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 localization of invadolysin. The level of invadolysin increased during both murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS), adipogenesis. Invadolysin displayed a dynamic localization 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.

Invadolysin acts genetically via the SAGA complex to modulate chromosome structure.

Identification of components essential to chromosome structure and behaviour remains a vibrant area of study. We have previously shown that invadolysin is essential in Drosophila, with roles in cell division and cell migration. Mitotic chromosomes are hypercondensed in length, but display an aberrant fuzzy appearance. We additionally demonstrated that in human cells, invadolysin is localized on the surface of lipid droplets, organelles that store not only triglycerides and sterols but also free histones H2A, H2Av and H2B. Is there a link between the storage of histones in lipid droplets and the aberrantly structured chromosomes of invadolysin mutants? We have identified a genetic interaction between invadolysin and nonstop, the de-ubiquitinating protease component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) chromatin-remodelling complex. invadolysin and nonstop mutants exhibit phenotypic similarities in terms of chromosome structure in both diploid and polyploid cells. Furthermore, IX-14(1)/not(1) transheterozygous animals accumulate mono-ubiquitinated histone H2B (ubH2B) and histone H3 tri-methylated at lysine 4 (H3K4me3). Whole mount immunostaining of IX-14(1)/not(1) transheterozygous salivary glands revealed that ubH2B accumulates surprisingly in the cytoplasm, rather than the nucleus. Over-expression of the Bre1 ubiquitin ligase phenocopies the effects of mutating either the invadolysin or nonstop genes. Intriguingly, nonstop and mutants of other SAGA subunits (gcn5, ada2b and sgf11) all suppress an invadolysin-induced rough eye phenotype. We conclude that the abnormal chromosome phenotype of invadolysin mutants is likely the result of disrupting the histone modification cycle, as accumulation of ubH2B and H3K4me3 is observed. We further suggest that the mislocalization of ubH2B to the cytoplasm has additional consequences on downstream components essential for chromosome behaviour. We therefore propose that invadolysin plays a crucial role in chromosome organization via its interaction with the SAGA complex.

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.

The conserved metalloprotease invadolysin localizes to the surface of lipid droplets.

Invadolysin is a metalloprotease conserved in many different organisms, previously shown to be essential in Drosophila with roles in cell division and cell migration. The gene seems to be ubiquitously expressed and four distinct splice variants have been identified in human cells but not in most other species examined. Immunofluorescent detection of human invadolysin in cultured cells reveals the protein to be associated with the surface of lipid droplets. By means of subcellular fractionation, we have independently confirmed the association of invadolysin with lipid droplets. We thus identify invadolysin as the first metalloprotease located on these dynamic organelles. In addition, analysis of larval fat-body morphological appearance and triglyceride levels in the Drosophila invadolysin mutant suggests that invadolysin plays a role in lipid storage or metabolism.

Depletion of Drad21/Scc1 in Drosophila cells leads to instability of the cohesin complex and disruption of mitotic progression.

BACKGROUND: The coordination of cell cycle events is necessary to ensure the proper duplication and dissemination of the genome. In this study, we examine the consequences of depleting Drad21 and SA, two non-SMC subunits of the cohesin complex, by dsRNA-mediated interference in Drosophila cultured cells. RESULTS: We have shown that a bona fide cohesin complex exists in Drosophila embryos. Strikingly, the Drad21/Scc1 and SA/Scc3 non-SMC subunits associate more intimately with one another than they do with the SMCs. We have observed defects in mitotic progression in cells from which Drad21 has been depleted: cells delay in prometaphase with normally condensed, but prematurely separated, sister chromatids and with abnormal spindle morphology. Much milder defects are observed when SA is depleted from cells. The dynamics of the chromosome passenger protein, INCENP, are affected after Drad21 depletion. We have also made the surprising observation that SA is unstable in the absence of Drad21; however, we have shown that the converse is not true. Interference with Drad21 in living Drosophila embryos also has deleterious effects on mitotic progression. CONCLUSIONS: We conclude that Drad21, as a member of a cohesin complex, is required in Drosophila cultured cells and embryos for proper mitotic progression. The protein is required in cultured cells for chromosome cohesion, spindle morphology, dynamics of a chromosome passenger protein, and stability of the cohesin complex, but apparently not for normal chromosome condensation. The observation of SA instability in the absence of Drad21 implies that the expression of cohesin subunits and assembly of the cohesin complex will be tightly regulated.

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.

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.

Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells.

BACKGROUND: The 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 FINDINGS: We 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/SIGNIFICANCE: These 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.

Drosophila cohesins DSA1 and Drad21 persist and colocalize along the centromeric heterochromatin during mitosis.

Sister chromatid cohesion in eukaryotes is maintained mainly by a conserved multiprotein complex termed cohesin. Drad21 and DSA1 are the Drosophila homologues of the yeast Scc1 and Scc3 cohesin subunits, respectively. We recently identified a Drosophila mitotic cohesin complex composed of Drad21/DSA1/DSMC1/DSMC3. Here we study the contribution of this complex to sister chromatid cohesion using immunofluorescence microscopy to analyze cell cycle chromosomal localization of DSA1 and Drad21 in S2 cells. We observed that DSA1 and Drad21 colocalize during all cell cycle stages in cultured cells. Both proteins remain in the centromere until metaphase, colocalizing at the centromere pairing domain that extends along the entire heterochromatin; the centromeric cohesion protein MEI-S332 is nonetheless reported in a distinct centromere domain. These results provide strong evidence that DSA1 and Drad21 are partners in a cohesin complex involved in the maintenance of sister chromatid arm and centromeric cohesion during mitosis in Drosophila.