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Invadolysin: a novel, conserved metalloprotease links mitotic structural rearrangements with cell migration.

McHugh B, Krause SA, Yu B, Deans AM, Heasman S, McLaughlin P, Heck MM - J. Cell Biol. (2004)

Bottom Line: Zymography reveals that a protease activity, present in wild-type larval brains, is missing from homozygous tissue, and we show that IX-14/invadolysin cleaves lamin in vitro.The IX-14/invadolysin protein is predominantly found in cytoplasmic structures resembling invadopodia in fly and human cells, but is dramatically relocalized to the leading edge of migrating cells.Strikingly, we find that the directed migration of germ cells is affected in Drosophila IX-14 mutant embryos.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
The cell cycle is widely known to be regulated by networks of phosphorylation and ubiquitin-directed proteolysis. Here, we describe IX-14/invadolysin, a novel metalloprotease present only in metazoa, whose activity appears to be essential for mitotic progression. Mitotic neuroblasts of Drosophila melanogaster IX-14 mutant larvae exhibit increased levels of nuclear envelope proteins, monopolar and asymmetric spindles, and chromosomes that appear hypercondensed in length with a surrounding halo of loosely condensed chromatin. Zymography reveals that a protease activity, present in wild-type larval brains, is missing from homozygous tissue, and we show that IX-14/invadolysin cleaves lamin in vitro. The IX-14/invadolysin protein is predominantly found in cytoplasmic structures resembling invadopodia in fly and human cells, but is dramatically relocalized to the leading edge of migrating cells. Strikingly, we find that the directed migration of germ cells is affected in Drosophila IX-14 mutant embryos. Thus, invadolysin identifies a new family of conserved metalloproteases whose activity appears to be essential for the coordination of mitotic progression, but which also plays an unexpected role in cell migration.

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Molecular characterization of l(3)IX-14. (A) Map of the DmIX-14 gene (CG3953), with exons depicted as black boxes and 5′ and 3′ UTRs as hatched boxes. The position of the P element insertion in l(3)IX-144Y7 is 40 bp upstream of the transcription start site (gray triangle). The first intron (8.6 kb, asterisk) is not shown to scale in this figure. (B) Northern blot of wild type and IX-14 heterozygous (+/−) and homozygous (−/−) total larval RNA probed with IX-14 full-length cDNA. The same blot probed for ribosomal protein RP49 mRNA is shown as a control. The IX-14 mRNA is undetectable in RNA obtained from homozygous IX-14 alleles. (C) T-COFFEE alignment showing homology between Drosophila melanogaster IX-14, metazoan orthologues, and Leishmanolysin, with the conserved HEXXHXXG (and third required H) zinc-metalloprotease motif boxed. Sequences shown are as follows: Ce, Caenorhabditis elegans (CAB16471); Hs, Homo sapiens (CAC42882); Mm, Mus musculus (NP 766411); Dm, Drosophila melanogaster (NP 652072); Lm, Leishmania major (AF039721). Although the homology between leishmanolysin and the other proteins appears to be largely limited to regions surrounding the metalloprotease motif, the placement of 14 cysteines (asterisks) is strikingly conserved. Nine regions shared among the higher eukaryotic orthologues (absent from leishmanolysin) are indicated by double-headed arrows. The gray triangles delimit the region of the leishmanolysin protein that is shown in D. (D) Stereo pair of the three-dimensional structure of leishmanolysin is shown (PDB accession code is 1LML). The black numbered spheres represent the higher eukaryotic “insertions” (relative to leishmanolysin), which all map to the surface of the leishmanolysin structure. The internal magenta sphere represents the zinc ion required for catalysis.
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fig4: Molecular characterization of l(3)IX-14. (A) Map of the DmIX-14 gene (CG3953), with exons depicted as black boxes and 5′ and 3′ UTRs as hatched boxes. The position of the P element insertion in l(3)IX-144Y7 is 40 bp upstream of the transcription start site (gray triangle). The first intron (8.6 kb, asterisk) is not shown to scale in this figure. (B) Northern blot of wild type and IX-14 heterozygous (+/−) and homozygous (−/−) total larval RNA probed with IX-14 full-length cDNA. The same blot probed for ribosomal protein RP49 mRNA is shown as a control. The IX-14 mRNA is undetectable in RNA obtained from homozygous IX-14 alleles. (C) T-COFFEE alignment showing homology between Drosophila melanogaster IX-14, metazoan orthologues, and Leishmanolysin, with the conserved HEXXHXXG (and third required H) zinc-metalloprotease motif boxed. Sequences shown are as follows: Ce, Caenorhabditis elegans (CAB16471); Hs, Homo sapiens (CAC42882); Mm, Mus musculus (NP 766411); Dm, Drosophila melanogaster (NP 652072); Lm, Leishmania major (AF039721). Although the homology between leishmanolysin and the other proteins appears to be largely limited to regions surrounding the metalloprotease motif, the placement of 14 cysteines (asterisks) is strikingly conserved. Nine regions shared among the higher eukaryotic orthologues (absent from leishmanolysin) are indicated by double-headed arrows. The gray triangles delimit the region of the leishmanolysin protein that is shown in D. (D) Stereo pair of the three-dimensional structure of leishmanolysin is shown (PDB accession code is 1LML). The black numbered spheres represent the higher eukaryotic “insertions” (relative to leishmanolysin), which all map to the surface of the leishmanolysin structure. The internal magenta sphere represents the zinc ion required for catalysis.

