"The methanol was replaced with fresh Merck ARISTAR methanol four times to remove any remaining formaldehyde or heptane and mixed after each replacement. Fixed embryos were stored in methanol at ÿ20° before processing for immunofluorescent detection as described (Therkauf and Heck 1999; Rothwell and Sullivan 2000). Developmental staging of embryos was assessed according to the numbering scheme introduced by Mary Bownes (Bownes 1975, 1982) and later refined by Eric Wieschaus and Christiane Nüsslein-Volhard (Wieschaus and Nüsslein-Volhard 1998). "
[Show abstract][Hide abstract] ABSTRACT: The condensin complex has been implicated in the higher-order organization of mitotic chromosomes in a host of model eukaryotes from yeasts to flies and vertebrates. Although chromosomes paradoxically appear to condense in condensin mutants, chromatids are not properly resolved, resulting in chromosome segregation defects during anaphase. We have examined the role of different condensin complex components in interphase chromatin function by examining the effects of various condensin mutations on position-effect variegation in Drosophila melanogaster. Surprisingly, most mutations affecting condensin proteins were often found to result in strong enhancement of variegation in contrast to what might be expected for proteins believed to compact the genome. This suggests either that the role of condensin proteins in interphase differs from their expected role in mitosis or that the way we envision condensin's activity needs to be modified to accommodate alternative possibilities.
"The IX-14 mutation in Drosophila causes cells to arrest in mitosis with hypercondensed mitotic chromosomes surrounded by poorly condensed chromatin, a phenotype that is clearly distinct from the chromosome hypercondensation commonly observed in mutations exhibiting mitotic delay (Heck et al., 1993; Theurkauf and Heck, 1999). Invadolysin also appears to have a role in chromosome architecture during interphase because mutations exhibit poorly structured polytene chromosomes and have compromised heterochromatin, as assayed by position effect variegation. "
[Show abstract][Hide abstract] 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.
The Journal of Cell Biology 11/2004; 167(4):673-686. · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During early embryogenesis of Drosophila melanogaster, mutations in the DNA-replication checkpoint lead to chromosome-segregation failures. Here we show that these segregation failures are associated with the assembly of an anastral microtubule spindle, a mitosis-specific loss of centrosome function, and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. The DNA-replication inhibitor aphidicolin and DNA-damaging agents trigger identical mitotic defects in wild-type embryos, indicating that centrosome inactivation is a checkpoint-independent and mitosis-specific response to damaged or incompletely replicated DNA. We propose that centrosome inactivation is part of a damage-control system that blocks chromosome segregation when replication/damage checkpoint control fails.
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