Pbl and Rho1 proteins interact in a yeast two-hybrid assay. (A) Summary of pairwise interactions between Pbl and Drosophila Rho1, Rac1, and Cdc42. Minus and plus signs indicate the absence or presence of the activity of the GAL–HIS and GAL–lacZ reporters. (B,C) Representative results of assays for histidine (B, on SC−His−Leu−Trp) and β-galactosidase (C, on SC−Leu−Trp) activity. (a) pAS2/Pbl and pACT2/Rho1; (b) pAS2/Pbl and pACT2/Rac1; (c) pAS2/Pbl and pACT2/Cdc42; (d) pAS2/ΔPbl and pACT2/Rho1; (e) pAS2/Rho1 and pACT2/Pbl; (f) pAS2/Rho1 and pACT2/ΔPbl; (g) pAS2/Cdc42 and pACT2/Pbl; (h) pAS2/Pbl and pACT2/Pbl. (AD) Activation domain; (DBD) DNA-binding domain; (Pbl) Pbl1-853; (PblΔDH), PblΔDH497–549; (ΔPbl) ΔPbl325–853.

pbl and Rho1 interact in vivo. Scanning electron micrographs (A–J) and methylene blue-stained transverse sections (K–O) of wild-type (A,F,K), GMR–GAL4, UAS–Pbl^3.2/+ (B,G,L), GMR–GAL4, UAS–Pbl^3.2/Rho1^72R (C,H,M), GMR–GAL4, UAS–PblΔDH497–549^{5B, 6A}/+ (D,I,N), and GMR–GAL4, UAS–PblΔDH497–549^5B,6A/Rho1^72R (E,J,O) flies. Overexpression of Pbl in the eye induces a disruption of the ordered array of ommatidia (B,G), as well as a disruption of the internal organization of rhabdomeres and a significant increase in the density and number of pigment cells (L). These defects are suppressed dominantly by mutations in Rho1 (C,H,M). Overexpression of PblΔDH497–549 induces disruption of the ordered structure of the eye, with fusions of ommatidia, loss of many bristles (D,I), and a decrease in the number of pigment cells (N). These defects are strongly enhanced in a dominant fashion by mutation in Rho1 (E,J,O). The eye phenotypes of Pbl and PblΔDH497–549 are completely suppressed by the coexpression of the UAS–antisensePbl (data not shown). Flies were raised at 25°C. Calibration bars: (A–E) 100 μm; (F–O) 20 μm.

Pbl is localized to the cell cortex and cleavage furrow during cytokinesis. (A–G) Wild-type embryos stained with anti-Pbl (A,D,G, red), anti-lamin (A,D,G, green) to mark nuclei, anti-α-spectrin (B,E,G, blue) to mark plasma membrane, and merged images (C,F). Upon initiation of cytokinesis, Pbl is cortical and cytoplasmic (A–C, the cell is outlined with a dotted line). As the cleavage furrow progresses (arrowheads in B,E), Pbl accumulates as two spots at the equator (arrows in D,F,G) and also in reforming nuclei (D,G). For comparison, images in C and F were not adjusted for brightness, contrast, or gamma. Embryo expressing full-length Pbl (prd–GAL4/pbl^EP3415) (H) and wild-type embryos (I, J) stained with anti-Pbl (red) and anti-lamin (H, green), anti-anillin (I, green), or anti-Pnut (J, green). Pbl accumulation at the equator between dividing cells (arrow in H) parallels accumulation of anillin (arrow in I) and Pnut (arrow in J) beneath the cleavage furrow in which the contractile ring is assembled. (K) Wild-type, (L) pbl² homozygous, and (M) pbl⁵ homozygous embryos stained with anti-Pbl (red) and anti-lamin (green). Unlike in a wild-type embryo (K), cycle 15 cells of the pbl² embryo have greatly reduced levels of Pbl expression (L) with few remaining speckles of staining scattered in nuclei of affected polyploid cells. Cycle 15 cells of pbl⁵ embryo (M) have levels of nuclear Pbl staining comparable with wild type.