Mentions: We mapped the original IX-14 allele to the 85E10-F16 region by crossing to deficiency lines with known breakpoints. We then generated a P-element insertion allele by local hopping a nearby P-element, l(3)04017. The P-element allele allowed cloning of adjacent genomic DNA by inverse PCR; hybridization of this fragment to a Drosophila P1 array refined our mapping to 85F14-15. A candidate IX-14 gene was cloned by identifying a 3.6-kb full-length Drosophila adult head library EST whose sequence overlapped with the ∼700-bp genomic fragment flanking the P-element. The gene was composed of nine exons, with the first exon separated from the remaining eight by a large (∼8.6 kb) intron (Fig. 4 A, asterisk). This gene has been designated CG3953 in the Drosophila genome annotation database.


Invadolysin: a novel, conserved metalloprotease links mitotic structural rearrangements with cell migration.

McHugh B, Krause SA, Yu B, Deans AM, Heasman S, McLaughlin P, Heck MM - J. Cell Biol. (2004)

Molecular characterization of l(3)IX-14. (A) Map of the DmIX-14 gene (CG3953), with exons depicted as black boxes and 5′ and 3′ UTRs as hatched boxes. The position of the P element insertion in l(3)IX-144Y7 is 40 bp upstream of the transcription start site (gray triangle). The first intron (8.6 kb, asterisk) is not shown to scale in this figure. (B) Northern blot of wild type and IX-14 heterozygous (+/−) and homozygous (−/−) total larval RNA probed with IX-14 full-length cDNA. The same blot probed for ribosomal protein RP49 mRNA is shown as a control. The IX-14 mRNA is undetectable in RNA obtained from homozygous IX-14 alleles. (C) T-COFFEE alignment showing homology between Drosophila melanogaster IX-14, metazoan orthologues, and Leishmanolysin, with the conserved HEXXHXXG (and third required H) zinc-metalloprotease motif boxed. Sequences shown are as follows: Ce, Caenorhabditis elegans (CAB16471); Hs, Homo sapiens (CAC42882); Mm, Mus musculus (NP 766411); Dm, Drosophila melanogaster (NP 652072); Lm, Leishmania major (AF039721). Although the homology between leishmanolysin and the other proteins appears to be largely limited to regions surrounding the metalloprotease motif, the placement of 14 cysteines (asterisks) is strikingly conserved. Nine regions shared among the higher eukaryotic orthologues (absent from leishmanolysin) are indicated by double-headed arrows. The gray triangles delimit the region of the leishmanolysin protein that is shown in D. (D) Stereo pair of the three-dimensional structure of leishmanolysin is shown (PDB accession code is 1LML). The black numbered spheres represent the higher eukaryotic “insertions” (relative to leishmanolysin), which all map to the surface of the leishmanolysin structure. The internal magenta sphere represents the zinc ion required for catalysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172566&req=5

