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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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DNA and RNA blot analyses show that two transcripts are expressed by the identified single copy Drosophila filamin gene. (a) Genomic DNA was digested with restriction enzymes EcoRI (E), BamHI (B), or HindIII (H), and probed with a 0.7-kb cDNA fragment derived from the 3′ portion of the filamin cDNA (bp 4,271–4,972). Two bands detected in lanes E and B are due to restriction sites present in an intron in the corresponding genomic DNA. (b) Total RNA isolated from ovaries (OV) and 8–24-h-old embryos (E) was blotted and hybridized with the cDNA probe used in the Southern analysis. A transcript of ∼7.5 kb is recognized in both tissues, and a second transcript of ∼3 kb is present in embryos. Size in kb is indicated on the left side of each panel.
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Figure 5: DNA and RNA blot analyses show that two transcripts are expressed by the identified single copy Drosophila filamin gene. (a) Genomic DNA was digested with restriction enzymes EcoRI (E), BamHI (B), or HindIII (H), and probed with a 0.7-kb cDNA fragment derived from the 3′ portion of the filamin cDNA (bp 4,271–4,972). Two bands detected in lanes E and B are due to restriction sites present in an intron in the corresponding genomic DNA. (b) Total RNA isolated from ovaries (OV) and 8–24-h-old embryos (E) was blotted and hybridized with the cDNA probe used in the Southern analysis. A transcript of ∼7.5 kb is recognized in both tissues, and a second transcript of ∼3 kb is present in embryos. Size in kb is indicated on the left side of each panel.

Mentions: During the characterization of the sko phenotype, we localized the Drosophila homologue of the human actin-binding protein α-filamin to the 89F polytene region using in situ hybridization with a fly filamin cDNA. This 3.2-kb cDNA clone, which encodes a portion of Drosophila filamin, was previously isolated from an ovary expression library using a polyclonal antiserum raised against partially purified fly actin-binding protein (Miller et al. 1989; Field and Alberts 1995). The cDNA represents the 3′ end of the filamin transcript, including a 513-bp 3′ UTR with a polyadenylation tail. It predicts a polypeptide that is homologous (51% identity) to the COOH-terminal domains of human α-filamin. Genomic Southern blot analysis indicates that this filamin cDNA is derived from a single copy gene in Drosophila (Fig. 5 a). However, on RNA blots the probe detects two transcripts, 3 and 7.5 kb, which are differentially expressed (Fig. 5 b). The 7.5-kb transcript is expressed in ovaries and embryos, while the 3-kb transcript is expressed in embryos, but not ovaries.


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)

DNA and RNA blot analyses show that two transcripts are expressed by the identified single copy Drosophila filamin gene. (a) Genomic DNA was digested with restriction enzymes EcoRI (E), BamHI (B), or HindIII (H), and probed with a 0.7-kb cDNA fragment derived from the 3′ portion of the filamin cDNA (bp 4,271–4,972). Two bands detected in lanes E and B are due to restriction sites present in an intron in the corresponding genomic DNA. (b) Total RNA isolated from ovaries (OV) and 8–24-h-old embryos (E) was blotted and hybridized with the cDNA probe used in the Southern analysis. A transcript of ∼7.5 kb is recognized in both tissues, and a second transcript of ∼3 kb is present in embryos. Size in kb is indicated on the left side of each panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169474&req=5

Figure 5: DNA and RNA blot analyses show that two transcripts are expressed by the identified single copy Drosophila filamin gene. (a) Genomic DNA was digested with restriction enzymes EcoRI (E), BamHI (B), or HindIII (H), and probed with a 0.7-kb cDNA fragment derived from the 3′ portion of the filamin cDNA (bp 4,271–4,972). Two bands detected in lanes E and B are due to restriction sites present in an intron in the corresponding genomic DNA. (b) Total RNA isolated from ovaries (OV) and 8–24-h-old embryos (E) was blotted and hybridized with the cDNA probe used in the Southern analysis. A transcript of ∼7.5 kb is recognized in both tissues, and a second transcript of ∼3 kb is present in embryos. Size in kb is indicated on the left side of each panel.
Mentions: During the characterization of the sko phenotype, we localized the Drosophila homologue of the human actin-binding protein α-filamin to the 89F polytene region using in situ hybridization with a fly filamin cDNA. This 3.2-kb cDNA clone, which encodes a portion of Drosophila filamin, was previously isolated from an ovary expression library using a polyclonal antiserum raised against partially purified fly actin-binding protein (Miller et al. 1989; Field and Alberts 1995). The cDNA represents the 3′ end of the filamin transcript, including a 513-bp 3′ UTR with a polyadenylation tail. It predicts a polypeptide that is homologous (51% identity) to the COOH-terminal domains of human α-filamin. Genomic Southern blot analysis indicates that this filamin cDNA is derived from a single copy gene in Drosophila (Fig. 5 a). However, on RNA blots the probe detects two transcripts, 3 and 7.5 kb, which are differentially expressed (Fig. 5 b). The 7.5-kb transcript is expressed in ovaries and embryos, while the 3-kb transcript is expressed in embryos, but not ovaries.

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