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Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli.

D'Alterio C, Tran DD, Yeung MW, Hwang MS, Li MA, Arana CJ, Mulligan VK, Kubesh M, Sharma P, Chase M, Tepass U, Godt D - J. Cell Biol. (2005)

Bottom Line: Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length.Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length.This study reveals Cad99C as a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Toronto, Toronto, Ontario, Canada, M5S 3G5.

ABSTRACT
Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. In this paper, we analyze the function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. This study reveals Cad99C as a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process.

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Loss of Cad99C causes defects in the microvillus brush border of follicle cells. All panels show a confocal section of the follicular epithelium of late stage 10a egg chambers. The oocyte is up. (A) In wild type, sandwiched between the F-actin–rich cortices of oocyte and follicle cells, are the actin filament bundles of follicle cell microvilli (arrowhead). (B) F-actin–rich protrusions on the apical surface of follicle cells are largely missing in a Cad99C21-8 mutant egg chamber. (C and C') Microvilli in phenotypically wild-type follicles (Cad99C21-8/+) are visualized by Cad99C (C, red) or CD8::GFP (C', green) and shown at higher magnification in insets. Expression of UAS-CD8::GFP was induced by Act5c>CD2>Gal4. (D and E) Examples of Cad99C21-8/21-6 mutant follicles stained with CD8::GFP (green), showing that microvilli, which are displayed at higher magnification in insets, are strongly reduced in size and irregular in shape and arrangement. (F) Top view showing regular microvilli tufts on the apical surface of normal (Cad99C21-8/+) follicle cells stained with CD8::GFP. (G) In contrast, the apical surface of Cad99C21-8/21-6 mutant follicle cells shows an irregular distribution of microvilli. (H) Cad99C dsRNA was coexpressed with CD8::GFP (green) in follicle cell clones. These cells do not express Cad99C (red) and show strongly reduced microvilli compared with neighboring Cad99C expressing wild-type cells. In wild type (I) and Cad99C21-8 mutants (J), Nudel protein (green) is secreted by follicle cells and located in the extracellular space between follicle cells and oocyte. Wild-type microvilli contain Cad99C (red). (K and L) The Nomarski image reveals a regular striped band of microvilli and vitelline bodies in wild type (K) but shows a disrupted irregular band in a Cad99C21-8 mutant (L). Accompanying confocal images show the presence (K) and absence (L) of Cad99C (red). An arrowhead points to the microvillus brush border in A–E, K, and L. wt, wild type; Cad99C−, Cad99C mutant. Bars, 10 μm.
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fig5: Loss of Cad99C causes defects in the microvillus brush border of follicle cells. All panels show a confocal section of the follicular epithelium of late stage 10a egg chambers. The oocyte is up. (A) In wild type, sandwiched between the F-actin–rich cortices of oocyte and follicle cells, are the actin filament bundles of follicle cell microvilli (arrowhead). (B) F-actin–rich protrusions on the apical surface of follicle cells are largely missing in a Cad99C21-8 mutant egg chamber. (C and C') Microvilli in phenotypically wild-type follicles (Cad99C21-8/+) are visualized by Cad99C (C, red) or CD8::GFP (C', green) and shown at higher magnification in insets. Expression of UAS-CD8::GFP was induced by Act5c>CD2>Gal4. (D and E) Examples of Cad99C21-8/21-6 mutant follicles stained with CD8::GFP (green), showing that microvilli, which are displayed at higher magnification in insets, are strongly reduced in size and irregular in shape and arrangement. (F) Top view showing regular microvilli tufts on the apical surface of normal (Cad99C21-8/+) follicle cells stained with CD8::GFP. (G) In contrast, the apical surface of Cad99C21-8/21-6 mutant follicle cells shows an irregular distribution of microvilli. (H) Cad99C dsRNA was coexpressed with CD8::GFP (green) in follicle cell clones. These cells do not express Cad99C (red) and show strongly reduced microvilli compared with neighboring Cad99C expressing wild-type cells. In wild type (I) and Cad99C21-8 mutants (J), Nudel protein (green) is secreted by follicle cells and located in the extracellular space between follicle cells and oocyte. Wild-type microvilli contain Cad99C (red). (K and L) The Nomarski image reveals a regular striped band of microvilli and vitelline bodies in wild type (K) but shows a disrupted irregular band in a Cad99C21-8 mutant (L). Accompanying confocal images show the presence (K) and absence (L) of Cad99C (red). An arrowhead points to the microvillus brush border in A–E, K, and L. wt, wild type; Cad99C−, Cad99C mutant. Bars, 10 μm.