fig4: Molecular characterization of l(3)IX-14. (A) Map of the DmIX-14 gene (CG3953), with exons depicted as black boxes and 5′ and 3′ UTRs as hatched boxes. The position of the P element insertion in l(3)IX-144Y7 is 40 bp upstream of the transcription start site (gray triangle). The first intron (8.6 kb, asterisk) is not shown to scale in this figure. (B) Northern blot of wild type and IX-14 heterozygous (+/−) and homozygous (−/−) total larval RNA probed with IX-14 full-length cDNA. The same blot probed for ribosomal protein RP49 mRNA is shown as a control. The IX-14 mRNA is undetectable in RNA obtained from homozygous IX-14 alleles. (C) T-COFFEE alignment showing homology between Drosophila melanogaster IX-14, metazoan orthologues, and Leishmanolysin, with the conserved HEXXHXXG (and third required H) zinc-metalloprotease motif boxed. Sequences shown are as follows: Ce, Caenorhabditis elegans (CAB16471); Hs, Homo sapiens (CAC42882); Mm, Mus musculus (NP 766411); Dm, Drosophila melanogaster (NP 652072); Lm, Leishmania major (AF039721). Although the homology between leishmanolysin and the other proteins appears to be largely limited to regions surrounding the metalloprotease motif, the placement of 14 cysteines (asterisks) is strikingly conserved. Nine regions shared among the higher eukaryotic orthologues (absent from leishmanolysin) are indicated by double-headed arrows. The gray triangles delimit the region of the leishmanolysin protein that is shown in D. (D) Stereo pair of the three-dimensional structure of leishmanolysin is shown (PDB accession code is 1LML). The black numbered spheres represent the higher eukaryotic “insertions” (relative to leishmanolysin), which all map to the surface of the leishmanolysin structure. The internal magenta sphere represents the zinc ion required for catalysis.
Mentions: We mapped the original IX-14 allele to the 85E10-F16 region by crossing to deficiency lines with known breakpoints. We then generated a P-element insertion allele by local hopping a nearby P-element, l(3)04017. The P-element allele allowed cloning of adjacent genomic DNA by inverse PCR; hybridization of this fragment to a Drosophila P1 array refined our mapping to 85F14-15. A candidate IX-14 gene was cloned by identifying a 3.6-kb full-length Drosophila adult head library EST whose sequence overlapped with the ∼700-bp genomic fragment flanking the P-element. The gene was composed of nine exons, with the first exon separated from the remaining eight by a large (∼8.6 kb) intron (Fig. 4 A, asterisk). This gene has been designated CG3953 in the Drosophila genome annotation database.

Bottom Line: Zymography reveals that a protease activity, present in wild-type larval brains, is missing from homozygous tissue, and we show that IX-14/invadolysin cleaves lamin in vitro.The IX-14/invadolysin protein is predominantly found in cytoplasmic structures resembling invadopodia in fly and human cells, but is dramatically relocalized to the leading edge of migrating cells.Strikingly, we find that the directed migration of germ cells is affected in Drosophila IX-14 mutant embryos.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
The cell cycle is widely known to be regulated by networks of phosphorylation and ubiquitin-directed proteolysis. Here, we describe IX-14/invadolysin, a novel metalloprotease present only in metazoa, whose activity appears to be essential for mitotic progression. Mitotic neuroblasts of Drosophila melanogaster IX-14 mutant larvae exhibit increased levels of nuclear envelope proteins, monopolar and asymmetric spindles, and chromosomes that appear hypercondensed in length with a surrounding halo of loosely condensed chromatin. Zymography reveals that a protease activity, present in wild-type larval brains, is missing from homozygous tissue, and we show that IX-14/invadolysin cleaves lamin in vitro. The IX-14/invadolysin protein is predominantly found in cytoplasmic structures resembling invadopodia in fly and human cells, but is dramatically relocalized to the leading edge of migrating cells. Strikingly, we find that the directed migration of germ cells is affected in Drosophila IX-14 mutant embryos. Thus, invadolysin identifies a new family of conserved metalloproteases whose activity appears to be essential for the coordination of mitotic progression, but which also plays an unexpected role in cell migration.

Show MeSH
Related in: MedlinePlus