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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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Restriction map of the genomic region of the Drosophila filamin gene. Dark lines a–d represent the positions of genomic DNA fragments used as probes described in the text. The filamin transcription unit spanning ∼30 kb is indicated by an open box. The triangle points to the position of the P-element insertion in the fly stock EP(3)3715. Open boxes indicate regions deleted in two deficiencies (Δ12.1, Δ5) generated by imprecise excisions of the P-element. The 3′ breakpoint of the deficiency Δ12.1 extends an undetermined distance beyond the filamin transcription unit.
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Figure 7: Restriction map of the genomic region of the Drosophila filamin gene. Dark lines a–d represent the positions of genomic DNA fragments used as probes described in the text. The filamin transcription unit spanning ∼30 kb is indicated by an open box. The triangle points to the position of the P-element insertion in the fly stock EP(3)3715. Open boxes indicate regions deleted in two deficiencies (Δ12.1, Δ5) generated by imprecise excisions of the P-element. The 3′ breakpoint of the deficiency Δ12.1 extends an undetermined distance beyond the filamin transcription unit.

Mentions: To characterize the genomic region of the Drosophila filamin gene, we used the filamin cDNA to isolate genomic clones from a cosmid library. A restriction map of the genomic region that contains the filamin gene is depicted in Fig. 7. Restriction fragments from across the region were subcloned, partially sequenced, and used in RNA blot experiments to define the filamin transcription unit. Both the 3- and 7.5-kb transcripts are detected with a genomic clone that contains the 3′ end of the gene (Fig. 7, probe a), while only the 7.5-kb transcript is recognized by the more 5′ genomic probes b and c. Probe c contains the 5′ end of the EST cDNA sequence. The genomic probe d fails to detect either the 7.5- or the 3-kb transcript, but does identify a neighboring transcript ∼4 kb upstream. These results allow us to define the Drosophila filamin gene within a ∼30-kb genomic region. However, our experiments have not determined the complete intron/exon organization of the gene. Since the 3-kb transcript is detected only by probes derived from the 3′ end of the filamin transcription unit, it may be expressed from an internal promotor, or as an alternatively spliced product that eliminates 5′ exons. Further sequence analysis of the 5′ end of this transcript will be required to distinguish between these possibilities. Significantly, the 3-kb transcript lacks the 5′ sequence that encodes the highly conserved actin-binding domain; it may encode a filamin isoform that provides a regulatory function.


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)

Restriction map of the genomic region of the Drosophila filamin gene. Dark lines a–d represent the positions of genomic DNA fragments used as probes described in the text. The filamin transcription unit spanning ∼30 kb is indicated by an open box. The triangle points to the position of the P-element insertion in the fly stock EP(3)3715. Open boxes indicate regions deleted in two deficiencies (Δ12.1, Δ5) generated by imprecise excisions of the P-element. The 3′ breakpoint of the deficiency Δ12.1 extends an undetermined distance beyond the filamin transcription unit.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Restriction map of the genomic region of the Drosophila filamin gene. Dark lines a–d represent the positions of genomic DNA fragments used as probes described in the text. The filamin transcription unit spanning ∼30 kb is indicated by an open box. The triangle points to the position of the P-element insertion in the fly stock EP(3)3715. Open boxes indicate regions deleted in two deficiencies (Δ12.1, Δ5) generated by imprecise excisions of the P-element. The 3′ breakpoint of the deficiency Δ12.1 extends an undetermined distance beyond the filamin transcription unit.
Mentions: To characterize the genomic region of the Drosophila filamin gene, we used the filamin cDNA to isolate genomic clones from a cosmid library. A restriction map of the genomic region that contains the filamin gene is depicted in Fig. 7. Restriction fragments from across the region were subcloned, partially sequenced, and used in RNA blot experiments to define the filamin transcription unit. Both the 3- and 7.5-kb transcripts are detected with a genomic clone that contains the 3′ end of the gene (Fig. 7, probe a), while only the 7.5-kb transcript is recognized by the more 5′ genomic probes b and c. Probe c contains the 5′ end of the EST cDNA sequence. The genomic probe d fails to detect either the 7.5- or the 3-kb transcript, but does identify a neighboring transcript ∼4 kb upstream. These results allow us to define the Drosophila filamin gene within a ∼30-kb genomic region. However, our experiments have not determined the complete intron/exon organization of the gene. Since the 3-kb transcript is detected only by probes derived from the 3′ end of the filamin transcription unit, it may be expressed from an internal promotor, or as an alternatively spliced product that eliminates 5′ exons. Further sequence analysis of the 5′ end of this transcript will be required to distinguish between these possibilities. Significantly, the 3-kb transcript lacks the 5′ sequence that encodes the highly conserved actin-binding domain; it may encode a filamin isoform that provides a regulatory function.

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