Limits...
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.

Show MeSH

Related in: MedlinePlus

The IX-14 protein exhibits protease activity. (A) Colloidal Coomassie blue–stained nondenaturing casein zymogram gel showing one brain equivalent of wild type (+/+) versus three brain equivalents of homozygous (IX-14) third instar larval brain extract. A doublet of protease activity is observed in wild-type extracts (asterisks), but is greatly depleted in mutant extracts. A band of protease activity is also present in the prestained molecular mass markers (arrowhead), which serves as an internal control in these experiments. (B) Coomassie blue–stained denaturing polyacrylamide gel showing equivalent loading of larval brain extracts (three mutant brains are a roughly equivalent amount to one wild-type brain). (C) Drosophila IX-14 cleaves Drosophila Dm0 lamin in vitro. In vitro transcribed and translated proteins were mixed and incubated for 60 min at 29°C (IX-14 and lamin alone are in the first two lanes). The cleavage of lamin was detected by immunoblotting with a mAb generated against the NH2-terminal head region (arrowhead). The addition of zinc at 2, 5, or 10 mM enhanced the cleavage reaction, whereas the addition of the 1,10-phenanthroline zinc chelator inhibited the cleavage of lamin by the IX-14 protease (asterisks).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172566&req=5

fig6: The IX-14 protein exhibits protease activity. (A) Colloidal Coomassie blue–stained nondenaturing casein zymogram gel showing one brain equivalent of wild type (+/+) versus three brain equivalents of homozygous (IX-14) third instar larval brain extract. A doublet of protease activity is observed in wild-type extracts (asterisks), but is greatly depleted in mutant extracts. A band of protease activity is also present in the prestained molecular mass markers (arrowhead), which serves as an internal control in these experiments. (B) Coomassie blue–stained denaturing polyacrylamide gel showing equivalent loading of larval brain extracts (three mutant brains are a roughly equivalent amount to one wild-type brain). (C) Drosophila IX-14 cleaves Drosophila Dm0 lamin in vitro. In vitro transcribed and translated proteins were mixed and incubated for 60 min at 29°C (IX-14 and lamin alone are in the first two lanes). The cleavage of lamin was detected by immunoblotting with a mAb generated against the NH2-terminal head region (arrowhead). The addition of zinc at 2, 5, or 10 mM enhanced the cleavage reaction, whereas the addition of the 1,10-phenanthroline zinc chelator inhibited the cleavage of lamin by the IX-14 protease (asterisks).

Mentions: To examine whether or not IX-14 has protease activity, suggested by its homology to the catalytic motif of leishmanolysin, we performed two complementary approaches. In the first, protein extracts prepared from wild-type and IX-14 mutant larval brains were examined for protease activity (Fig. 6 A). Protease activity was assayed in zymogram gels containing casein as substrate. Whole larvae harbor a high level of protease activity at numerous molecular masses when assessed by zymography (unpublished data). Therefore, we prepared protein extracts from larval brains only, as the phenotypes were clearly apparent in this proliferating tissue. Wild-type brain lysates had two visible bands of protease activity, migrating at ∼120 and 135 kD (Fig. 6 A, asterisks). Remarkably, these two bands of activity were almost completely absent in IX-14 brains, indicating that mutant extracts were indeed missing proteolytic activity. A separate stained gel documents that wild-type and mutant lanes were equivalently loaded (Fig. 6 B). As these zymogram gels are nondenaturing, the molecular mass of the observed activity is not necessarily related to predicted molecular mass, but may suggest that IX-14 migrates as a multimer or part of a complex. The lack of protease activity in mutant brains further suggests that the IX-14 gene product is responsible not only for the mutant phenotypes but also for the protease activity observed under these conditions (either directly or potentially through the activation of other proteases).


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)

The IX-14 protein exhibits protease activity. (A) Colloidal Coomassie blue–stained nondenaturing casein zymogram gel showing one brain equivalent of wild type (+/+) versus three brain equivalents of homozygous (IX-14) third instar larval brain extract. A doublet of protease activity is observed in wild-type extracts (asterisks), but is greatly depleted in mutant extracts. A band of protease activity is also present in the prestained molecular mass markers (arrowhead), which serves as an internal control in these experiments. (B) Coomassie blue–stained denaturing polyacrylamide gel showing equivalent loading of larval brain extracts (three mutant brains are a roughly equivalent amount to one wild-type brain). (C) Drosophila IX-14 cleaves Drosophila Dm0 lamin in vitro. In vitro transcribed and translated proteins were mixed and incubated for 60 min at 29°C (IX-14 and lamin alone are in the first two lanes). The cleavage of lamin was detected by immunoblotting with a mAb generated against the NH2-terminal head region (arrowhead). The addition of zinc at 2, 5, or 10 mM enhanced the cleavage reaction, whereas the addition of the 1,10-phenanthroline zinc chelator inhibited the cleavage of lamin by the IX-14 protease (asterisks).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The IX-14 protein exhibits protease activity. (A) Colloidal Coomassie blue–stained nondenaturing casein zymogram gel showing one brain equivalent of wild type (+/+) versus three brain equivalents of homozygous (IX-14) third instar larval brain extract. A doublet of protease activity is observed in wild-type extracts (asterisks), but is greatly depleted in mutant extracts. A band of protease activity is also present in the prestained molecular mass markers (arrowhead), which serves as an internal control in these experiments. (B) Coomassie blue–stained denaturing polyacrylamide gel showing equivalent loading of larval brain extracts (three mutant brains are a roughly equivalent amount to one wild-type brain). (C) Drosophila IX-14 cleaves Drosophila Dm0 lamin in vitro. In vitro transcribed and translated proteins were mixed and incubated for 60 min at 29°C (IX-14 and lamin alone are in the first two lanes). The cleavage of lamin was detected by immunoblotting with a mAb generated against the NH2-terminal head region (arrowhead). The addition of zinc at 2, 5, or 10 mM enhanced the cleavage reaction, whereas the addition of the 1,10-phenanthroline zinc chelator inhibited the cleavage of lamin by the IX-14 protease (asterisks).
Mentions: To examine whether or not IX-14 has protease activity, suggested by its homology to the catalytic motif of leishmanolysin, we performed two complementary approaches. In the first, protein extracts prepared from wild-type and IX-14 mutant larval brains were examined for protease activity (Fig. 6 A). Protease activity was assayed in zymogram gels containing casein as substrate. Whole larvae harbor a high level of protease activity at numerous molecular masses when assessed by zymography (unpublished data). Therefore, we prepared protein extracts from larval brains only, as the phenotypes were clearly apparent in this proliferating tissue. Wild-type brain lysates had two visible bands of protease activity, migrating at ∼120 and 135 kD (Fig. 6 A, asterisks). Remarkably, these two bands of activity were almost completely absent in IX-14 brains, indicating that mutant extracts were indeed missing proteolytic activity. A separate stained gel documents that wild-type and mutant lanes were equivalently loaded (Fig. 6 B). As these zymogram gels are nondenaturing, the molecular mass of the observed activity is not necessarily related to predicted molecular mass, but may suggest that IX-14 migrates as a multimer or part of a complex. The lack of protease activity in mutant brains further suggests that the IX-14 gene product is responsible not only for the mutant phenotypes but also for the protease activity observed under these conditions (either directly or potentially through the activation of other proteases).

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