Alternative polyadenylation is a determinant of oncogenic Ras function.

Alternative polyadenylation of mRNA has important but poorly understood roles in development and cancer. Activating mutations in the Ras oncogene are common drivers of many human cancers. From a screen for enhancers of activated Ras (let-60) in Caenorhabditis elegans, we identified cfim-1, a subunit of the alternative polyadenylation machinery. Ablation of cfim-1 increased penetrance of the multivulva phenotype in let-60/Ras gain-of-function (gf) mutants. Depletion of the human cfim-1 ortholog CFIm25/NUDT21 in cancer cells with KRAS mutations increased their migration and stimulated an epithelial-to-mesenchymal transition. CFIm25-depleted cells and cfim-1 mutants displayed biased placement of poly(A) tails to more proximal sites in many conserved transcripts. Functional analysis of these transcripts identified the multidrug resistance protein mrp-5/ABCC1 as a previously unidentified regulator of C. elegans vulva development and cell migration in human cells through alternative 3'UTR usage. Our observations demonstrate a conserved functional role for alternative polyadenylation in oncogenic Ras function.

A conserved CCM complex promotes apoptosis non-autonomously by regulating zinc homeostasis.

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1-/- zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.

Interrogating the ccm-3 Gene Network.

Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by mutations in one of three genes (CCM1-3). Loss of CCM3 causes the poorest prognosis, and little is known about how it regulates vascular integrity. The C. elegans ccm-3 gene regulates the development of biological tubes that resemble mammalian vasculature, and in a genome-wide reverse genetic screen, we identified more than 500 possible CCM-3 pathway genes. With a phenolog-like approach, we generated a human CCM signaling network and identified 29 genes in common, of which 14 are required for excretory canal extension and membrane integrity, similar to ccm-3. Notably, depletion of the MO25 ortholog mop-25.2 causes severe defects in tube integrity by preventing CCM-3 localization to apical membranes. Furthermore, loss of MO25 phenocopies CCM3 ablation by causing stress fiber formation in endothelial cells. This work deepens our understanding of how CCM3 regulates vascular integrity and may help identify therapeutic targets for treating CCM3 patients.

CCM-3 Promotes C. elegans Germline Development by Regulating Vesicle Trafficking Cytokinesis and Polarity.

Cerebral cavernous malformations (CCMs) are vascular defects of the CNS that arise from loss of integrity of the endothelial cells lining blood capillaries, causing leakage of blood into the brain [1]. This results in headaches, seizures, and/or hemorrhagic stroke, depending on the location of the lesion. CCM affects 0.5% of the population and follows an autosomal dominant inheritance pattern caused by mutations in one of the three genes: CCM1 (gene name KRIT1), CCM2 (also known as malcavernin or OSM), and CCM3 (gene name PDCD10) [2, 3], with the earliest onset and most severe prognosis occurring in CCM3 patients [4]. The three CCM genes encode structurally distinct scaffold proteins that function in multiple complexes [5-9]. Using the C. elegans germline as a model of multicellular tube development, we show here that CCM-3 is enriched at the luminal membrane of the germline and the contractile ring of dividing cells in the embryo. Loss of ccm-3 results in defective RAB-11-mediated endocytic recycling, which in turn is necessary for gonadal lumen (rachis) formation, completion of cytokinesis, and localization of cell-surface receptors. CCM-3-mediated localization of anillin and non-muscle myosin to the lateral surfaces of germ cells is required for proper cytoskeletal organization, subsequent oocyte growth, and localization of polarity proteins. Biochemical analysis reveals conservation of the STRIPAK complex and distinct roles for GCK-1 (germinal center kinase III family protein) and striatin/CASH-1 in controlling the localization and function of CCM-3. Taken together, our data establish CCM-3 as a novel regulator of rachis lumenization and polarity establishment during embryogenesis.

CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.

The mechanisms governing apical membrane assembly during biological tube development are poorly understood. Here, we show that extension of the C. elegans excretory canal requires cerebral cavernous malformation 3 (CCM-3), independent of the CCM1 orthologue KRI-1. Loss of ccm-3 causes canal truncations and aggregations of canaliculular vesicles, which form ectopic lumen (cysts). We show that CCM-3 localizes to the apical membrane, and in cooperation with GCK-1 and STRIPAK, promotes CDC-42 signalling, Golgi stability and endocytic recycling. We propose that endocytic recycling is mediated through the CDC-42-binding kinase MRCK-1, which interacts physically with CCM-3-STRIPAK. We further show canal membrane integrity to be dependent on the exocyst complex and the actin cytoskeleton. This work reveals novel in vivo roles of CCM-3·STRIPAK in regulating tube extension and membrane integrity through small GTPase signalling and vesicle dynamics, which may help explain the severity of CCM3 mutations in patients.

