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Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization.

Sisson JC, Field C, Ventura R, Royou A, Sullivan W - J. Cell Biol. (2000)

Bottom Line: Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated.Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190.We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, Sinsheimer Labs, University of California at Santa Cruz, Santa Cruz, California 95064, USA. sisson@darwin.ucsc.edu

ABSTRACT
Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

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Molecular characterization of Lva. (A) The predicted gene structure shows exons (black rectangles) and introns (gaps). Exons are numbered 1–8. The gene prediction analysis was performed with the program FGENESH (available online from Baylor College of Medicine, Waco, TX). The positions of the putative start codon (bent arrow), stop codon (straight arrow), and the 5′ extent of the cDNA sequence (vertical arrowhead) are as indicated. (B) Lva is predicted to be globular at its termini (shaded) and form a coiled-coil along its central region (black). Arrowheads indicate the junctions between amino acids (a.a.) encoded by adjacent exons and the black marks below the diagram indicate the position of peptides matching MS/MS data.
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Figure 2: Molecular characterization of Lva. (A) The predicted gene structure shows exons (black rectangles) and introns (gaps). Exons are numbered 1–8. The gene prediction analysis was performed with the program FGENESH (available online from Baylor College of Medicine, Waco, TX). The positions of the putative start codon (bent arrow), stop codon (straight arrow), and the 5′ extent of the cDNA sequence (vertical arrowhead) are as indicated. (B) Lva is predicted to be globular at its termini (shaded) and form a coiled-coil along its central region (black). Arrowheads indicate the junctions between amino acids (a.a.) encoded by adjacent exons and the black marks below the diagram indicate the position of peptides matching MS/MS data.

Mentions: We have focused our initial efforts on characterizing the novel protein, Lva. The mass spectra obtained from Lva perfectly match tryptic peptides derived from the translation product of a single predicted Drosophila gene, indicating that it encodes Lva (Fig. 2 B; CG#6450). The mass spectra match 64 tryptic peptides from seven of the eight predicted exons (Fig. 2A and Fig. B). Tryptic peptides including the NH2-terminal 18 amino acids, predicted to be encoded by exon 1, were not detected by MS/MS. However, the four nucleotides just upstream of the putative start codon of exon 1 are a good match to the Drosophila translational consensus sequence (Cavener 1987), containing the invariant adenine at the −2 position, and stop codons exist in all reading frames just upstream of the putative start codon. cDNA sequence derived from a contiguous set of overlapping clones confirms the genomic DNA sequence from nucleotide 842 within exon 4 to the start of a poly(A) tail, 334 nucleotides downstream of the putative stop codon (Fig. 2 A). In addition, the cDNA sequence and MS/MS confirm the predicted intron/exon borders between exons 4–8 and 2–7, respectively. A full-length cDNA clone has not yet been identified.


Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization.

Sisson JC, Field C, Ventura R, Royou A, Sullivan W - J. Cell Biol. (2000)

Molecular characterization of Lva. (A) The predicted gene structure shows exons (black rectangles) and introns (gaps). Exons are numbered 1–8. The gene prediction analysis was performed with the program FGENESH (available online from Baylor College of Medicine, Waco, TX). The positions of the putative start codon (bent arrow), stop codon (straight arrow), and the 5′ extent of the cDNA sequence (vertical arrowhead) are as indicated. (B) Lva is predicted to be globular at its termini (shaded) and form a coiled-coil along its central region (black). Arrowheads indicate the junctions between amino acids (a.a.) encoded by adjacent exons and the black marks below the diagram indicate the position of peptides matching MS/MS data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169433&req=5

Figure 2: Molecular characterization of Lva. (A) The predicted gene structure shows exons (black rectangles) and introns (gaps). Exons are numbered 1–8. The gene prediction analysis was performed with the program FGENESH (available online from Baylor College of Medicine, Waco, TX). The positions of the putative start codon (bent arrow), stop codon (straight arrow), and the 5′ extent of the cDNA sequence (vertical arrowhead) are as indicated. (B) Lva is predicted to be globular at its termini (shaded) and form a coiled-coil along its central region (black). Arrowheads indicate the junctions between amino acids (a.a.) encoded by adjacent exons and the black marks below the diagram indicate the position of peptides matching MS/MS data.
Mentions: We have focused our initial efforts on characterizing the novel protein, Lva. The mass spectra obtained from Lva perfectly match tryptic peptides derived from the translation product of a single predicted Drosophila gene, indicating that it encodes Lva (Fig. 2 B; CG#6450). The mass spectra match 64 tryptic peptides from seven of the eight predicted exons (Fig. 2A and Fig. B). Tryptic peptides including the NH2-terminal 18 amino acids, predicted to be encoded by exon 1, were not detected by MS/MS. However, the four nucleotides just upstream of the putative start codon of exon 1 are a good match to the Drosophila translational consensus sequence (Cavener 1987), containing the invariant adenine at the −2 position, and stop codons exist in all reading frames just upstream of the putative start codon. cDNA sequence derived from a contiguous set of overlapping clones confirms the genomic DNA sequence from nucleotide 842 within exon 4 to the start of a poly(A) tail, 334 nucleotides downstream of the putative stop codon (Fig. 2 A). In addition, the cDNA sequence and MS/MS confirm the predicted intron/exon borders between exons 4–8 and 2–7, respectively. A full-length cDNA clone has not yet been identified.

Bottom Line: Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated.Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190.We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, Sinsheimer Labs, University of California at Santa Cruz, Santa Cruz, California 95064, USA. sisson@darwin.ucsc.edu

ABSTRACT
Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

Show MeSH
Related in: MedlinePlus