Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of filamin protein in ovaries and adults. (a) Western blots of protein extracts from wild-type and mutant ovaries and whole flies, probed with anti-filamin antibody 4-3D. In ovaries (left), the ∼250 kD protein seen in wild-type (+/+) extracts is not detected in the sko or cheerio1 mutants. Extracts of whole male adults (right panel) show a 97-kD protein in addition to the 250-kD species in wild-type flies. Neither polypeptide is detected in sko mutant extracts; in cheerio1 mutants, the 97-kD species is detected but the 250-kD species is not. Identical results were obtained using whole female adults (not shown). S, supernatant; P, pellet; equal total protein is loaded in each lane. (b) Localization of filamin in ring canals. Egg chambers from wild-type and sko animals were probed with antibody 4-3D and visualized by confocal microscopy. In the stage 3 egg chambers shown, filamin is seen in ring canal structures in wild-type (left panel) but not in the sko mutant (right panel). Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169474&req=5

Figure 8: Expression of filamin protein in ovaries and adults. (a) Western blots of protein extracts from wild-type and mutant ovaries and whole flies, probed with anti-filamin antibody 4-3D. In ovaries (left), the ∼250 kD protein seen in wild-type (+/+) extracts is not detected in the sko or cheerio1 mutants. Extracts of whole male adults (right panel) show a 97-kD protein in addition to the 250-kD species in wild-type flies. Neither polypeptide is detected in sko mutant extracts; in cheerio1 mutants, the 97-kD species is detected but the 250-kD species is not. Identical results were obtained using whole female adults (not shown). S, supernatant; P, pellet; equal total protein is loaded in each lane. (b) Localization of filamin in ring canals. Egg chambers from wild-type and sko animals were probed with antibody 4-3D and visualized by confocal microscopy. In the stage 3 egg chambers shown, filamin is seen in ring canal structures in wild-type (left panel) but not in the sko mutant (right panel). Bars, 50 μm.

Mentions: To investigate whether the sko mutation represents an allele of Drosophila filamin, we looked at the expression of filamin protein(s) in wild-type and sko mutant flies. A polyclonal antiserum, 4-3D, was raised against a bacterial fusion protein expressing the COOH-terminal 90 residues of filamin. Extracts from whole flies and tissues of sko and cher1 mutants, as well as from wild-type flies, were analyzed by immunoblotting. As shown in Fig. 8 a, in wild-type ovary extracts the antibody predominantly reacts with a large ∼250-kD polypeptide. The size of this polypeptide correlates well with the single 7.5-kb filamin transcript expressed in ovary RNA, and is comparable in size to human α-filamin. Significantly, the 250-kD filamin polypeptide is not detected in ovaries derived from sterile sko/sko females, nor in homozygous cher1 ovaries. A less abundant polypeptide of ∼140 kD is detected by the 4-3D antiserum in wild-type ovary extracts, and is not eliminated in the sko or cheerio mutant backgrounds. These results demonstrate that the sko and cher1 mutations disrupt the expression of a 250-kD filamin product in ovaries. The loss of the filamin polypeptide coincides with the disruption of actin organization and female sterility.


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)

Expression of filamin protein in ovaries and adults. (a) Western blots of protein extracts from wild-type and mutant ovaries and whole flies, probed with anti-filamin antibody 4-3D. In ovaries (left), the ∼250 kD protein seen in wild-type (+/+) extracts is not detected in the sko or cheerio1 mutants. Extracts of whole male adults (right panel) show a 97-kD protein in addition to the 250-kD species in wild-type flies. Neither polypeptide is detected in sko mutant extracts; in cheerio1 mutants, the 97-kD species is detected but the 250-kD species is not. Identical results were obtained using whole female adults (not shown). S, supernatant; P, pellet; equal total protein is loaded in each lane. (b) Localization of filamin in ring canals. Egg chambers from wild-type and sko animals were probed with antibody 4-3D and visualized by confocal microscopy. In the stage 3 egg chambers shown, filamin is seen in ring canal structures in wild-type (left panel) but not in the sko mutant (right panel). Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Expression of filamin protein in ovaries and adults. (a) Western blots of protein extracts from wild-type and mutant ovaries and whole flies, probed with anti-filamin antibody 4-3D. In ovaries (left), the ∼250 kD protein seen in wild-type (+/+) extracts is not detected in the sko or cheerio1 mutants. Extracts of whole male adults (right panel) show a 97-kD protein in addition to the 250-kD species in wild-type flies. Neither polypeptide is detected in sko mutant extracts; in cheerio1 mutants, the 97-kD species is detected but the 250-kD species is not. Identical results were obtained using whole female adults (not shown). S, supernatant; P, pellet; equal total protein is loaded in each lane. (b) Localization of filamin in ring canals. Egg chambers from wild-type and sko animals were probed with antibody 4-3D and visualized by confocal microscopy. In the stage 3 egg chambers shown, filamin is seen in ring canal structures in wild-type (left panel) but not in the sko mutant (right panel). Bars, 50 μm.
Mentions: To investigate whether the sko mutation represents an allele of Drosophila filamin, we looked at the expression of filamin protein(s) in wild-type and sko mutant flies. A polyclonal antiserum, 4-3D, was raised against a bacterial fusion protein expressing the COOH-terminal 90 residues of filamin. Extracts from whole flies and tissues of sko and cher1 mutants, as well as from wild-type flies, were analyzed by immunoblotting. As shown in Fig. 8 a, in wild-type ovary extracts the antibody predominantly reacts with a large ∼250-kD polypeptide. The size of this polypeptide correlates well with the single 7.5-kb filamin transcript expressed in ovary RNA, and is comparable in size to human α-filamin. Significantly, the 250-kD filamin polypeptide is not detected in ovaries derived from sterile sko/sko females, nor in homozygous cher1 ovaries. A less abundant polypeptide of ∼140 kD is detected by the 4-3D antiserum in wild-type ovary extracts, and is not eliminated in the sko or cheerio mutant backgrounds. These results demonstrate that the sko and cher1 mutations disrupt the expression of a 250-kD filamin product in ovaries. The loss of the filamin polypeptide coincides with the disruption of actin organization and female sterility.

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