Mentions: To analyze microvillus formation in the absence of Cad99C, we examined the distribution of F-actin and the membrane marker CD8-GFP. In wild type, F-actin is seen in microvilli of follicle cells and is enriched in the cortex of follicle cells and oocyte (Fig. 5 A). In Cad99C mutant follicles, F-actin staining is strongly reduced in the space between follicle cells and oocyte, suggesting defective microvilli (Fig. 5 B). CD8::GFP labeling revealed a regular microvillus brush border in wild-type follicle cells (Fig. 5, C' and F), whereas Cad99C mutant follicle cells displayed a range of defects of microvilli (Fig. 5, D, E, and G). In all mutant follicles, microvilli appeared substantially shorter than in wild type (Fig. 5, D and E). In many cases, apical protrusions appeared reduced in number, and they formed an irregular spiky pattern, suggesting that microvilli are abnormally shaped and may be clumped. In other cases, no obvious protrusions were detected or only few microvilli with an abnormal, wavy shape protruded from the apical surface (Fig. 5, E and G). Expression of Cad99C dsRNA caused the same type of microvilli defects as Cad99C mutations (Fig. 5 H). The persistent strong CD8::GFP labeling of the apical membrane domain even in cases where no protrusions were detected indicates that a substantial amount of membrane material is retained apically. These findings argue against a complete loss of microvilli and suggest that microvilli are strongly shortened and may also be collapsed against the apical cell surface. Together, these data show that Cad99C is essential to form microvilli of normal length and organization in follicle cells.


Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli.

D'Alterio C, Tran DD, Yeung MW, Hwang MS, Li MA, Arana CJ, Mulligan VK, Kubesh M, Sharma P, Chase M, Tepass U, Godt D - J. Cell Biol. (2005)

