Perturbation of invadolysin disrupts cell migration in zebrafish (Danio rerio)

Invadolysin is an essential, conserved metalloprotease which links cell division with cell migration and is intriguingly associated with lipid droplets. In this work we examine the expression pattern, protein localisation and gross anatomical consequences of depleting invadolysin in the teleost Danio rerio. We observe that invadolysin plays a significant role in cell migration during development. When invadolysin is depleted by targeted morpholino injection, the appropriate deposition of neuromast clusters and distribution of melanophores are both disrupted. We also observe that blood vessels generated via angiogenesis are affected in invadolysin morphant fish while those formed by vasculogenesis appear normal, demonstrating an unanticipated role for invadolysin in vessel formation. Our results thus highlight a common feature shared by, and a requirement for invadolysin in, these distinct morphological events dependent on cell migration

Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling

Multi-cellular organisms need to successfully link cell growth and metabolism to environmental cues during development. Insulin receptor–target of rapamycin (InR–TOR) signalling is a highly conserved pathway that mediates this link. Herein, we describe poly, an essential gene in Drosophila that mediates InR–TOR signalling. Loss of poly results in lethality at the third instar larval stage, but only after a stage of extreme larval longevity. Analysis in Drosophila demonstrates that Poly and InR interact and that poly mutants show an overall decrease in InR–TOR signalling, as evidenced by decreased phosphorylation of Akt, S6K and 4E-BP. Metabolism is altered in poly mutants, as revealed by microarray expression analysis and a decreased triglyceride : protein ratio in mutant animals. Intriguingly, the cellular distribution of Poly is dependent on insulin stimulation in both Drosophila and human cells, moving to the nucleus with insulin treatment, consistent with a role in InR–TOR signalling. Together, these data reveal that Poly is a novel, conserved (from flies to humans) mediator of InR signalling that promotes an increase in cell growth and metabolism. Furthermore, homology to small subunits of Elongator demonstrates a novel, unexpected role for this complex in insulin signalling

Heart on a Plate: Histological and Functional Assessment of Isolated Adult Zebrafish Hearts Maintained in Culture

The zebrafish is increasingly used for cardiovascular genetic and functional studies. We present a novel protocol to maintain and monitor whole isolated beating adult zebrafish hearts in culture for long-term experiments. Excised whole adult zebrafish hearts were transferred directly into culture dishes containing optimized L-15 Leibovitz growth medium and maintained for 5 days. Hearts were assessed daily using video-edge analysis of ventricle function using low power microscopy images. High-throughput histology techniques were used to assess changes in myocardial architecture and cell viability. Mean spontaneous Heart rate (HR, min−1) declined significantly between day 0 and day 1 in culture (96.7±19.5 to 45.2±8.2 min−1, mean±SD, p = 0.001), and thereafter declined more slowly to 27.6±7.2 min−1 on day 5. Ventricle wall motion amplitude (WMA) did not change until day 4 in culture (day 0, 46.7±13.0 µm vs day 4, 16.9±1.9 µm, p = 0.08). Contraction velocity (CV) declined between day 0 and day 3 (35.6±14.8 vs 15.2±5.3 µms−1, respectively, p = 0.012) while relaxation velocity (RV) declined quite rapidly (day 0, 72.5±11.9 vs day 1, 29.5±5.8 µms−1, p = 0.03). HR and WMA responded consistently to isoproterenol from day 0 to day 5 in culture while CV and RV showed less consistent responses to beta-agonist. Cellular architecture and cross-striation pattern of cardiomyocytes remained unchanged up to day 3 in culture and thereafter showed significant deterioration with loss of striation pattern, pyknotic nuclei and cell swelling. Apoptotic markers within the myocardium became increasingly frequent by day 3 in culture. Whole adult zebrafish hearts can be maintained in culture-medium for up to 3 days. However, after day-3 there is significant deterioration in ventricle function and heart rate accompanied by significant histological changes consistent with cell death and loss of cardiomyocyte cell integrity. Further studies are needed to assess whether this preparation can be optimised for longer term survival

Differential Requirements for MCM Proteins in DNA Replication in Drosophila S2 Cells

BackgroundThe MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo.Methodology/Principle FindingsWe performed a systematic depletion of the MCM proteins in Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95–99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. We also depleted MCM8. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5.Conclusions/SignificanceThese studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells

A role for the metalloprotease invadolysin in insulin signaling and adipogenesis

Invadolysin is a novel metalloprotease conserved amongst metazoans that is essential for life in Drosophila. We previously showed that invadolysin was essential for the cell cycle and cell migration, linking to metabolism through a role in lipid storage and interaction with mitochondrial proteins. In this study we demonstrate that invadolysin mutants exhibit increased autophagy and decreased glycogen storage - suggestive of a role for invadolysin in insulin signaling in Drosophila. Consistent with this, effectors of insulin signaling were decreased in invadolysin mutants. In addition, we discovered that invadolysin was deposited on newly synthesized lipid droplets in a PKC-dependent manner. We examined two in vitro models of adipogenesis for the expression and localisation of invadolysin. The level of invadolysin increased during both murine 3T3-L1 and human SGBS adipogenesis. Invadolysin displayed a dynamic localisation to lipid droplets over the course of adipogenesis, which may be due to the differential expression of distinct invadolysin variants. Pharmacological inhibition of adipogenesis abrogated the increase in invadolysin. In summary, our results on in vivo and in vitro systems highlight an important role for invadolysin in insulin signaling and adipogenesis

Loss of Cell Cycle Checkpoint Control in Drosophila Rfc4 Mutants

Two alleles of the Drosophila melanogaster Rfc4(DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these twoDmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes