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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01382

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Citron Kinase is an essential effector of the Pbl-activated Rho signalling pathway in Drosophila melanogaster

¹ ARC Special Research Centre for the Molecular Genetics of Development, Adelaide University, Adelaide, SA 5005, Australia
² School of Molecular and Biomedical Science, Adelaide University, Adelaide, SA 5005, Australia
³ Molecular Genetics and Evolution, Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia

View larger version (12K): [in a new window]   Fig. 1. Evolutionary conservation of the Citron Kinase family of proteins. (A) The family of Drosophila proteins containing the same conserved domains as vertebrate Citron. Each line is a scale representation of the protein sequence (scale bar represents 100 amino acids), with each conserved domain marked as a different shape. S/T Kinase, protein kinase C-class Serine/Threonine kinase domain; C1, protein kinase C-type diacylglycerol binding domain; PH, Pleckstrin homology phospholipid binding domain; CNH, Citron homology domain of unknown function; PBD, p21-like Cdc42 binding domain. Numbers between the conserved domains indicate the percentage of amino acid identity between the corresponding domains. Arrowed lines indicate the regions known to be required for binding to Rho. (B) A C-terminal fragment of Citron (Citron 4, amino acids 1439 to 1854) interacts specifically with constitutively active RhoA-G14V. LexA-Citron fusion proteins 1 to 4, represented by the lines below the domain structure of Citron in A, were assayed for interaction with VP16-RhoA-G14V fusion protein by activation of a lacZ reporter gene in a yeast two-hybrid assay. The strength of interaction is indicated to the right of each Citron fragment, with (–) indicating no interaction and (+++) indicating a strong interaction.

View larger version (122K): [in a new window]   Fig. 2. citron is expressed maternally and in proliferating tissues during Drosophila development. Whole-mount in situ hybridisation with a Drosophila citron DIG-labelled RNA probe. (A) A stage 10A oocyte showing citron transcripts in the nurse cells. (B) Uniform distribution of citron transcripts in a blastoderm embryo. Maternally provided citron mRNA has degraded by stage 9, compare wild-type (C) and homozygous Df(3)iro-2 mutant (D) embryos. (E-H) Zygotic tissue-specific expression of citron in the proliferating CNS (arrow) and PNS (arrowhead) starts during germ-band retraction. Anterior is to the left, dorsal to the top.

View larger version (61K): [in a new window]   Fig. 3. Subcellular localisation of Citron-GFP to the contractile ring during cytokinesis depends on the normal activation of Rho signalling. (A-H) Citron-GFP was expressed in the 3-6 hour embryonic epidermis using paired-Gal4. DNA is stained with Hoechst 33258 (B,F), Citron-GFP is stained with anti-GFP (C,G), and microtubules are stained with anti--tubulin antibodies (D,H). (A,E) Merged images with Citron-GFP stained green, DNA stained blue and microtubules stained red. (A-D) A wild-type embryo showing Citron-GFP in the contractile ring (arrow) as it constricts around the central spindle microtubules. Citron-GFP is not localised in the adjacent anaphase cell that is yet to constrict (arrowhead). (E-G) An embryo mutant for the Rho activator pebble showing typical tetranucleate and binucleate cells and bipolar and tripolar spindles of cells failing cytokinesis. Citron-GFP shows no localisation to the contractile ring in pebble mutant telophase cells. It is found diffusely through the cytoplasm of telophase cells and not at the positions where contractile rings would normally form (arrowheads). (I-J) Drosophila Schneider line 2 cultured cells stained for DNA (green) and actin (red). Incubation with citron dsRNA (I) or dsRNA corresponding to the Rho activator pebble (J) causes the formation of multinucleate cells.

View larger version (75K): [in a new window]   Fig. 4. Citron function during cytokinesis in the Drosophila embryonic PNS depends on Rho activation. Flat preparations of the PNS from abdominal segments of stage 16 embryos stained for the neuronal specific antibody 22C10 (A,A',B,B',D,D'). (C-C''') Embryonic PNS cells stained with: 22C10 (C, blue in C'''), nuclear envelope marker anti-Lamin (C', red in C''') and plasma membrane marker anti- Spectrin (C'', green in C'''). Anterior is to the left and dorsal up. (A,B,D) Dorsal external sensory (DES) cluster area. (A',B',C-C''',D') Lateral chordotonal organ (CH) cluster area. (A,A') w1118 (control) embryos showing typical organization of the DES and CH cell clusters. (B,B',C-C''') cit2/Df(3)iro-2 mutant embryos exhibit variable abnormalities, such as absence of cells and multinucleate cells (arrows). The number of nuclei in these cells was estimated by presence of Lamin-positive nuclear envelopes within -Spectrin delineated cellular membrane. A general disorganisation of both clusters and axonal misrouting was also observed. (D,D') cit2/Df(3)iro-2 mutant embryos that are also heterozygous for pbl3, showing an increase in the number of multinucleate cells (arrow). (E) To quantitate the effect of loss of one copy of the pbl gene on the cit phenotype, the number of mono- and multinucleate PNS cells in a defined region was scored for each genotype (cells were scored in 30 identical frames). Bars represent the percentage of multinucleate cells in each genotype. Error bars represent one standard deviation.