Analysis of apoptosis in Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans has provided researchers with a wealth of information on the molecular mechanisms controlling programmed cell death (apoptosis). Its genetic tractability, optical clarity, and relatively short lifespan are key advantages for rapid assessment of apoptosis in vivo. The use of forward and reverse genetics methodology, coupled with in vivo imaging, has provided deep insights into how a multicellular organism orchestrates the self-destruction of specific cells during development and in response to exogenous stresses. Strains of C. elegans carrying mutations in the core elements of the apoptotic pathway, or in tissue-specific regulators of apoptosis, can be used for genetic analyses to reveal conserved mechanisms by which apoptosis is regulated in the somatic and reproductive (germline) tissue. Here we present an introduction to the study of apoptosis in C. elegans, including current techniques for visualization, analysis, and screening.

Immunostaining for markers of apoptosis in the Caenorhabditis elegans germline.

The transparency of Caenorhabditis elegans makes it an ideal organism for visualizing proteins by immunofluorescence microscopy; however, the tough cuticle of worms and the egg shell surrounding embryos pose challenges in achieving effective fixation so that antibodies can diffuse into cells. In this protocol, we describe immunostaining of apoptosis-related proteins in the C. elegans adult germline using fluorescent reagents. Protein localization and abundance can be determined in various mutant backgrounds and under a variety of conditions, such as exposure to genotoxic stress. The number of antibodies specific to C. elegans proteins is quite limited compared with other organisms, but there is a growing list of immunological reagents directed against proteins in other organisms that cross-react with the homologous C. elegans proteins.

Fluorescent visualization of germline apoptosis in living Caenorhabditis elegans.

Visualization of apoptosis using fluorescent tools is quite straightforward in living Caenorhabditis elegans. Several transgenic lines are available that mark dying cells with fluorescent proteins, making it possible to quantify kinetics at various stages of the apoptotic process. Proteins required for the engulfment of cell corpses are particularly useful for detecting early apoptotic stages using this approach. For example, expression of the engulfment protein CED-1 fused to green fluorescent protein (CED-1::GFP) creates a halo around cells during early apoptosis, before their refractile morphology can be detected by differential interference contrast (DIC) optics. In addition, vital dyes such as acridine orange (AO) and SYTO-12 are selectively retained in apoptotic cells and can be used to visualize apoptosis in the germlines of living animals. It is also possible to use vital dyes in combination with transgenic strains expressing fluorescent markers of cell corpses to examine, in detail, multiple stages of apoptosis in vivo. Because of the high optical contrast of fluorescent reagents, apoptosis can be visualized even at low magnification, facilitating the use of screening platforms to identify apoptosis regulators. This protocol describes multiple uses of fluorescent reagents for visualization of germline apoptosis in living C. elegans, including AO staining, time-course studies using fluorescent proteins, and low-throughput screening of cell death genes using RNA interference (RNAi).

High-throughput RNAi screening for germline apoptosis genes in Caenorhabditis elegans.

Among the greatest tools that Caenorhabditis elegans can provide researchers are the capabilities to perform high-throughput, genome-wide screens. Using bacterial RNAi libraries, which cover the majority (>85%) of the worm genome, genes can be rapidly and systematically evaluated for apoptosis phenotypes in the germline. Screens can be designed to directly assess the levels of apoptotic corpses under normal physiological conditions using transgenic strains expressing fluorescent reporters that mark apoptotic bodies. Vital dyes that are selectively retained in apoptotic cells, such as acridine orange (AO), can also be used to screen for genes that regulate germline apoptosis. Using these reagents, screens can be performed in wild-type worms or mutant backgrounds that suppress or enhance apoptosis phenotypes. This protocol describes methods for designing and carrying out high-throughput or targeted RNAi screens for germline apoptosis regulators.

Induction of germline apoptosis in Caenorhabditis elegans.

RNA interference (RNAi) is an incredibly powerful tool for rapid and efficient knockdown of gene expression. This technology can be used to induce apoptosis in the germline of Caenorhabditis elegans. Genotoxic stressors such as ionizing radiation (IR), ultraviolet light, chemical mutagens (e.g., N-ethyl-N-nitrosourea [ENU]), and DNA cross-linking reagents can also be used to stimulate apoptosis. These approaches, described here, combined with the powers of in vivo imaging methods, should keep C. elegans apoptosis researchers busy for several years, sorting out how various signaling pathways influence life and death decisions in this organism.

Visualizing apoptosis in embryos and the germline of Caenorhabditis elegans.

Visualizing apoptosis in developing embryos or the germline of Caenorhabditis elegans is remarkably easy because of the transparency of the organism. The invariant pattern of cell division and programmed cell death during development makes it possible to quantify small but reproducible changes in apoptosis, which are easy to detect by light microscopy because of the refractile properties of dying cells. Although apoptotic death is easy to visualize and quantify in the germline of adult hermaphrodites, the pattern of cell death is variable, especially when triggered by stress. The most convenient method for visualization of apoptosis in vivo is light microscopy, which requires immobilizing live embryos or adult animals on slides. This protocol describes the basic methods for visualizing and analyzing apoptosis in living animals.

