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Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.

Li MG, Serr M, Edwards K, Ludmann S, Yamamoto D, Tilney LG, Field CM, Hays TS - J. Cell Biol. (1999)

Bottom Line: In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes.Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility.The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell and Developmental Biology, University of Minnesota, St. Paul, Minnesota 55108, USA.

ABSTRACT
The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

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(a) Amino acid alignment of the actin binding domains in Drosophila and vertebrate filamins. The Drosophila filamin sequence is on top (“fly,” accession number GH12209). In human, three filamin isoforms, ABP280 (or α-filamin), β-filamin, and γ-filamin, have been reported and are encoded by separate genes located at different chromosome positions (Takafuta et al. 1998). Sequence alignment shows that the deduced NH2 terminus of fly filamin matches very well with the human β- and γ-filamins. The start codon of the third human isoform has not been defined, but if the human ABP280 polypeptide initiates at the second ATG, the resultant NH2 terminus matches those of the other human and fly filamins. Human-a represents human ABP280 (α-filamin) encoded by the gene on the X chromosome (accession number 113001); human-b indicates β-filamin on the third chromosome (accession number 328597); human-g represents γ-filamin derived from chromosome 7 (accession number 4218955) and chicken, chicken filamin (accession number 1079404). Residues identical to the fly sequence are denoted by dots. (b) Schematic of a filamin dimer, comparing the predicted human and Drosophila monomers. The drawing is based on previous biochemical and electron microscopy characterizations of human ABP280 structure (Gorlin et al. 1990). Filamin is a homodimer with an elongated subunit structure (160 nm). The self-association of partner subunits occurs at the COOH-terminal end of the molecule within repeat 24, while the actin binding domain (ABD) lies at the opposite free end of each monomeric subunit. The 24 repeats extend from the ABD to the COOH terminus and are interrupted by two hinge regions (hinge 1 and 2). Sequence alignment predicts that repeats 6–9 are missing from the Drosophila filamin. The percentage of amino acid conservation between the fly and human sequences is shown at the right.
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Figure 6: (a) Amino acid alignment of the actin binding domains in Drosophila and vertebrate filamins. The Drosophila filamin sequence is on top (“fly,” accession number GH12209). In human, three filamin isoforms, ABP280 (or α-filamin), β-filamin, and γ-filamin, have been reported and are encoded by separate genes located at different chromosome positions (Takafuta et al. 1998). Sequence alignment shows that the deduced NH2 terminus of fly filamin matches very well with the human β- and γ-filamins. The start codon of the third human isoform has not been defined, but if the human ABP280 polypeptide initiates at the second ATG, the resultant NH2 terminus matches those of the other human and fly filamins. Human-a represents human ABP280 (α-filamin) encoded by the gene on the X chromosome (accession number 113001); human-b indicates β-filamin on the third chromosome (accession number 328597); human-g represents γ-filamin derived from chromosome 7 (accession number 4218955) and chicken, chicken filamin (accession number 1079404). Residues identical to the fly sequence are denoted by dots. (b) Schematic of a filamin dimer, comparing the predicted human and Drosophila monomers. The drawing is based on previous biochemical and electron microscopy characterizations of human ABP280 structure (Gorlin et al. 1990). Filamin is a homodimer with an elongated subunit structure (160 nm). The self-association of partner subunits occurs at the COOH-terminal end of the molecule within repeat 24, while the actin binding domain (ABD) lies at the opposite free end of each monomeric subunit. The 24 repeats extend from the ABD to the COOH terminus and are interrupted by two hinge regions (hinge 1 and 2). Sequence alignment predicts that repeats 6–9 are missing from the Drosophila filamin. The percentage of amino acid conservation between the fly and human sequences is shown at the right.

Mentions: We subsequently identified and sequenced an expressed sequence tag (EST) cDNA clone (data are available from GenBank/EMBL/DDBJ under accession number GH12209), derived from adult head mRNA, that potentially encodes the full-length Drosophila filamin protein. The 7,536-bp cDNA contains the original 3′ cDNA sequence and includes a polyadenylation tail. An uninterrupted open reading frame begins from the first nucleotide of the cDNA; however, the first ATG codon is located 401 bp downstream. Although the flanking sequence of this ATG differs from the consensus sequence for translation initiation in Drosophila (Cavener 1987), conceptual translation from this initial ATG codon predicts a 2343 residue polypeptide with a deduced mass (∼250 kD) and NH2-terminal sequence that are highly similar to human filamins (Fig. 6) (Takafuta et al. 1998). Amino acid sequence comparisons indicate that the Drosophila filamin is ∼50% identical and ∼60% similar to human and chicken filamins along the entire length of the protein. Although we have not unequivocally defined the 5′ end of the transcript, the data suggest that the fly EST cDNA clone represents a full-length copy of the identified 7.5-kb filamin transcript.


Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.