Ectopic expression of amino-terminally truncated Pbl or Ect2 blocks cytokinesis. (A) Stage 9 UAS–Pbl^3.2/+, prd–GAL4/+ embryo expressing full-length Pbl in a prd-like pattern stained with anti-α-spectrin (red) to mark plasma membrane, anti-lamin (green) to reveal nuclei, and anti-Pbl (blue). Expression of full-length Pbl does not cause a phenotype during cell division, despite high levels of expression of exogenous Pbl in the affected segment (arrows). Note a pair of young postmitotic cells (arrowhead) with high levels of Pbl in the nuclei. High levels of exogenous Pbl and its apparent association with the nuclear envelope cause the nuclear laminae to appear blue. (B,C) Stage 9 UAS–ΔPbl325–853^S4–11/+, prd–GAL4/+ embryo expressing amino-terminally truncated Pbl stained with anti-α-spectrin (red) and anti-lamin (green). (D) A similar embryo stained with propidium iodide (red) to mark DNA and anti-actin to mark contractile ring (green). Expression of amino-terminally truncated Pbl leads to a block of cytokinesis. Most cells within the affected segment (arrows in B) become polyploid. During late anaphase, the cleavage furrow fails to initiate (arrowheads in C) and actin does not redistribute to the equatorial region in which the contractile ring is assembled (arrowheads in D). The ΔPbl325–853 protein is cytoplasmic and cortical (data not shown). (E) Stage 9 prd–GAL4/UAS–ΔEct2^S2-1A embryo expressing amino-terminally truncated Ect2 stained with anti-α-spectrin (red) and anti-lamin (green). (F) A similar embryo stained with anti-anillin (red) to mark the contractile ring and anti-lamin (green). Most cells within the segment expressing ΔEct2 (arrows in E) become binucleate. Failure of cytokinesis during later divisions leads to formation of multinucleate cells (arrowhead in E). Similar to ΔPbl325–853, there are no signs of a cleavage furrow and anillin does not accumulate at the equatorial region in late anaphase (arrowheads in F). Dotted lines in A,B, and E denote ectodermal segments expressing Pbl or Ect2.

Absence of a contractile ring and failure of cytokinesis in pbl mutants. Wild-type (A) and pbl⁵/pbl² (B) embryos stained with anti-α-spectrin (red) to reveal plasma membrane and anti-lamin (green) to mark nuclei. Ectodermal cells of a wild-type embryo (A) contain only one nuclear lamina (nucleus) per cell, whereas several nuclei can be found in pbl mutant cells (B). Absence of cytokinesis in cell divisions following mitotic cycle 14 results in accumulation of multiple nuclei per cell (arrows). Wild-type (C) and pbl⁵/pbl² (D) embryos stained with anti-actin (red) to mark plasma membrane and anti-phosphohistone H3 (green) to mark chromosomes during mitosis. Wild-type cells (C) form a single mitotic apparatus per cell and initiate contraction of the cleavage furrow (arrow) in late anaphase. In polyploid pbl mutant cells (D), there are no signs of cleavage, and during mitotic cycle 15, two mitotic apparatuses are formed (arrows) that independently enter anaphase. Wild-type (E,H,J) and pbl⁵/pbl² (F,G,I,K) embryos stained with anti-actin (E–G, green), anti-anillin (H,I, red), and anti-Pnut (J,K, red) as markers of a contractile ring. Embryos in E–G were counterstained with propidium iodide (red) to mark DNA, and embryos in H–K were counterstained with anti-lamin (green) to reveal nuclei. In dividing wild-type cells, actin (E), anillin (H), and Pnut (J) accumulate at the equatorial region in which the contractile ring is assembled during late anaphase. They remain enriched at the equator through telophase (arrows in E,H,J). During cycle 14 in pbl mutant cells, actin (F), anillin (I), and Pnut (K) do not accumulate at the equatorial region and remain associated with plasma membrane. Similarly, during later mitotic cycles (G) there are no signs of contractile ring formation.