View larger version (85K): [in a new window]   Fig. 5. Loss of Drosophila citron leads to failure of cytokinesis and hyperploidy in the larval brain. Wild-type (A) and cit2/Df(3)iro-2 mutant (B-H'') brain lobe tissues from third instar larvae. (A,B) Immunochemical staining of whole mount brain lobes with anti-phospho Histone H3 (PH3), to detect cells in mitosis, showing large nuclei (arrow) in cit2/Df(3)iro-2 cells (B) compared to wild-type (A). (C) Binucleate cells dissociated from cit2/Df(3)iro-2 mutant brain lobes stained with Hoechst 33258 to detect DNA (white) overlaid on the phase-contrast image of the cells. (D-F) Mitotic figures from cit2/Df(3)iro-2 mutant cells, which were squashed and stained with Giemsa dye to detect chromosomes. (D) Tetraploid cell at metaphase. (E) Hyperploid cell showing effective chromosome separation at anaphase. (F) Very highly hyperploid cell. (G-G'', H-H'') High magnification view of individual polyploid cells from mutant brain lobes stained with anti--Tubulin (G,H) and anti-PH3 (G',H'). (G'',H'') Merged images. (G-G'') Abnormal metaphase. Three spindle poles are detected by anti--Tubulin staining (cf pebble mutants in Fig. 3E). (H-H'') The cell exhibits a highly polyploid content of condensed DNA and there are eight spindle poles (four in the field of view) associated with defective microtubule spindles that fail to assemble chromosomes at the cell equator (H').

View larger version (83K): [in a new window]   Fig. 6. Loss of Drosophila citron gene function results in a dramatic reduction in imaginal disc size. Imaginal discs from wild-type (A,C,E,G) and cit2/Df(3)iro-2 mutant (B,D,F,H) third instar larvae labelled with Hoechst 33258 to detect DNA (A,B) or with anti-PH3 to detect cells in mitosis (C,D, shown at high magnification) or labelled using TUNEL to detect cells undergoing apoptosis (E,F and high magnification in G,H). Transheterozygous mutant imaginal discs exhibit a dramatic reduction in size (e.g. leg discs shown in B, compare with wild type in A), contain fewer cells progressing through mitosis (compare D with C) and many more cells undergoing apoptosis (compare mutant wing disc in F and H with wild type in E and G). Scale bars: 25 µm.

View larger version (79K): [in a new window]   Fig. 7. Reduction of Pebble function in wings by RNA interference causes cytokinetic failure. (A,C,E) Low magnification images of whole adult wings. (A',C',E') Corresponding high magnification images of the posterior region of the wing (framed in A) that was used to evaluate phenotypes (see Fig. 8E). (A,A') A wild-type wing showing normal size and shape of the wing (A) and ordered arrangement of hairs (A'). (B,B') A high magnification of a wild-type pupal wing stained with Phalloidin to detect F-actin (red) and Hoechst 33258 for DNA (blue). Each individual wild-type cell has one nucleus (B) and one prehair F-actin bundle (B'). (C,C') An en-GAL4>UAS-pblRNAi wing showing diminution of Engrailed-domain of the wing (C) and wing hair phenotype (C') resulting from reduced pbl activity. Many en-GAL4>UAS-pblRNAi adult wing cells have more that one hair. (D,D') A high magnification view of an en-GAL4> UAS-pblRNAi mutant pupal wing stained with Phalloidin for F-actin (red) and Hoechst 33258 for DNA (blue). Mutant cells show more than one nucleus (D) and prehair F-actin bundles (D'). (E,E') Loss of one copy of RhoA72R further reduces the Engrailed-specific region of the en-GAL4>UAS-pblRNAi wing (E) and increases the number of multihaired cells (E'). The multiple haired cell phenotype is quantified in Fig. 8E.

View larger version (82K): [in a new window]   Fig. 8. Citron acts as a positive factor in the Pebble-Rho signalling pathway. Images of whole adult wings. (A) An en-GAL4>UAS-pblRNAi wing in a wild-type background. (B) An en-GAL4>UAS-pblRNAi wing heterozygous for cit1 shows significant enhancement of the pblRNAi phenotype. (C) An en-GAL4>UAS-pblRNAi wing heterozygous for rok2 does not appear be significantly modified. (D) Co-expression of a gain-of-function allele of citron (UAS-cit2) and en-GAL4>UAS-pblRNAi slightly rescues the pblRNAi phenotype. (E) The number of multiple haired wing cells in a defined region (posterior to L5, framed in Fig. 7A) was scored for each genotype shown in Fig. 7 and Fig. 8 in order to quantitate the effect. Multiple haired cells were scored in 14 adult wings for each genotype. Bars represent the percentage of multihaired cells in each genotype. Error bars represent one standard deviation. Statistical significance of the difference between two pairs of genotypes was determined using a 2 2x2 contingency test (P<0.001). Significant enhancement of the en-GAL4>UAS-pblRNAi phenotype was seen when flies were heterozygous for RhoA72R, cit1 or Df(3)iro-2 alleles.