Generation and analysis of zebrafish melanoma models.

The rapid emergence of the zebrafish as a cancer model has been aided by advances in genetic, chemical, and imaging technologies. Melanoma in particular highlights both the power and challenges associated with cancer modeling in zebrafish. This chapter focuses on the lessons that have emerged from the melanoma models as paradigmatic of what will apply to nearly all cancer models in the zebrafish system. We specifically focus on methodologies related to germline and mosaic transgenic melanoma generation, and how these can be used to deeply interrogate additional cooperating oncogenes or tumor suppressors. These transgenic tumors can in turn be used to generate zebrafish-specific, stable melanoma cell lines which can be fluorescently labeled, modified by cDNA/CRISPR techniques, and used for detailed in vivo imaging of cancer progression in real time. These zebrafish melanoma models are beginning to elucidate both cell intrinsic and microenvironmental factors in melanoma that have broader implications for human disease. We envision that nearly all of the techniques described here can be applied to other zebrafish cancer models, and likely expanded beyond what we describe here.

Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays.

3D printing has emerged as a rapid and cost-efficient manufacturing technique to enable the fabrication of bespoke, complex prototypes. If the technology is to have a significant impact in biomedical applications, such as drug discovery and molecular diagnostics, the devices produced must be biologically compatible to enable their use with established reference assays and protocols. In this work we demonstrate that we can adapt the Fish Embryo Test (FET) as a new method to quantify the toxicity of 3D printed microfluidic devices. We assessed the biocompatibility of four commercially available 3D printing polymers (VisiJetCrystal EX200, Watershed 11122XC, Fototec SLA 7150 Clear and ABSplus P-430), through the observation of key developmental markers in the developing zebrafish embryos. Results show all of the photopolymers to be highly toxic to the embryos, resulting in fatality, although we do demonstrate that post-printing treatment of Fototec 7150 makes it suitable for zebrafish culture within the FET.

The art and design of genetic screens: zebrafish.

Inventive genetic screens in zebrafish are revealing new genetic pathways that control vertebrate development, disease and behaviour. By exploiting the versatility of zebrafish, biological processes that had been previously obscured can be visualized and many of the responsible genes can be isolated. Coupled with gene knockdown and overexpression technologies, and small-molecule-induced phenotypes, genetic screens in zebrafish provide a powerful system by which to dissect vertebrate gene function and gene networks.

SCF(Met30)-mediated control of the transcriptional activator Met4 is required for the G(1)-S transition.

Progression through the cell cycle requires the coordination of basal metabolism with the cell cycle and growth machinery. Repression of the sulfur gene network is mediated by the ubiquitin ligase SCF(Met30), which targets the transcription factor Met4p for degradation. Met30p is an essential protein in yeast. We have found that a met4Deltamet30Delta double mutant is viable, suggesting that the essential function of Met30p is to control Met4p. In support of this hypothesis, a Met4p mutant unable to activate transcription does not cause inviability in a met30Delta strain. Also, overexpression of an unregulated Met4p mutant is lethal in wild-type cells. Under non-permissive conditions, conditional met30Delta strains arrest as large, unbudded cells with 1N DNA content, at or shortly after the pheromone arrest point. met30Delta conditional mutants fail to accumulate CLN1 and CLN2, but not CLN3 mRNAs, even when CLN1 and CLN2 are expressed from strong heterologous promoters. One or more genes under the regulation of Met4p may delay the progression from G(1) into S phase through specific regulation of critical G(1) phase mRNAs.

Feedback-regulated degradation of the transcriptional activator Met4 is triggered by the SCF(Met30 )complex.