Mutations in Rho1 or expression of a dominant–negative Rho1 abolish cytokinesis. Rho1^72O homozygous (A, stage 16), Rho1^72R homozygous (B, stage 13) embryos, and an embryo expressing Rho1^N19 in a paired-like pattern (C,D, UAS–Rho1^N19/+, prd–GAL4/+, stage 9) stained with anti-α-spectrin (red in A–C, blue in D) to mark plasma membrane, anti-lamin (green) to reveal nuclei, and anti-Pbl (red in D). (A,B) Null mutations in Rho1 block cytokinesis in affected cells. Many cells in the head region of the embryo (A) become polyploid and contain two nuclei per cell (arrows). In addition, polyploid cells are found occasionally in thoracic or abdominal segments of Rho1 embryos (arrow in B, T2 segment). Homozygous Rho1 embryos were identified on the basis of their anterior open phenotype. (C,D) Ectopic expression of a dominant–negative Rho1 leads to formation of polyploid cells. Almost every cell within the affected segments (arrows in C) becomes binucleate. Despite high levels of nuclear Pbl in telophase, cleavage furrow fails (arrows in D) and cytokinesis is not initiated. (E,F) Embryo expressing Pbl and Rho1^N19 (E, UAS–Pbl^3.2/+, prd–GAL4/UAS–Rho1^N19) and embryo ex pressing Pbl alone (F, UAS–Pbl^3.2/+, prd–GAL4/+) stained with anti-Pbl (red) and anti-lamin (green). (E) Coexpression of Pbl and Rho1^N19 results in accumulation of Pbl at the cortex in telophase (arrow) and especially in interphase (arrowheads) cells. (F) In contrast, distribution of Pbl in cells ectopically expressing Pbl alone is similar to wild-type cells with protein localized to the nuclei during interphase (arrowheads) and to the cortex in early anaphase (inset). (G) Proposed signaling pathway initiating cytokinesis. (Dia) Diaphanous; (F-actin) fibrous actin; (G-actin) globular actin; (Rho1–GDP) inactive GDP-bound Rho1; (Rho1–GTP) active GTP-bound Rho1. For details see Discussion.

pbl encodes an exchange factor for small G proteins of the Rho family. (A) Domain structure of Pbl and Ect2. The two proteins are represented schematically with functional domains shown in color. The lengths of Pbl and Ect2 are 853 and 738 amino acids, respectively. Overall, the two proteins are 40% identical and 61% similar. Percent identity/similarity between respective domains is shown below the Ect2 protein. Location of mutations and amino acid changes in pbl alleles are indicated by arrows. Lines below the Pbl protein represent constructs used to generate new alleles of pbl in transgenic flies. (BRCT) BRCA1 carboxyl terminus; (DH) Dbl homology; (NLS) nuclear localization signal; (PH) pleckstrin homology. (B) Alignment of amino acid sequences of Pbl, mouse Ect2, and predicted ORF from human EST185199. Black boxes indicate identical amino acids. Common functional domains of Pbl and Ect2 are underlined. The NLS and PEST sequences of Pbl are marked above its sequence. All domains are indicated with the same colors as in A. Amino acids 66–157 of Pbl show 42% identity (56% similarity) to a predicted amino acid sequence of EST185199 (nucleotides 39–314) from human colon carcinoma cell line. In addition, amino acids 44–185 of Pbl show 27% identity (49% similarity) to a predicted amino acid sequence of human EST zs58b08.r1 (nucleotides 248–661; data not shown). Positions of protein truncation caused by nonsense mutations in the pbl² and pbl³ alleles (Q37Ter and W185Ter, respectively) are marked with red triangles. The red letter D denotes the V531D mutation within the DH domain in the pbl⁵ allele. The GenBank accession nos. are L11316 (ect2), AA313301 (EST185199), and AA287172 (EST zs58b08.r1). The BRCT domains and DH and PH domains of Ect2 were defined according to Callebaut and Mornon (1997) and Schultz et al. (1998), respectively. (C) Mitotic figures in cycle 14 domains of a wild-type embryo stained with anti-Pbl (red) and anti-phosphohistone H3 (yellow) to mark chromosomes during mitosis. Pbl protein is not detected during late prophase, metaphase, and anaphase. Pbl has the highest levels of expression in telophase and interphase nuclei.