Li MG, Serr M, Edwards K, Ludmann S, Yamamoto D, Tilney LG, Field CM, Hays TS - J. Cell Biol. (1999)

(a) Amino acid alignment of the actin binding domains in Drosophila and vertebrate filamins. The Drosophila filamin sequence is on top (“fly,” accession number GH12209). In human, three filamin isoforms, ABP280 (or α-filamin), β-filamin, and γ-filamin, have been reported and are encoded by separate genes located at different chromosome positions (Takafuta et al. 1998). Sequence alignment shows that the deduced NH2 terminus of fly filamin matches very well with the human β- and γ-filamins. The start codon of the third human isoform has not been defined, but if the human ABP280 polypeptide initiates at the second ATG, the resultant NH2 terminus matches those of the other human and fly filamins. Human-a represents human ABP280 (α-filamin) encoded by the gene on the X chromosome (accession number 113001); human-b indicates β-filamin on the third chromosome (accession number 328597); human-g represents γ-filamin derived from chromosome 7 (accession number 4218955) and chicken, chicken filamin (accession number 1079404). Residues identical to the fly sequence are denoted by dots. (b) Schematic of a filamin dimer, comparing the predicted human and Drosophila monomers. The drawing is based on previous biochemical and electron microscopy characterizations of human ABP280 structure (Gorlin et al. 1990). Filamin is a homodimer with an elongated subunit structure (160 nm). The self-association of partner subunits occurs at the COOH-terminal end of the molecule within repeat 24, while the actin binding domain (ABD) lies at the opposite free end of each monomeric subunit. The 24 repeats extend from the ABD to the COOH terminus and are interrupted by two hinge regions (hinge 1 and 2). Sequence alignment predicts that repeats 6–9 are missing from the Drosophila filamin. The percentage of amino acid conservation between the fly and human sequences is shown at the right.
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Related In: Results  -  Collection

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Figure 6: (a) Amino acid alignment of the actin binding domains in Drosophila and vertebrate filamins. The Drosophila filamin sequence is on top (“fly,” accession number GH12209). In human, three filamin isoforms, ABP280 (or α-filamin), β-filamin, and γ-filamin, have been reported and are encoded by separate genes located at different chromosome positions (Takafuta et al. 1998). Sequence alignment shows that the deduced NH2 terminus of fly filamin matches very well with the human β- and γ-filamins. The start codon of the third human isoform has not been defined, but if the human ABP280 polypeptide initiates at the second ATG, the resultant NH2 terminus matches those of the other human and fly filamins. Human-a represents human ABP280 (α-filamin) encoded by the gene on the X chromosome (accession number 113001); human-b indicates β-filamin on the third chromosome (accession number 328597); human-g represents γ-filamin derived from chromosome 7 (accession number 4218955) and chicken, chicken filamin (accession number 1079404). Residues identical to the fly sequence are denoted by dots. (b) Schematic of a filamin dimer, comparing the predicted human and Drosophila monomers. The drawing is based on previous biochemical and electron microscopy characterizations of human ABP280 structure (Gorlin et al. 1990). Filamin is a homodimer with an elongated subunit structure (160 nm). The self-association of partner subunits occurs at the COOH-terminal end of the molecule within repeat 24, while the actin binding domain (ABD) lies at the opposite free end of each monomeric subunit. The 24 repeats extend from the ABD to the COOH terminus and are interrupted by two hinge regions (hinge 1 and 2). Sequence alignment predicts that repeats 6–9 are missing from the Drosophila filamin. The percentage of amino acid conservation between the fly and human sequences is shown at the right.
Mentions: We subsequently identified and sequenced an expressed sequence tag (EST) cDNA clone (data are available from GenBank/EMBL/DDBJ under accession number GH12209), derived from adult head mRNA, that potentially encodes the full-length Drosophila filamin protein. The 7,536-bp cDNA contains the original 3′ cDNA sequence and includes a polyadenylation tail. An uninterrupted open reading frame begins from the first nucleotide of the cDNA; however, the first ATG codon is located 401 bp downstream. Although the flanking sequence of this ATG differs from the consensus sequence for translation initiation in Drosophila (Cavener 1987), conceptual translation from this initial ATG codon predicts a 2343 residue polypeptide with a deduced mass (∼250 kD) and NH2-terminal sequence that are highly similar to human filamins (Fig. 6) (Takafuta et al. 1998). Amino acid sequence comparisons indicate that the Drosophila filamin is ∼50% identical and ∼60% similar to human and chicken filamins along the entire length of the protein. Although we have not unequivocally defined the 5′ end of the transcript, the data suggest that the fly EST cDNA clone represents a full-length copy of the identified 7.5-kb filamin transcript.

Bottom Line: In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes.Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility.The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell and Developmental Biology, University of Minnesota, St. Paul, Minnesota 55108, USA.

ABSTRACT
The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

Show MeSH
Related in: MedlinePlus