Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking.

The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.

Regulation of the error-prone DNA polymerase Polκ by oncogenic signaling and its contribution to drug resistance.

The DNA polymerase Polκ plays a key role in translesion synthesis, an error-prone replication mechanism. Polκ is overexpressed in various tumor types. Here, we found that melanoma and lung and breast cancer cells experiencing stress from oncogene inhibition up-regulated the expression of Polκ and shifted its localization from the cytoplasm to the nucleus. This effect was phenocopied by inhibition of the kinase mTOR, by induction of ER stress, or by glucose deprivation. In unstressed cells, Polκ is continually transported out of the nucleus by exportin-1. Inhibiting exportin-1 or overexpressing Polκ increased the abundance of nuclear-localized Polκ, particularly in response to the BRAFV600E-targeted inhibitor vemurafenib, which decreased the cytotoxicity of the drug in BRAFV600E melanoma cells. These observations were analogous to how Escherichia coli encountering cell stress and nutrient deprivation can up-regulate and activate DinB/pol IV, the bacterial ortholog of Polκ, to induce mutagenesis that enables stress tolerance or escape. However, we found that the increased expression of Polκ was not excessively mutagenic, indicating that noncatalytic or other functions of Polκ could mediate its role in stress responses in mammalian cells. Repressing the expression or nuclear localization of Polκ might prevent drug resistance in some cancer cells.

Probing RNA Structure in Liquid-Liquid Phase Separation Using SHAPE-MaP.

RNA is an integral component of many biological condensates. A variety of features of RNAs are linked to their function in biological phase separation. Length and negative charge provide fairly generic chemical inputs that drive condensation while sequence has been shown to influence both the molecular identity and biophysical properties of droplets. mRNA sequence guides the secondary structure of the polymers and RNA secondary structure licenses-specific RNA-RNA interactions and the recruitment of RNA-binding proteins. Here, we describe a method for directly probing the structure of mRNAs in the context of RNP-droplets formed via LLPS.

A New Lens for RNA Localization: Liquid-Liquid Phase Separation.

RNA localization mechanisms have been intensively studied and include localized protection of mRNA from degradation, diffusion-coupled local entrapment of mRNA, and directed transport of mRNAs along the cytoskeleton. While it is well understood how cells utilize these three mechanisms to organize mRNAs within the cytoplasm, a newly appreciated mechanism of RNA localization has emerged in recent years in which mRNAs phase-separate and form liquid-like droplets. mRNAs both contribute to condensation of proteins into liquid-like structures and are themselves regulated by being incorporated into membraneless organelles. This ability to condense into droplets is in many instances contributing to previously appreciated mRNA localization phenomena. Here we review how phase separation enables mRNAs to selectively and efficiently colocalize and be coregulated, allowing control of gene expression in time and space.

Principles of Systems Biology, No. 30.

This month: two examples of door-opening, innovative microscopy (Garcia and also Benzinger et al.), expanding our functional knowledge of bacteria by over 10,000 genes (Deutschbauer), and probing how RNA structure dictates inclusion in liquid-like droplets in vivo (Langdon and Gladfelter).

mRNA structure determines specificity of a polyQ-driven phase separation.

RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here, we report that secondary structure allows messenger RNAs (mRNAs) to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

Melanoma genome evolution across species.

BACKGROUND: Cancer genomes evolve in both space and time, which contributes to the genetic heterogeneity that underlies tumor progression and drug resistance. In human melanoma, identifying mechanistically important events in tumor evolution is hampered due to the high background mutation rate from ultraviolet (UV) light. Cross-species oncogenomics is a powerful tool for identifying these core events, in which transgenically well-defined animal models of cancer are compared to human cancers to identify key conserved alterations. RESULTS: We use a zebrafish model of tumor progression and drug resistance for cross-species genomic analysis in melanoma. Zebrafish transgenic tumors are initiated with just 2 genetic lesions, BRAFV600E and p53-/-, yet take 4-6 months to appear, at which time whole genome sequencing demonstrated >3,000 new mutations. An additional 4-month exposure to the BRAF inhibitor vemurafenib resulted in a highly drug resistant tumor that showed 3 additional new DNA mutations in the genes BUB1B, PINK1, and COL16A1. These genetic changes in drug resistance are accompanied by a massive reorganization of the transcriptome, with differential RNA expression of over 800 genes, centered on alterations in cAMP and PKA signaling. By comparing both the DNA and mRNA changes to a large panel of human melanomas, we find that there is a highly significant enrichment of these alterations in human patients with vemurafenib resistant disease. CONCLUSIONS: Our results suggest that targeting of alterations that are conserved between zebrafish and humans may offer new avenues for therapeutic intervention. The approaches described here will be broadly applicable to the diverse array of cancer models available in the zebrafish, which can be used to inform human cancer genomics.

