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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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Molecular analysis of Cad99C. (A) Genomic map of the Cad99C transcription unit. The ORF is in red. Deletions derived from P element insert GE21034 are shown in blue and those from GE23478 in green. A dotted line indicates the uncertainty interval of a breakpoint. Triangles indicate primers used to map the deletion breakpoints. A black bar indicates the region used for RNAi. TM, transmembrane domain. (B) Cad99C and PCDH15 have a similar protein structure characterized by 11 CDs (blue), a single transmembrane domain (yellow), and a PDZ domain–binding site at the COOH terminus (green). Potential Ca2+-binding sites between CDs are shown in red. Arrows point to linker regions that presumably cannot bind Ca2+. Black bars indicate regions used for generation of antibodies. (C) Immunoblot analysis of Cad99C expression in ovaries. Cad99C antibodies (extracellular epitope; B) reveal a major protein of ∼217 KD (arrowhead) in wild type that is not detected in Cad99C mutants and severely reduced after Cad99C RNAi. The two smaller proteins (195 and 172 kD) found in wild type but not in mutant ovaries may represent degraded or otherwise modified Cad99C products. Only the Cad99C23-2 allele, which still contains exon 1 in contrast to other alleles (A), produces detectable amounts of protein, albeit of reduced size.
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fig2: Molecular analysis of Cad99C. (A) Genomic map of the Cad99C transcription unit. The ORF is in red. Deletions derived from P element insert GE21034 are shown in blue and those from GE23478 in green. A dotted line indicates the uncertainty interval of a breakpoint. Triangles indicate primers used to map the deletion breakpoints. A black bar indicates the region used for RNAi. TM, transmembrane domain. (B) Cad99C and PCDH15 have a similar protein structure characterized by 11 CDs (blue), a single transmembrane domain (yellow), and a PDZ domain–binding site at the COOH terminus (green). Potential Ca2+-binding sites between CDs are shown in red. Arrows point to linker regions that presumably cannot bind Ca2+. Black bars indicate regions used for generation of antibodies. (C) Immunoblot analysis of Cad99C expression in ovaries. Cad99C antibodies (extracellular epitope; B) reveal a major protein of ∼217 KD (arrowhead) in wild type that is not detected in Cad99C mutants and severely reduced after Cad99C RNAi. The two smaller proteins (195 and 172 kD) found in wild type but not in mutant ovaries may represent degraded or otherwise modified Cad99C products. Only the Cad99C23-2 allele, which still contains exon 1 in contrast to other alleles (A), produces detectable amounts of protein, albeit of reduced size.

Mentions: Gene annotation and cDNA analysis predict that Cad99C (CG310034; Celniker et al., 2002) encodes a single-pass transmembrane protein with 11 cadherin domains (CDs; Fig. 2 B). The cytoplasmic tail of Cad99C is asparagine rich and contains the motif SEVETTTEL at the COOH terminus, in which the underlined residues fit the consensus S/T–X–L/V of class 1 PDZ domain–binding sites (Sheng and Sala, 2001). Cad99C is conserved in Anopheles gambiae and Apis mellifora, showing 62 and 52% identity in the extracellular region and 44 and 32% in the cytoplasmic tail, respectively. The closest relative in humans is PCDH15. Cad99C and PCDH15 have very similar protein architectures (Fig. 2 B). They show 30% identity across the 11 extracellular CDs (expect value is e−121; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507072/DC1). In comparison, there is 24% identity between the 11 CDs of Cad99C and the first 11 CDs or next 11 CDs of human fat3 (e−57 and 3e−63, respectively), a cadherin that gives the second best score in a Blast search. PCDH15 also contains a COOH-terminal PDZ-binding site (SQSTSL; Adato et al., 2005); otherwise, no significant similarity was identified in the cytoplasmic tail. It is noteworthy that the cytoplasmic tail is substantially diverged even between mouse and human PCDH15, showing only 57% identity, in contrast to 94% identity in the extracellular portion.