Saccharomyces cerevisiae SCF(Met30) ubiquitin-protein ligase controls cell cycle function and sulfur amino acid metabolism. We report here that the SCF(Met30 )complex mediates the transcriptional repression of the MET gene network by triggering degradation of the transcriptional activator Met4p when intracellular S-adenosylmethionine (AdoMet) increases. This AdoMet-induced Met4p degradation is dependent upon the 26S proteasome function. Unlike Met4p, the other components of the specific transcriptional activation complexes that are assembled upstream of the MET genes do not appear to be regulated at the protein level. We provide evidence that the interaction between Met4p and the F-box protein Met30p occurs irrespective of the level of intracellular AdoMet, suggesting that the timing of Met4p degradation is not controlled by its interaction with the SCF(Met30) complex. We also demonstrate that Met30p is a short-lived protein, which localizes within the nucleus. Furthermore, transcription of the MET30 gene is regulated by intracellular AdoMet levels and is dependent upon the Met4p transcription activation function. Thus Met4p appears to control its own degradation by regulating the amount of assembled SCF(Met30) ubiquitin ligase.

Yeast G1 cyclins are unstable in G1 phase.

In most eukaryotes, commitment to cell division occurs in late G1 phase at an event called Start in the yeast Saccharomyces cerevisiae, and called the restriction point in mammalian cells. Start is triggered by the cyclin-dependent kinase Cdc28 and three rate-limiting activators, the G1 cyclins Cln1, Cln2 and Cln3. Cyclin accumulation in G1 is driven in part by the cell-cycle-regulated transcription of CLN1 and CLN2, which peaks at Start. CLN transcription is modulated by physiological signals that regulate G1 progression, but it is unclear whether Cln protein stability is cell-cycle-regulated. It has been suggested that once cells pass Start, Cln proteolysis is triggered by the mitotic cyclins Clb1, 2, 3 and 4. But here we show that G1 cyclins are unstable in G1 phase, and that Clb-Cdc28 activity is not needed fgr G1 cyclin turnover. Cln instability thus provides a means to couple Cln-Cdc28 activity to transcriptional regulation and protein synthetic rate in pre-Start G1 cells.

Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis.

The ubiquitin-dependent proteolytic pathway targets many key regulatory proteins for rapid intracellular degradation. Specificity in protein ubiquitination derives from E3 ubiquitin protein ligases, which recognize substrate proteins. Recently, analysis of the E3s that regulate cell division has revealed common themes in structure and function. One particularly versatile class of E3s, referred to as Skp1p-Cdc53p-F-box protein (SCF) complexes, utilizes substrate-specific adaptor subunits called F-box proteins to recruit various substrates to a core ubiquitination complex. A vast array of F-box proteins have been revealed by genome sequencing projects, and the early returns from genetic analysis in several organisms promise that F-box proteins will participate in the regulation of many processes, including cell division, transcription, signal transduction and development.

Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box proteincomplexes that regulate cell division and methionine biosynthesis in yeast.

In budding yeast, ubiquitination of the cyclin-dependent kinase (Cdk) inhibitor Sic1 is catalyzed by the E2 ubiquitin conjugating enzyme Cdc34 in conjunction with an E3 ubiquitin ligase complex composed of Skp1, Cdc53 and the F-box protein, Cdc4 (the SCFCdc4 complex). Skp1 binds a motif called the F-box and in turn F-box proteins appear to recruit specific substrates for ubiquitination. We find that Skp1 interacts with Cdc53 in vivo, and that Skp1 bridges Cdc53 to three different F-box proteins, Cdc4, Met30, and Grr1. Cdc53 contains independent binding sites for Cdc34 and Skp1 suggesting it functions as a scaffold protein within an E2/E3 core complex. F-box proteins show remarkable functional specificity in vivo: Cdc4 is specific for degradation of Sic1, Grr1 is specific for degradation of the G1 cyclin Cln2, and Met30 is specific for repression of methionine biosynthesis genes. In contrast, the Cdc34-Cdc53-Skp1 E2/E3 core complex is required for all three functions. Combinatorial control of SCF complexes may provide a basis for the regulation of diverse cellular processes.

Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway.

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.