Methods for detection and analysis of apoptosis signaling in the C. elegans germline.

This review assesses current and emerging methods for the detection, and analysis of apoptosis in the Caenorhabditis elegans germline. The nematode worm C. elegans is highly tractable to genetic manipulation, making it an excellent model for elucidating mechanisms of apoptosis signaling in a multicellular setting. Here we profile the most efficacious fluorescent tools to visualize and quantify germline apoptosis. We focus specifically on the application of fluorescent markers to screen by RNAi for genes and pathways that regulate germline apoptosis under normal conditions or in response to genotoxic stress. We also present the limitations of these methods, and suggest complimentary techniques in order that researchers new to the field can comprehensively assess apoptosis phenotypes in the C. elegans germline.

Metabolic mechanisms for anoxia tolerance and freezing survival in the intertidal gastropod, Littorina littorea.

The gastropod mollusk, Littorina littorea L., is a common inhabitant of the intertidal zone along rocky coastlines of the north Atlantic. This species has well-developed anoxia tolerance and freeze tolerance and is extensively used as a model for exploring the biochemical adaptations that support these tolerances as well as for toxicological studies aimed at identifying effective biomarkers of aquatic pollution. This article highlights our current understanding of the molecular mechanisms involved in anaerobiosis and freezing survival of periwinkles, particularly with respect to anoxia-induced metabolic rate depression. Analysis of foot muscle and hepatopancreas metabolism includes anoxia-responsive changes in enzyme regulation, signal transduction, gene expression, post-transcriptional regulation of mRNA, control of translation, and cytoprotective strategies including chaperones and antioxidant defenses. New studies describe the regulation of glucose-6-phosphate dehydrogenase by reversible protein phosphorylation, the role of microRNAs in suppressing mRNA translation in the hypometabolic state, modulation of glutathione S-transferase isozyme patterns, and the regulation of the unfolded protein response.

Glucose-6-Phosphate Dehydrogenase Regulation in Anoxia Tolerance of the Freshwater Crayfish Orconectes virilis.

Glucose-6-phosphate dehydrogenase (G6PDH), the enzyme which catalyzes the rate determining step of the pentose phosphate pathway (PPP), controls the production of nucleotide precursor molecules (R5P) and powerful reducing molecules (NADPH) that support multiple biosynthetic functions, including antioxidant defense. G6PDH from hepatopancreas of the freshwater crayfish (Orconectes virilis) showed distinct kinetic changes in response to 20 h anoxic exposure. K(m) values for both substrates decreased significantly in anoxic crayfish; K(m) NADP(+) dropped from 0.015 ± 0.008 mM to 0.012 ± 0.008 mM, and K(m) G6P decreased from 0.13 ± 0.02 mM to 0.08 ± 0.007 mM. Two lines of evidence indicate that the mechanism involved is reversible phosphorylation. In vitro incubations that stimulated protein kinase or protein phosphatase action mimicked the effects on anoxia on K(m) values, whereas DEAE-Sephadex chromatography showed the presence of two enzyme forms (low- and high-phosphate) whose proportions changed during anoxia. Incubation studies implicated protein kinase A and G in mediating the anoxia-responsive changes in G6PDH kinetic properties. In addition, the amount of G6PDH protein (measured by immunoblotting) increased by ∼60% in anoxic hepatopancreas. Anoxia-induced phosphorylation of G6PDH could contribute to modifying carbon flow through the PPP under anoxic conditions, potentially maintaining NADPH supply for antioxidant defense during prolonged anoxia-induced hypometabolism.

An overview of stress response and hypometabolic strategies in Caenorhabditis elegans: conserved and contrasting signals with the mammalian system.

Studies of the molecular mechanisms that are involved in stress responses (environmental or physiological) have long been used to make links to disease states in humans. The nematode model organism, Caenorhabditis elegans, undergoes a state of hypometabolism called the 'dauer' stage. This period of developmental arrest is characterized by a significant reduction in metabolic rate, triggered by ambient temperature increase and restricted oxygen/ nutrients. C. elegans employs a number of signal transduction cascades in order to adapt to these unfavourable conditions and survive for long times with severely reduced energy production. The suppression of cellular metabolism, providing energetic homeostasis, is critical to the survival of nematodes through the dauer period. This transition displays molecular mechanisms that are fundamental to control of hypometabolism across the animal kingdom. In general, mammalian systems are highly inelastic to environmental stresses (such as extreme temperatures and low oxygen), however, there is a great deal of conservation between the signal transduction pathways of nematodes and mammals. Along with conserving many of the protein targets in the stress response, many of the critical regulatory mechanisms are maintained, and often differ only in their level of expression. Hence, the C. elegans model outlines a framework of critical molecular mechanisms that may be employed in the future as therapeutic targets for addressing disease states.