Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

BackgroundCabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein.Methodology/Principal FindingsTo determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2.Conclusions/SignificanceOur results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins

The Cabut zinc finger region is not essential for nuclear localization in CHO-K1 cells.

<p>(A) Schematic representation of the eGFPCbt fusion constructs used to transfect CHO-K1 cells. The locations of the SR region (black rectangle) and the zinc fingers (1, 2, 3, grey rectangles) are indicated. (B–G) Localization of eGFP fusion proteins in CHO-K1 cells transiently transfected with the constructs shown in (A). Cells were immunostained with anti-eGFP (green, first panel) and DAPI (blue; second panel). Differential interference contrast (DIC) was used to visualize cell boundaries (third panel). The overlay panel shows DIC and anti-eGFP staining. The localization of the fusion proteins is shown in the fourth panel (C, cytoplasmic; N, nuclear). Wild-type eGFP was located in the cytoplasm and the nucleus (B), but the eGFPCbt fusion protein translocates to the nucleus (C). With the exception of the peGFPCbt_262–428 fusion protein, which lacks the N-terminal region of the Cbt protein (G), all Cbt deletions affecting the zinc finger region showed cytoplasmic localization (D–F). Scale bar: 10 µm. (H) Western blots of protein extracts from CHO-K1 cells transfected with the constructs shown in (A) and stained with anti-GFP. (1) Non-transfected cells, (2) empty peGFP vector (∼27 kDa), (3) peGFPCbt_1–428 (∼70 kDa), (4) peGFPCbt_1–322 (∼60 kDa), (5) peGFPCbt_1–292 (∼60 kDa) and (6) peGFPCbt_1–262 (∼50 kDa).</p

The ₇₁PNKKPRL₇₇ sequence is the NLS of the Cabut protein.

<p>(A) Schematic representation of the β-GalCbt fusion constructs transfected into S2 cells to determine whether the ₁₆₂KMNRKRAAEVALPPVQTPETPVAKLVTPP₁₉₀ and/or ₇₁PNKKPRL₇₇ sequences are functional NLSs. The Serine-rich (SR) domain is shown in black and the PNKKPRL sequence in fuchsia. (B–J) Localization of β-Gal fusion proteins in S2 cells transiently transfected with the constructs shown in (A). Cells were stained with anti-β-Gal (red; first panel) and anti-Lam (green; second panel) to mark nuclear membranes. The overlay panel depicts double staining of cells with both antibodies, and the localization of the fusion proteins is shown in the fourth panel (C, cytoplasmic; N, nuclear). Note that fusion proteins lacking or containing a mutated ₇₁PNKKPRL₇₇ sequence (E and J) were cytoplasmic. The ₇₁PNKKPRL₇₇ sequence was able to translocate β-Gal to the nucleus when fused to either the N- or the C-terminus of the protein (H and I). Scale bar: 10 µm. (K) Western blots of protein extracts from S2 cells transfected with the constructs shown in (A) and stained with anti-β-Gal. (1) pIE-β-GalCbt_1–141 (∼140 kDa), (2) pIE-β-GalCbt_1–108 (∼140 kDa), (3) and (7) pIE-β-GalCbt_1–77 (∼135 kDa), (4) pIE-β-GalCbt_1–70 (∼135 kDa), (5) pIE-_PNKKPRL-β-Gal (∼120 kDa), (6) pIE-β-Gal-_PNKKPRL (∼120 kDa) and (8) pIE-β-Gal-Cbt_K73N–K74N (∼135 kDa).</p

Cabut functions as a transcriptional repressor in S2 cells and regulates its own transcription.

