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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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Localization of GFP-tagged IX-14 in Drosophila S2 and HeLa cells. (A) Drosophila S2 cells transiently transfected with constructs expressing EGFP vector only (top) and DmIX-14 tagged at the NH2 (middle) and COOH termini (bottom). EGFP, green; DAPI, red. DmIX-14 tagged with EGFP at either end appears to be concentrated in cytoplasmic foci, in contrast to EGFP alone, which shows diffuse localization throughout the cytoplasm and nucleus. Bar, 5 μm. (B) HeLa cells transiently transfected with constructs expressing EGFP vector only (left) or HsIX-14 tagged at the COOH terminus (right). EGFP alone is localized diffusely throughout both nucleus and cytoplasm, HsIX-14~EGFP is localized to cytoplasmic ring-like structures. Bar, 5 μm.
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fig7: Localization of GFP-tagged IX-14 in Drosophila S2 and HeLa cells. (A) Drosophila S2 cells transiently transfected with constructs expressing EGFP vector only (top) and DmIX-14 tagged at the NH2 (middle) and COOH termini (bottom). EGFP, green; DAPI, red. DmIX-14 tagged with EGFP at either end appears to be concentrated in cytoplasmic foci, in contrast to EGFP alone, which shows diffuse localization throughout the cytoplasm and nucleus. Bar, 5 μm. (B) HeLa cells transiently transfected with constructs expressing EGFP vector only (left) or HsIX-14 tagged at the COOH terminus (right). EGFP alone is localized diffusely throughout both nucleus and cytoplasm, HsIX-14~EGFP is localized to cytoplasmic ring-like structures. Bar, 5 μm.

Mentions: Several approaches were used to determine the subcellular localization of the IX-14 protein. Drosophila S2 cells transiently transfected with expression plasmids tagging either the NH2 or COOH terminus with EGFP showed cytoplasmic localization, whereas vector alone localized to both the nucleus and cytoplasm (Fig. 7 A). The human genome contains a single IX-14 gene, which we have shown by preliminary siRNA analysis to be essential for viability (unpublished data). Due to the limit of resolution with the relatively small and generally nonadherent S2 cells, we turned to examining IX-14 localization in human cells. A COOH-terminal EGFP fusion construct of HsIX-14 in HeLa cells was also localized in the cytoplasm, often as unusual ring-like structures (Fig. 7 B). A control transfection with EGFP alone localized throughout both the nucleus and cytoplasm. From these experiments we concluded that the IX-14 protein localized predominantly in the cytoplasm of fly and human cells.


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)

Localization of GFP-tagged IX-14 in Drosophila S2 and HeLa cells. (A) Drosophila S2 cells transiently transfected with constructs expressing EGFP vector only (top) and DmIX-14 tagged at the NH2 (middle) and COOH termini (bottom). EGFP, green; DAPI, red. DmIX-14 tagged with EGFP at either end appears to be concentrated in cytoplasmic foci, in contrast to EGFP alone, which shows diffuse localization throughout the cytoplasm and nucleus. Bar, 5 μm. (B) HeLa cells transiently transfected with constructs expressing EGFP vector only (left) or HsIX-14 tagged at the COOH terminus (right). EGFP alone is localized diffusely throughout both nucleus and cytoplasm, HsIX-14~EGFP is localized to cytoplasmic ring-like structures. Bar, 5 μm.
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Related In: Results  -  Collection

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fig7: Localization of GFP-tagged IX-14 in Drosophila S2 and HeLa cells. (A) Drosophila S2 cells transiently transfected with constructs expressing EGFP vector only (top) and DmIX-14 tagged at the NH2 (middle) and COOH termini (bottom). EGFP, green; DAPI, red. DmIX-14 tagged with EGFP at either end appears to be concentrated in cytoplasmic foci, in contrast to EGFP alone, which shows diffuse localization throughout the cytoplasm and nucleus. Bar, 5 μm. (B) HeLa cells transiently transfected with constructs expressing EGFP vector only (left) or HsIX-14 tagged at the COOH terminus (right). EGFP alone is localized diffusely throughout both nucleus and cytoplasm, HsIX-14~EGFP is localized to cytoplasmic ring-like structures. Bar, 5 μm.
Mentions: Several approaches were used to determine the subcellular localization of the IX-14 protein. Drosophila S2 cells transiently transfected with expression plasmids tagging either the NH2 or COOH terminus with EGFP showed cytoplasmic localization, whereas vector alone localized to both the nucleus and cytoplasm (Fig. 7 A). The human genome contains a single IX-14 gene, which we have shown by preliminary siRNA analysis to be essential for viability (unpublished data). Due to the limit of resolution with the relatively small and generally nonadherent S2 cells, we turned to examining IX-14 localization in human cells. A COOH-terminal EGFP fusion construct of HsIX-14 in HeLa cells was also localized in the cytoplasm, often as unusual ring-like structures (Fig. 7 B). A control transfection with EGFP alone localized throughout both the nucleus and cytoplasm. From these experiments we concluded that the IX-14 protein localized predominantly in the cytoplasm of fly and human cells.

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