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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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dsRNA-mediated interference of IX-14 in Drosophila S2 cells phenocopies the mutation. (A) Spindles of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for α-tubulin (green), P~H3 (red), and DAPI (blue). The normal bipolar spindle in the control S2 cell is to be contrasted with the disorganized spindle shown in the treated cell. This is similar to that observed in IX-14 homozygous mutant alleles. Bar, 5 μm. (B) Centrosomes of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for CP190 (green) and DAPI (red). The treated cells show the centrosome separation defect (arrow) similar to that observed in l(3)IX-14 homozygous mutant alleles. Bar, 5 μm.
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fig5: dsRNA-mediated interference of IX-14 in Drosophila S2 cells phenocopies the mutation. (A) Spindles of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for α-tubulin (green), P~H3 (red), and DAPI (blue). The normal bipolar spindle in the control S2 cell is to be contrasted with the disorganized spindle shown in the treated cell. This is similar to that observed in IX-14 homozygous mutant alleles. Bar, 5 μm. (B) Centrosomes of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for CP190 (green) and DAPI (red). The treated cells show the centrosome separation defect (arrow) similar to that observed in l(3)IX-14 homozygous mutant alleles. Bar, 5 μm.

Mentions: As further evidence that the gene we identified is responsible for the defects observed in IX-14 larval tissues, we performed dsRNA-mediated interference of IX-14 in Drosophila Schneider 2 (S2) cells. The abnormal spindle phenotypes seen in the mutant alleles, typically monopolar or disorganized, were phenocopied in roughly 25% of the IX-14 RNAi mitotic cells (Fig. 5 A). In addition, mitotic figures from RNAi cultures were frequently observed to have single centrosomes or what appeared to be two closely apposed centrosomes (Fig. 5 B). Although control S2 cells have a low level of aneuploidy and occasional abnormal numbers of centrosomes, neither the aberrant spindles nor unseparated centrosomes were observed in control cells (Fig. 5, −dsRNA panels). Therefore, we are confident that loss of the IX-14 protein is responsible for the observed cellular phenotypes.


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)

dsRNA-mediated interference of IX-14 in Drosophila S2 cells phenocopies the mutation. (A) Spindles of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for α-tubulin (green), P~H3 (red), and DAPI (blue). The normal bipolar spindle in the control S2 cell is to be contrasted with the disorganized spindle shown in the treated cell. This is similar to that observed in IX-14 homozygous mutant alleles. Bar, 5 μm. (B) Centrosomes of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for CP190 (green) and DAPI (red). The treated cells show the centrosome separation defect (arrow) similar to that observed in l(3)IX-14 homozygous mutant alleles. Bar, 5 μm.
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Related In: Results  -  Collection

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

fig5: dsRNA-mediated interference of IX-14 in Drosophila S2 cells phenocopies the mutation. (A) Spindles of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for α-tubulin (green), P~H3 (red), and DAPI (blue). The normal bipolar spindle in the control S2 cell is to be contrasted with the disorganized spindle shown in the treated cell. This is similar to that observed in IX-14 homozygous mutant alleles. Bar, 5 μm. (B) Centrosomes of S2 cells: control (top) and 72 h after dsRNA treatment (bottom). Cells are stained for CP190 (green) and DAPI (red). The treated cells show the centrosome separation defect (arrow) similar to that observed in l(3)IX-14 homozygous mutant alleles. Bar, 5 μm.
Mentions: As further evidence that the gene we identified is responsible for the defects observed in IX-14 larval tissues, we performed dsRNA-mediated interference of IX-14 in Drosophila Schneider 2 (S2) cells. The abnormal spindle phenotypes seen in the mutant alleles, typically monopolar or disorganized, were phenocopied in roughly 25% of the IX-14 RNAi mitotic cells (Fig. 5 A). In addition, mitotic figures from RNAi cultures were frequently observed to have single centrosomes or what appeared to be two closely apposed centrosomes (Fig. 5 B). Although control S2 cells have a low level of aneuploidy and occasional abnormal numbers of centrosomes, neither the aberrant spindles nor unseparated centrosomes were observed in control cells (Fig. 5, −dsRNA panels). Therefore, we are confident that loss of the IX-14 protein is responsible for the observed cellular phenotypes.

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