Voluntary exercise improves hypothalamic and metabolic function in obese mice.

Exercise plays a critical role in regulating glucose homeostasis and body weight. However, the mechanism of exercise on metabolic functions associated with the CNS has not been fully understood. C57BL6 male mice (n=45) were divided into three groups: normal chow diet, high-fat diet (HFD) treatment, and HFD along with voluntary running wheel exercise training for 12 weeks. Metabolic function was examined by the Comprehensive Lab Animal Monitoring System and magnetic resonance imaging; phenotypic analysis included measurements of body weight, food intake, glucose and insulin tolerance tests, as well as insulin and leptin sensitivity studies. By immunohistochemistry, the amount changes in the phosphorylation of signal transducer and activator of transcription 3, neuronal proliferative maker Ki67, apoptosis positive cells as well as pro-opiomelanocortin (POMC)-expressing neurons in the arcuate area of the hypothalamus was identified. We found that 12 weeks of voluntary exercise training partially reduced body weight gain and adiposity induced by an HFD. Insulin and leptin sensitivity were enhanced in the exercise training group verses the HFD group. Furthermore, the HFD-impaired POMC-expressing neuron is remarkably restored in the exercise training group. The restoration of POMC neuron number may be due to neuroprotective effects of exercise on POMC neurons, as evidenced by altered proliferation and apoptosis. In conclusion, our data suggest that voluntary exercise training improves metabolic symptoms induced by HFD, in part through protected POMC-expressing neuron from HFD and enhanced leptin signaling in the hypothalamus that regulates whole-body energy homeostasis.

RNA Controls PolyQ Protein Phase Transitions.

Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophysical properties of phase-separated compartments.

A Quantitative System for Studying Metastasis Using Transparent Zebrafish.

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models.

BACKGROUND: Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process. RESULTS: To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAF(V600E) or NRAS(Q61K) driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAF(V600E) and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway. CONCLUSION: This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.

The histone methyltransferase SUV39H1 suppresses embryonal rhabdomyosarcoma formation in zebrafish.

Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses chromatin modifications on both the DNA and histones and is as important as the DNA sequence itself. Chromatin-modifying factors are playing an increasingly important role in tumorigenesis, particularly among pediatric rhabdomyosarcomas (RMS), revealing potential novel therapeutic targets. We performed an overexpression screen of chromatin-modifying factors in a KRAS(G12D)-driven zebrafish model for RMS. Here, we describe the identification of a histone H3 lysine 9 histone methyltransferase, SUV39H1, as a suppressor of embryonal RMS formation in zebrafish. This suppression is specific to the histone methyltransferase activity of SUV39H1, as point mutations in the SET domain lacked the effect. SUV39H1-overexpressing and control tumors have a similar proliferation rate, muscle differentiation state, and tumor growth rate. Strikingly, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. We demonstrate that the delayed tumor onset occurs between 5 and 7 days post fertilization. Gene expression profiling at these stages revealed that in the context of KRAS(G12D) overexpression, SUV39H1 may suppress cell cycle progression. Our studies provide evidence for the role of SUV39H1 as a tumor suppressor.

The transcriptional landscape of hematopoietic stem cell ontogeny.

Transcriptome analysis of adult hematopoietic stem cells (HSCs) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSCs purified from >2,500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network-biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knockdown in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSCs, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.

DHODH modulates transcriptional elongation in the neural crest and melanoma.

Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.