myc transcripts (J) but not crb^intra-myc-wo (K) transcripts (green) localize apically in FCs. In J and K, both endogenous and exogenous transcripts are detected. Crb^intra-myc protein (green) shows apical localization in wt FCs (L). The majority of Crb^intra-myc-wo protein (green) is apically localized in wt FCs (M). In 5% of cells, Crb^intra-myc-wo (green) shows cytoplasmic localization (N). Crb^intra-myc (O, red) apically localizes in the crb mutant. Crb^intra-myc-wo (P, red) is largely cytoplasmic in the crb mutant. In crb mutant FCs, apical localization of Sdt (Q and R, red) is restored by expression of crb^intra-myc but not the crb^intra-myc-wo transgene (also note the multilayering of mutant cells in R). In Q and R, GFP is in green and mutant cells do not express GFP. Bars, 5 μm.

Figure 4.

Crb and Sdt interact on the apical cortex. Sdt (red) localizes on the apical cortex in wt FCs (A). Sdt localization in newly induced (B) and aged dynein mutant clones (C). Patj/Dlt (red) localizes on the apical domain in wt FCs (D) and gradually localizes into the cytoplasm in the dynein mutant (E, a mutant clone shortly after induction; F, an aged mutant clone). Sdt gradually delocalizes in crb mutant cells (G, early clone; H and I, aged clone). Sdt (red) largely colocalizes with Crb^intra-myc (green) on the apical domain in wt FCs (J). Both Sdt and Crb^intra-myc become cytoplasmic and do not colocalize in colchicine-treated FCs, which mimic dynein mutant FCs (K). Equal protein loadings of total lysates from wt (left lane) and colchicine-treated ovary (right lane) were probed with an anti-Crb antibody (L); loading control was probed with an anti–α-tubulin antibody (L'). Crb^intra-myc and Sdt only form a complex when both localize on the apical cortex (M). Western blot is probed with Sdt. (lane 1) wt (crb^intra-myc expressed in wt background) input (10%). (lane 2) Anti-Myc immunoprecipitation from wt sample. (lane 3) Anti-Flag immunoprecipitation (negative control) from wt sample. (lane 4) Anti-Myc immunoprecipitation from a colchicine-treated sample. (lane 5) Anti-Flag immunoprecipitation (control) from a colchicine-treated sample. Bars, 5 μm.