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)

Molecular analysis of Cad99C. (A) Genomic map of the Cad99C transcription unit. The ORF is in red. Deletions derived from P element insert GE21034 are shown in blue and those from GE23478 in green. A dotted line indicates the uncertainty interval of a breakpoint. Triangles indicate primers used to map the deletion breakpoints. A black bar indicates the region used for RNAi. TM, transmembrane domain. (B) Cad99C and PCDH15 have a similar protein structure characterized by 11 CDs (blue), a single transmembrane domain (yellow), and a PDZ domain–binding site at the COOH terminus (green). Potential Ca2+-binding sites between CDs are shown in red. Arrows point to linker regions that presumably cannot bind Ca2+. Black bars indicate regions used for generation of antibodies. (C) Immunoblot analysis of Cad99C expression in ovaries. Cad99C antibodies (extracellular epitope; B) reveal a major protein of ∼217 KD (arrowhead) in wild type that is not detected in Cad99C mutants and severely reduced after Cad99C RNAi. The two smaller proteins (195 and 172 kD) found in wild type but not in mutant ovaries may represent degraded or otherwise modified Cad99C products. Only the Cad99C23-2 allele, which still contains exon 1 in contrast to other alleles (A), produces detectable amounts of protein, albeit of reduced size.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171266&req=5

fig2: Molecular analysis of Cad99C. (A) Genomic map of the Cad99C transcription unit. The ORF is in red. Deletions derived from P element insert GE21034 are shown in blue and those from GE23478 in green. A dotted line indicates the uncertainty interval of a breakpoint. Triangles indicate primers used to map the deletion breakpoints. A black bar indicates the region used for RNAi. TM, transmembrane domain. (B) Cad99C and PCDH15 have a similar protein structure characterized by 11 CDs (blue), a single transmembrane domain (yellow), and a PDZ domain–binding site at the COOH terminus (green). Potential Ca2+-binding sites between CDs are shown in red. Arrows point to linker regions that presumably cannot bind Ca2+. Black bars indicate regions used for generation of antibodies. (C) Immunoblot analysis of Cad99C expression in ovaries. Cad99C antibodies (extracellular epitope; B) reveal a major protein of ∼217 KD (arrowhead) in wild type that is not detected in Cad99C mutants and severely reduced after Cad99C RNAi. The two smaller proteins (195 and 172 kD) found in wild type but not in mutant ovaries may represent degraded or otherwise modified Cad99C products. Only the Cad99C23-2 allele, which still contains exon 1 in contrast to other alleles (A), produces detectable amounts of protein, albeit of reduced size.
Mentions: Gene annotation and cDNA analysis predict that Cad99C (CG310034; Celniker et al., 2002) encodes a single-pass transmembrane protein with 11 cadherin domains (CDs; Fig. 2 B). The cytoplasmic tail of Cad99C is asparagine rich and contains the motif SEVETTTEL at the COOH terminus, in which the underlined residues fit the consensus S/T–X–L/V of class 1 PDZ domain–binding sites (Sheng and Sala, 2001). Cad99C is conserved in Anopheles gambiae and Apis mellifora, showing 62 and 52% identity in the extracellular region and 44 and 32% in the cytoplasmic tail, respectively. The closest relative in humans is PCDH15. Cad99C and PCDH15 have very similar protein architectures (Fig. 2 B). They show 30% identity across the 11 extracellular CDs (expect value is e−121; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507072/DC1). In comparison, there is 24% identity between the 11 CDs of Cad99C and the first 11 CDs or next 11 CDs of human fat3 (e−57 and 3e−63, respectively), a cadherin that gives the second best score in a Blast search. PCDH15 also contains a COOH-terminal PDZ-binding site (SQSTSL; Adato et al., 2005); otherwise, no significant similarity was identified in the cytoplasmic tail. It is noteworthy that the cytoplasmic tail is substantially diverged even between mouse and human PCDH15, showing only 57% identity, in contrast to 94% identity in the extracellular portion.

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