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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 IX-14/invadolysin in human cells. (A) HsIX-14 localization in HeLa cells, detected with a rabbit antibody generated to HsIX-14 (amino acids 327–629). HsIX-14, green; α-tubulin, red; DAPI, blue. The HsIX-14 staining is seen as discrete ring-like structures in the cytoplasm of interphase cells, in addition to a nuclear pool of the protein. This staining pattern becomes more diffuse in mitosis, and is similar in Jurkat and CF-PAC cells (not depicted). Bar, 5 μm. (B) HsIX-14 localization in normal stationary human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI blue. Bar, 5 μm. (C) HsIX-14 localization in normal migrating human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI, blue. Note that all of the IX-14/invadolysin has now strikingly relocalized to the leading edge of the cells.
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fig8: Localization of IX-14/invadolysin in human cells. (A) HsIX-14 localization in HeLa cells, detected with a rabbit antibody generated to HsIX-14 (amino acids 327–629). HsIX-14, green; α-tubulin, red; DAPI, blue. The HsIX-14 staining is seen as discrete ring-like structures in the cytoplasm of interphase cells, in addition to a nuclear pool of the protein. This staining pattern becomes more diffuse in mitosis, and is similar in Jurkat and CF-PAC cells (not depicted). Bar, 5 μm. (B) HsIX-14 localization in normal stationary human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI blue. Bar, 5 μm. (C) HsIX-14 localization in normal migrating human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI, blue. Note that all of the IX-14/invadolysin has now strikingly relocalized to the leading edge of the cells.

Mentions: Two protein sequences for the human orthologue possibly representing alternatively spliced forms have been submitted: CAC42883–version 1 and CAC42882–version 2 (Fig. 4 B). These forms differ in their NH2-terminal regions (upstream of the residues VINK) and by the presence of a 37–amino acid sequence in the COOH-terminal half of version 2 found in all the other eukaryotic orthologues so far (between the residues EDTG:RQML). We generated an antibody to amino acids 327 to 629 of HsIX-14.v1 (downstream of the catalytic motif). As this region is fully present in both predicted human versions, the antibody should recognize the two forms if they indeed both exist. Fig. 8 A shows a typical staining pattern observed with the HsIX-14327-629 antibody in HeLa cells. We observed unusual ring-like structures similar to those seen with the EGFP-tagged protein. These striking structures were also observed in two other human transformed cell lines, Jurkat and CF-PAC (unpublished data). These structures were observed in all interphase cells, and although their size remained fairly constant (<1 μm in diameter), the number of the structures varied on a cell to cell basis. Z-series of sections through cells showed that these structures were located in the lower third of the cells. Because the ring-like structures were dispersed in mitosis in all cell types examined, we believe that the localization (and/or activity) of IX-14 may be regulated during the cell cycle.


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 IX-14/invadolysin in human cells. (A) HsIX-14 localization in HeLa cells, detected with a rabbit antibody generated to HsIX-14 (amino acids 327–629). HsIX-14, green; α-tubulin, red; DAPI, blue. The HsIX-14 staining is seen as discrete ring-like structures in the cytoplasm of interphase cells, in addition to a nuclear pool of the protein. This staining pattern becomes more diffuse in mitosis, and is similar in Jurkat and CF-PAC cells (not depicted). Bar, 5 μm. (B) HsIX-14 localization in normal stationary human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI blue. Bar, 5 μm. (C) HsIX-14 localization in normal migrating human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI, blue. Note that all of the IX-14/invadolysin has now strikingly relocalized to the leading edge of the cells.
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Related In: Results  -  Collection

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fig8: Localization of IX-14/invadolysin in human cells. (A) HsIX-14 localization in HeLa cells, detected with a rabbit antibody generated to HsIX-14 (amino acids 327–629). HsIX-14, green; α-tubulin, red; DAPI, blue. The HsIX-14 staining is seen as discrete ring-like structures in the cytoplasm of interphase cells, in addition to a nuclear pool of the protein. This staining pattern becomes more diffuse in mitosis, and is similar in Jurkat and CF-PAC cells (not depicted). Bar, 5 μm. (B) HsIX-14 localization in normal stationary human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI blue. Bar, 5 μm. (C) HsIX-14 localization in normal migrating human macrophages cultured in vitro. HsIX-14, green; rhodamine-phalloidin/actin, red; DAPI, blue. Note that all of the IX-14/invadolysin has now strikingly relocalized to the leading edge of the cells.
Mentions: Two protein sequences for the human orthologue possibly representing alternatively spliced forms have been submitted: CAC42883–version 1 and CAC42882–version 2 (Fig. 4 B). These forms differ in their NH2-terminal regions (upstream of the residues VINK) and by the presence of a 37–amino acid sequence in the COOH-terminal half of version 2 found in all the other eukaryotic orthologues so far (between the residues EDTG:RQML). We generated an antibody to amino acids 327 to 629 of HsIX-14.v1 (downstream of the catalytic motif). As this region is fully present in both predicted human versions, the antibody should recognize the two forms if they indeed both exist. Fig. 8 A shows a typical staining pattern observed with the HsIX-14327-629 antibody in HeLa cells. We observed unusual ring-like structures similar to those seen with the EGFP-tagged protein. These striking structures were also observed in two other human transformed cell lines, Jurkat and CF-PAC (unpublished data). These structures were observed in all interphase cells, and although their size remained fairly constant (<1 μm in diameter), the number of the structures varied on a cell to cell basis. Z-series of sections through cells showed that these structures were located in the lower third of the cells. Because the ring-like structures were dispersed in mitosis in all cell types examined, we believe that the localization (and/or activity) of IX-14 may be regulated during the cell cycle.

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