Loss of Cad99C causes defects in the microvillus brush border of follicle cells. All panels show a confocal section of the follicular epithelium of late stage 10a egg chambers. The oocyte is up. (A) In wild type, sandwiched between the F-actin–rich cortices of oocyte and follicle cells, are the actin filament bundles of follicle cell microvilli (arrowhead). (B) F-actin–rich protrusions on the apical surface of follicle cells are largely missing in a Cad99C21-8 mutant egg chamber. (C and C') Microvilli in phenotypically wild-type follicles (Cad99C21-8/+) are visualized by Cad99C (C, red) or CD8::GFP (C', green) and shown at higher magnification in insets. Expression of UAS-CD8::GFP was induced by Act5c>CD2>Gal4. (D and E) Examples of Cad99C21-8/21-6 mutant follicles stained with CD8::GFP (green), showing that microvilli, which are displayed at higher magnification in insets, are strongly reduced in size and irregular in shape and arrangement. (F) Top view showing regular microvilli tufts on the apical surface of normal (Cad99C21-8/+) follicle cells stained with CD8::GFP. (G) In contrast, the apical surface of Cad99C21-8/21-6 mutant follicle cells shows an irregular distribution of microvilli. (H) Cad99C dsRNA was coexpressed with CD8::GFP (green) in follicle cell clones. These cells do not express Cad99C (red) and show strongly reduced microvilli compared with neighboring Cad99C expressing wild-type cells. In wild type (I) and Cad99C21-8 mutants (J), Nudel protein (green) is secreted by follicle cells and located in the extracellular space between follicle cells and oocyte. Wild-type microvilli contain Cad99C (red). (K and L) The Nomarski image reveals a regular striped band of microvilli and vitelline bodies in wild type (K) but shows a disrupted irregular band in a Cad99C21-8 mutant (L). Accompanying confocal images show the presence (K) and absence (L) of Cad99C (red). An arrowhead points to the microvillus brush border in A–E, K, and L. wt, wild type; Cad99C−, Cad99C mutant. Bars, 10 μm.
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fig5: Loss of Cad99C causes defects in the microvillus brush border of follicle cells. All panels show a confocal section of the follicular epithelium of late stage 10a egg chambers. The oocyte is up. (A) In wild type, sandwiched between the F-actin–rich cortices of oocyte and follicle cells, are the actin filament bundles of follicle cell microvilli (arrowhead). (B) F-actin–rich protrusions on the apical surface of follicle cells are largely missing in a Cad99C21-8 mutant egg chamber. (C and C') Microvilli in phenotypically wild-type follicles (Cad99C21-8/+) are visualized by Cad99C (C, red) or CD8::GFP (C', green) and shown at higher magnification in insets. Expression of UAS-CD8::GFP was induced by Act5c>CD2>Gal4. (D and E) Examples of Cad99C21-8/21-6 mutant follicles stained with CD8::GFP (green), showing that microvilli, which are displayed at higher magnification in insets, are strongly reduced in size and irregular in shape and arrangement. (F) Top view showing regular microvilli tufts on the apical surface of normal (Cad99C21-8/+) follicle cells stained with CD8::GFP. (G) In contrast, the apical surface of Cad99C21-8/21-6 mutant follicle cells shows an irregular distribution of microvilli. (H) Cad99C dsRNA was coexpressed with CD8::GFP (green) in follicle cell clones. These cells do not express Cad99C (red) and show strongly reduced microvilli compared with neighboring Cad99C expressing wild-type cells. In wild type (I) and Cad99C21-8 mutants (J), Nudel protein (green) is secreted by follicle cells and located in the extracellular space between follicle cells and oocyte. Wild-type microvilli contain Cad99C (red). (K and L) The Nomarski image reveals a regular striped band of microvilli and vitelline bodies in wild type (K) but shows a disrupted irregular band in a Cad99C21-8 mutant (L). Accompanying confocal images show the presence (K) and absence (L) of Cad99C (red). An arrowhead points to the microvillus brush border in A–E, K, and L. wt, wild type; Cad99C−, Cad99C mutant. Bars, 10 μm.
Mentions: To analyze microvillus formation in the absence of Cad99C, we examined the distribution of F-actin and the membrane marker CD8-GFP. In wild type, F-actin is seen in microvilli of follicle cells and is enriched in the cortex of follicle cells and oocyte (Fig. 5 A). In Cad99C mutant follicles, F-actin staining is strongly reduced in the space between follicle cells and oocyte, suggesting defective microvilli (Fig. 5 B). CD8::GFP labeling revealed a regular microvillus brush border in wild-type follicle cells (Fig. 5, C' and F), whereas Cad99C mutant follicle cells displayed a range of defects of microvilli (Fig. 5, D, E, and G). In all mutant follicles, microvilli appeared substantially shorter than in wild type (Fig. 5, D and E). In many cases, apical protrusions appeared reduced in number, and they formed an irregular spiky pattern, suggesting that microvilli are abnormally shaped and may be clumped. In other cases, no obvious protrusions were detected or only few microvilli with an abnormal, wavy shape protruded from the apical surface (Fig. 5, E and G). Expression of Cad99C dsRNA caused the same type of microvilli defects as Cad99C mutations (Fig. 5 H). The persistent strong CD8::GFP labeling of the apical membrane domain even in cases where no protrusions were detected indicates that a substantial amount of membrane material is retained apically. These findings argue against a complete loss of microvilli and suggest that microvilli are strongly shortened and may also be collapsed against the apical cell surface. Together, these data show that Cad99C is essential to form microvilli of normal length and organization in follicle cells.

Bottom Line: Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length.Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length.This study reveals Cad99C as a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Toronto, Toronto, Ontario, Canada, M5S 3G5.

ABSTRACT
Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. In this paper, we analyze the function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. This study reveals Cad99C as a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process.

Show MeSH
Related in: MedlinePlus