<p>(A) Expression of Gal4Cbt_1–428 led to repression of luciferase activity levels relative to a control Gal4 protein. Luciferase activity was measured 48 h after transfection. pRENILLA was used to normalize for cell number, transfection efficiency, and general effects on transcription (luciferase activity = firefly luciferase/renilla luciferase). (B) S2 cells transfected with <i>Prom1-2</i>-GFP as a control or co-transfected with <i>Prom1-2</i>-GFP and MT-<i>cbt</i>. The MT promoter was induced by exposing the cells to medium containing copper (upper picture, no copper; lower picture, plus copper). Fluorescence levels were reduced following transcriptional induction of Cbt by copper in cells co-transfected with <i>Prom1-2</i>-GPF and MT-<i>cbt</i> (left panels). Fluorescence was measured 48 h after induction (right panel). pCHERRY was used for normalization (Fluorescence levels = GFP/CHERRY). In A and B, data are presented as the mean ± SD of three replicates.</p

PCR-generated constructs and associated primers.

<p>Sequences recognized by restriction enzymes (in parentheses) are underlined. The start and stop codons are in bold. F, forward primer; R, reverse primer.</p

The Cabut zinc finger region is not essential for nuclear localization in S2 cells.

<p>(A) Schematic representation of the <i>Drosophila</i> Cbt protein, in which the locations of the Ser-rich (SR) region and the DBD are indicated. Sequences, coordinates and locations (arrows) of the predicted NLSs are also indicated. (B) Multiple alignment of the second and third zinc fingers of murine TIEG1 and TIEG3, human TIEG1 and TIEG2 and <i>Drosophila</i> Cbt. Red lines below the alignment indicate the amino acids included in the murine and human TIEG NLSs. (C) Schematic representation of the β-GalCbt fusion constructs used to transfect S2 cells. The locations of the SR region and the DBD are indicated by boxes. (D–F) Localization of β-Gal fusion proteins in S2 cells transiently transfected with the constructs shown in (C). Cells were stained with anti-β-Gal (red; first panel) and anti-Lam (green; second panel) to mark nuclear membranes. The overlay panel depicts double staining of cells with both antibodies, and the localization of the fusion proteins is shown in the fourth panel (C, cytoplasmic; N, nuclear). Wild-type β-Gal was located in the cytoplasm (D), but the β-GalCbt fusion protein translocated to the nucleus (E). However, the Cbt zinc finger region alone was not able to translocate β-Gal to the nucleus (F). Scale bar: 10 µm. (G) Western blot of protein extracts from S2 cells transfected with the constructs shown in (C) and stained with anti-β-Gal. (1) Non-transfected cells, (2) empty pIE-β-Gal vector (∼122 kDa), (3) pIE-β-GalCbt_1–428 (∼160 kDa) and (4) pIE-β-GalCbt_262–428 (∼140 kDa). Cells transfected with the pIE-β-GalCbt_1–428 construct presented some degradation that did not affect the subcellular localization of the fusion protein.</p

Identification of domains required for Cabut's transcriptional repressor activity.

<p>(A) Multiple alignment of the Sin3A interacting domains (SID) from <i>Drosophila</i> Cbt and several vertebrate TFs such as the MAD protein and mebers of the Sp1 family (TIEG, BTEB). Note that this domain is highly conserved in sequence (marked by a pink rectangle and residues in bold) but not with respect to location within the protein. (B) Schematic representation of the CbtGal4 fusion constructs transfected into S2 cells to identify transcriptional regulatory domains in the Cbt protein. The AAEVAL sequence is indicated in red and the C₂H₂ zinc fingers in blue. (C) Degree of <i>luciferase</i> repression obtained in UAS/Gal4 assay in S2 cells transiently transfected with the constructs shown in (B). Repression rate = luciferase activity of pIE-Gal4/luciferase activity of tested construct. pRENILLA was used for normalization as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032004#pone-0032004-g001" target="_blank">Fig. 1</a>. Data are presented as the mean ± SE (n≥5). Note that removal of the AAEVAL sequence completely abolishes the repressor activity of the Cbt protein (asterisks indicate p-value<0.01, t-Student's test).</p