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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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Related in: MedlinePlus

Chromosome defects in l(3)IX-14. (A–D) Mitotic chromosome spreads from wild-type (wt) and homozygous l(3)IX-14 mutant (IX-14) third instar larval brains. “+ col” denotes images from the representative genotype after treatment with colchicine for 90 min. Note that even with colchicine treatment, the IX-14 chromosomes are unable to condense as tightly as the wild-type chromosomes. Bar, 5 μm. (E and F) Polytene chromosome spreads from wild-type and IX-14 homozygous mutant third instar larval salivary glands. Banding and a chromocenter are not as obvious in the mutant chromosomes. Bar, 10 μm. (G) Position effect variegation assay using “white” as a reporter gene (wm4). Flies were sorted into categories based on redness of the adult eyes (white bars, 0–25% redness; yellow bars, 26–50% redness; orange bars, 51–75% redness; red bars, 76–100% redness). The distribution of eye color is shown for the original wm4 stock as well as for wm4 stocks containing one copy of each of the two mutant l(3)IX-14 alleles. The majority of wm4;l(3)IX-14 (both alleles) flies grouped toward the red end of the spectrum, implying that the mutant alleles act as suppressors of variegation (enhancing expression of the reporter gene) and, conversely, that the wild-type gene product acts to compact chromatin.
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fig1: Chromosome defects in l(3)IX-14. (A–D) Mitotic chromosome spreads from wild-type (wt) and homozygous l(3)IX-14 mutant (IX-14) third instar larval brains. “+ col” denotes images from the representative genotype after treatment with colchicine for 90 min. Note that even with colchicine treatment, the IX-14 chromosomes are unable to condense as tightly as the wild-type chromosomes. Bar, 5 μm. (E and F) Polytene chromosome spreads from wild-type and IX-14 homozygous mutant third instar larval salivary glands. Banding and a chromocenter are not as obvious in the mutant chromosomes. Bar, 10 μm. (G) Position effect variegation assay using “white” as a reporter gene (wm4). Flies were sorted into categories based on redness of the adult eyes (white bars, 0–25% redness; yellow bars, 26–50% redness; orange bars, 51–75% redness; red bars, 76–100% redness). The distribution of eye color is shown for the original wm4 stock as well as for wm4 stocks containing one copy of each of the two mutant l(3)IX-14 alleles. The majority of wm4;l(3)IX-14 (both alleles) flies grouped toward the red end of the spectrum, implying that the mutant alleles act as suppressors of variegation (enhancing expression of the reporter gene) and, conversely, that the wild-type gene product acts to compact chromatin.

Mentions: Whole mount preparations of IX-14 third instar larval brains and imaginal discs showed that mutant tissues had proliferation defects resulting in much-reduced brain size and missing imaginal discs (unpublished data). Fig. 1 shows typical DAPI-stained mitotic figures observed in wild-type (Fig. 1 A) and in IX-14 mutant (Fig. 1 C) neuroblasts. The IX-14 neuroblasts show the length-wise hypercondensation of mitotic chromosomes initially observed after orcein staining, but additionally demonstrate that the mutant chromosomes appear loosely condensed with a ragged periphery. This phenotype differs from the extreme hypercondensation in other mutations that produce a mitotic arrest phenotype (Heck et al., 1993) or treatment of wild-type cells with microtubule poisons such as colchicine (Fig. 1 B). Allowing more time in mitosis with colchicine does not rescue the lateral condensation defect in IX-14 mutant neuroblasts (Fig. 1 D), thus we believe the phenotype represents a specific condensation defect rather than “conventional” hypercondensation in response to mitotic delay.


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)

Chromosome defects in l(3)IX-14. (A–D) Mitotic chromosome spreads from wild-type (wt) and homozygous l(3)IX-14 mutant (IX-14) third instar larval brains. “+ col” denotes images from the representative genotype after treatment with colchicine for 90 min. Note that even with colchicine treatment, the IX-14 chromosomes are unable to condense as tightly as the wild-type chromosomes. Bar, 5 μm. (E and F) Polytene chromosome spreads from wild-type and IX-14 homozygous mutant third instar larval salivary glands. Banding and a chromocenter are not as obvious in the mutant chromosomes. Bar, 10 μm. (G) Position effect variegation assay using “white” as a reporter gene (wm4). Flies were sorted into categories based on redness of the adult eyes (white bars, 0–25% redness; yellow bars, 26–50% redness; orange bars, 51–75% redness; red bars, 76–100% redness). The distribution of eye color is shown for the original wm4 stock as well as for wm4 stocks containing one copy of each of the two mutant l(3)IX-14 alleles. The majority of wm4;l(3)IX-14 (both alleles) flies grouped toward the red end of the spectrum, implying that the mutant alleles act as suppressors of variegation (enhancing expression of the reporter gene) and, conversely, that the wild-type gene product acts to compact chromatin.
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Related In: Results  -  Collection

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fig1: Chromosome defects in l(3)IX-14. (A–D) Mitotic chromosome spreads from wild-type (wt) and homozygous l(3)IX-14 mutant (IX-14) third instar larval brains. “+ col” denotes images from the representative genotype after treatment with colchicine for 90 min. Note that even with colchicine treatment, the IX-14 chromosomes are unable to condense as tightly as the wild-type chromosomes. Bar, 5 μm. (E and F) Polytene chromosome spreads from wild-type and IX-14 homozygous mutant third instar larval salivary glands. Banding and a chromocenter are not as obvious in the mutant chromosomes. Bar, 10 μm. (G) Position effect variegation assay using “white” as a reporter gene (wm4). Flies were sorted into categories based on redness of the adult eyes (white bars, 0–25% redness; yellow bars, 26–50% redness; orange bars, 51–75% redness; red bars, 76–100% redness). The distribution of eye color is shown for the original wm4 stock as well as for wm4 stocks containing one copy of each of the two mutant l(3)IX-14 alleles. The majority of wm4;l(3)IX-14 (both alleles) flies grouped toward the red end of the spectrum, implying that the mutant alleles act as suppressors of variegation (enhancing expression of the reporter gene) and, conversely, that the wild-type gene product acts to compact chromatin.
Mentions: Whole mount preparations of IX-14 third instar larval brains and imaginal discs showed that mutant tissues had proliferation defects resulting in much-reduced brain size and missing imaginal discs (unpublished data). Fig. 1 shows typical DAPI-stained mitotic figures observed in wild-type (Fig. 1 A) and in IX-14 mutant (Fig. 1 C) neuroblasts. The IX-14 neuroblasts show the length-wise hypercondensation of mitotic chromosomes initially observed after orcein staining, but additionally demonstrate that the mutant chromosomes appear loosely condensed with a ragged periphery. This phenotype differs from the extreme hypercondensation in other mutations that produce a mitotic arrest phenotype (Heck et al., 1993) or treatment of wild-type cells with microtubule poisons such as colchicine (Fig. 1 B). Allowing more time in mitosis with colchicine does not rescue the lateral condensation defect in IX-14 mutant neuroblasts (Fig. 1 D), thus we believe the phenotype represents a specific condensation defect rather than “conventional” hypercondensation in response to mitotic delay.

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