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First published online 28 February 2006
doi: 10.1242/jcs.02831

Journal of Cell Science 119, 1184-1195 (2006)
Published by The Company of Biologists 2006
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Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells

Karin Schlichting*, Michaela Wilsch-Bräuninger*, Fabio Demontis* and Christian Dahmann{ddagger}

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany


Figure 1
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  		Fig. 1. Cad99C protein is not detected in ovaries of Cad99C mutant flies. (A) Extent of the deletions in Cad99C mutant alleles. The positions of the EP elements GE21034 and GE23478, which were used to generate the Cad99C mutant alleles, and the exon-intron structure of the Cad99C gene are indicated. The allele Cad99C248A also removes the two predicted genes CG15510 and CG31033 to the left of Cad99C. (B) Western blot analysis of ovaries with an anti-Cad99C antiserum. Ovaries were isolated from heterozygous Cad99C57A/+, Cad99C51C/+, Cad99C120B/+, and Cad99C248A/+ (lanes 1, 3, 5, 7) and homozygous mutant Cad99C57A/57A, Cad99C51C/51C, Cad99C120B/120B, and Cad99C248A/248A (lanes 2, 4, 6, 8) flies. The anti-Cad99C antiserum detects a protein of 184 kDa (arrow), the expected molecular weight of Cad99C, in extracts of heterozygous, but not homozygous, Cad99C mutant ovaries. Subsequent blotting with an anti-{alpha}-tubulin antibody (lower panel) shows the loading of similar amounts of protein in each lane. Molecular weights are indicated to the left (in kDa).

 

Figure 2
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  		Fig. 2. Eggs laid by Cad99C mutant flies are permeable to the dye Neutral Red. (A-E) Eggs derived from (A) heterozygous Cad99C57A/+ or (B-E) homozygous mutant Cad99C57A/57A flies were dechorionated and incubated in the dye Neutral Red. Eggs derived from homozygous mutant flies showed a range of Neutral Red stainings from (B) undetectable to (C) uniform weak, (D) partially strong and (E) uniformly strong. (F) Quantification of the percentage of eggs with no, weak, partial and strong Neutral Red staining. The genotypes of the flies from which the eggs were derived are indicated. GE21034 is the parental fly line used to generate the Cad99C57A and Cad99C248A mutants. At least 750 eggs per genotype were analyzed.

 

Figure 3
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  		Fig. 3. The vitelline membrane of Cad99C mutant eggshells is unevenly deposited and displays holes. (A,B) Ultrathin sections of stage-13 egg chambers of (A) heterozygous Cad99C57A/+ or (B) homozygous mutant Cad99C57A/57A flies stained for the vitelline membrane protein sV17. Control egg chambers display a continuous line of sV17 staining, whereas mutant egg chambers display sites where the sV17 staining is not detected (asterisk). (C,D) Electron micrographs of sections through stage-11 egg chambers of (C) heterozygous Cad99C57A/+ or (D) homozygous mutant Cad99C57A/57A flies. The vitelline membrane (Vm) is unevenly deposited in the mutant egg chambers and is not detected at some sites (asterisks). Oo, oocyte; Fc, follicle cells. Bars, 2 µm (C,D).

 

Figure 4
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  		Fig. 4. Cad99C is expressed in follicle cells surrounding the oocyte. (A,B) Wild-type ovaries hybridized with a Cad99C-specific RNA probe. (C-E') Ovaries of (C-D') heterozygous Cad99C57A/+ or (E,E') homozygous mutant Cad99C57A/57A flies stained with an anti-Cad99C antiserum (green) and Rhodamine-Phalloidin (red). In (C,D,E) only the Cad99C staining is shown. Bars, 20 µm (C); 50 µm (D-E). St., stage.

 

Figure 5
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  		Fig. 5. Cad99C protein localizes to microvilli of follicle cells. (A-C) Follicle cells of a stage-10 wild-type egg chamber stained for (A) DE-cadherin and (C) Cad99C. (B) Merge of (A) and (C). DE-cadherin is shown in green and Cad99C in red. Apical is to the top. (D-F) Follicle cells of a stage-10 egg chamber expressing the membrane marker CD8-GFP (CY2, UAS-CD8-GFP) stained for (D) GFP and (F) Cad99C. (E) Merge of (D) and (F). Cad99C colocalizes with CD8-GFP to apical protrusions. Apical is to the top. (G,H) Electron micrographs of anti-Cad99C immunogold-labeled sections of stage-10 egg chambers of (G) heterozygous Cad99C57A/+ or (H) homozygous mutant Cad99C57A/57A flies. Insets show higher magnification views. 10 nm immunogold particles are detected on microvilli of heterozygous Cad99C57A/+ follicle cells, but not homozygous mutant Cad99C57A/57A follicle cells. Note that, in homozygous mutant Cad99C57A/57A flies, microvilli (arrowheads) are observed more frequently between the vitelline bodies and the planar follicle cell surface compared with control flies. Oo, oocyte; Fc, follicle cells; Yg, Yolk granule; Vb, Vitelline body. Bars, 5 µm (A-F); 500 nm (G,H).

 

Figure 6
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  		Fig. 6. Follicle cell microvilli are abnormal in Cad99C mutants. (A-C) Follicle cells of stage-10 (A) heterozygous Cad99C51C/+, (B) mutant Cad99C57A/51C, or (C) mutant Cad99C57A/120B egg chambers expressing CD8-GFP (CY2, UAS-CD8-GFP) stained for GFP. Cad99C mutant flies have abnormal CD8-GFP-labeled protrusions compared with control flies. Apical is to the top. (D-F) Clones of homozygous mutant Cad99C57A/57A follicle cells of stage-10 egg chambers stained with Rhodamine-Phalloidin marked by the absence of Cad99C. Cad99C staining is shown in (D) and Rhodamine-Phalloidin staining in (F). (E) Merge of (D) and (F). Phalloidin staining detects microvilli in between control follicle cells and the oocyte (arrowhead), but not between mutant follicle cells and the oocyte (arrow). Note that the distance between the oocyte and the Cad99C57A/57A follicle cells is increased. Oo, oocyte; Fc, Follicle cells. Bars, 5 µm (A-D).

 

Figure 7
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  		Fig. 7. Overexpression of Cad99C leads to large bundles of microvilli and vitelline membrane defects. (A,B) Follicle cells of stage-10 egg chambers expressing (A) CD8-GFP (CY2, UAS-CD8-GFP) or (B) CD8-GFP and Cad99C-HA (CY2, UAS-CD8-GFP, UAS-Cad99C-HA) stained for GFP. The CD8-GFP-labeled protrusions in follicle cells expressing Cad99C-HA form roof-like structures (asterisk). Apical is to the top. (C,D) Electron micrographs of anti-Cad99C immunogold-labeled sections of stage-10 (C) control egg chambers and (D) egg chambers expressing Cad99C-GFP (Act5C>GAL4, UAS-Cad99C-GFP) in clones of follicle cells. Overexpression of Cad99C-GFP results in large Cad99C-labeled bundles of follicle cell microvilli located in between vitelline bodies (Vb). Cad99C-labeled microvilli that are parallel and close to the oocyte surface are marked by an asterisk. (E,F) Electron micrographs of anti-sV17 10 nm immunogold-labeled sections of stage-12 (E) control egg chambers and (F) egg chambers expressing Cad99C-GFP (Act5C>GAL4, UAS-Cad99C-GFP) in clones of follicle cells. Long microvilli persist in follicle cells expressing Cad99C-GFP and the vitelline membrane (Vm) is disrupted (asterisk). Oo, oocyte; Fc, follicle cells. Bars, 5 µm (A,B); 500 nm (C-F).

 

Figure 8
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  		Fig. 8. The extracellular region fused to the transmembrane domain of Cad99C is sufficient to promote the formation of large bundles of follicle cell microvilli. (A) Scheme of Cad99C-HA, Cad99C-EXTRA-HA and Cad99C-INTRA-HA. Cad99C-EXTRA-HA lacks the entire cytoplasmic region, whereas Cad99C-INTRA-HA lacks most of the extracellular region. Full-length and deletion mutants of Cad99C all have a triple HA tag at their C-terminus. Sp, signal peptide; TM, transmembrane domain; PDZ-BS, putative PDZ domain-binding site. (B-E) Electron micrographs of anti-HA 12 nm immunogold-labeled sections of stage-10 (B) control egg chambers (Act5C>GAL4) and egg chambers expressing (C) Cad99C-HA (Act5C>GAL4, UAS-Cad99C-HA), (D) Cad99C-INTRA-HA (Act5C>GAL4, UAS-Cad99C-INTRA-HA), and (E) Cad99C-EXTRA-HA (Act5C>GAL4, UAS-Cad99C-EXTRA-HA) in clones of follicle cells. (C-E) Insets show higher magnification views. Overexpression of Cad99C-HA or Cad99C-EXTRA-HA results in the formation of large HA-labeled bundles of follicle cell microvilli. Oo, oocyte; Fc, follicle cells. Bars, 500 nm (B-E).

 

Figure 9
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  		Fig. 9. Drosophila Cad99C is closely related to human PCDH15. (A) Drosophila melanogaster (Dm) Cad99C, Anopheles gambiae (Ag) Cad99C, Mus musculus (Mm) Pcdh15 and Homo sapiens (Hs) PCDH15 share a similar protein domain organization. They are single-span transmembrane (TM) proteins containing a signal peptide (Sp) and 11 cadherin repeats in their extracellular regions. The cytoplasmic region has a conserved PDZ domain-binding site (PDZ-BS). The available Anopheles Cad99C sequence is incomplete at its N-terminus. (B) Conservation of a C-terminal PDZ domain-binding site between the four proteins. The cytoplasmic regions of Drosophila and Anopheles Cad99C, and mouse Pcdh15 and human PCDH15 contain conserved C-terminal class I PDZ domain-binding sites (underlined) with the consensus sequence (S/T)XL (Sheng and Sala, 2001Go). (C) Phylogenetic tree built with the UPGMA method. Dm Cad99C, Ag Cad99C, Mm Pcdh15 and Hs PCDH15 segregate into the same clade (box), indicating that Cad99C and PCDH15 share a common evolutionary history and are more related to each other than to any other of the cadherins considered. The non-classical cadherins Dm and Ag Cad88C appear to be related to Hs cadherin 23 (CDH23) and Mm Cdh23; like PCDH15, CDH23 is etiologically associated with Usher syndrome type I.

 

Figure 10
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  		Fig. 10. Model for Cad99C function. Cad99C is present on follicle cell microvilli and, through its extracellular cadherin repeats, could establish molecular links to the extracellular matrix, to Cad99C or to a different heterophilic binding partner on a neighboring microvillus, or to a protein located on the oocyte surface. These molecular links could stabilize or promote the assembly of microvilli by providing physical adhesion of the microvilli to a target and/or signaling to the cytoskeleton.

 

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© The Company of Biologists Ltd 2006