CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present C. elegans light-induced co-clustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Co-localization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.

Visual detection of binary, ternary, and quaternary protein interactions in fission yeast by Pil1 co-tethering assay

Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein's function. We present C. elegans light-induced co-clustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Co-localization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.

Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay

Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins, transmembrane proteins, and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

High-lipid prey reduce juvenile survivorship and delay egg laying in a small linyphiid spider Hylyphantes graminicola

ABSTRACTPrey proteins and lipids greatly impact predator life-history traits. However, life-history plasticity offers predators the opportunity to tune the life-history traits in response to the limited macronutrients to allocate among traits. A fast-growing predator species with a strict maturation time may be more likely to consume nutritionally imbalanced prey. Here, we tested this hypothesis by examining the effect of the protein-to-lipid ratio in prey on a small sheet web-building spider, Hylyphantes graminicola, with a short life span, using adult Drosophila melanogaster as the prey. By manipulating the macronutrient content of the prey to generate three prey types with different protein-to-lipid ratios (i.e. high, intermediate and low), we demonstrated that the majority of the spiders that consumed only these flies could reach full maturity. However, juvenile spiders that consumed high-lipid (low protein-to-lipid ratio) flies had a higher rate of mortality than those consuming medium-protein and high-protein flies. The prey protein-to-lipid ratio had no significant effects on the developmental duration and size at maturity. Although the prey protein-to-lipid ratio had no significant influence on mating behaviour and female fecundity, females reared on high-lipid flies exhibited a significant delay in oviposition compared with those reared on high-protein flies. We conclude that high-lipid prey has negative effects on the survival and reproductive function of H. graminicola. Our study thus provides clear evidence that low plasticity with fast development to a certain size means a high nutritional requirement for protein at a cost of lower survival and prolonged time to egg laying when prey have low protein-to-lipid content in H. graminicola.

Highly Modular Protein Micropatterning Sheds Light on the Role of Clathrin-Mediated Endocytosis for the Quantitative Analysis of Protein-Protein Interactions in Live Cells

Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g., the receptor) in regular micropatterns within the plasma membrane and the monitoring of the lateral distribution of the bait’s interaction partner (the prey, e.g., the cytosolic downstream molecule) enables the in-depth examination of protein-protein interactions in a live cell context. This study reports on potential pitfalls and difficulties in data interpretation based on the enrichment of clathrin, which is a protein essential for clathrin-mediated receptor endocytosis. Using a highly modular micropatterning approach based on large-area micro-contact printing and streptavidin-biotin-mediated surface functionalization, clathrin was found to form internalization hotspots within the patterned areas, which, potentially, leads to unspecific bait/prey protein co-recruitment. We discuss the consequences of clathrin-coated pit formation on the quantitative analysis of relevant protein-protein interactions, describe controls and strategies to prevent the misinterpretation of data, and show that the use of DNA-based linker systems can lead to the improvement of the technical platform.

Amplification of Snake Venom Toxicity by Endogenous Signaling Pathways

The active components of snake venoms encompass a complex and variable mixture of proteins that produce a diverse, but largely stereotypical, range of pharmacologic effects and toxicities. Venom protein diversity and host susceptibilities determine the relative contributions of five main pathologies: neuromuscular dysfunction, inflammation, coagulopathy, cell/organ injury, and disruption of homeostatic mechanisms of normal physiology. In this review, we describe how snakebite is not only a condition mediated directly by venom, but by the amplification of signals dysregulating inflammation, coagulation, neurotransmission, and cell survival. Although venom proteins are diverse, the majority of important pathologic events following envenoming follow from a small group of enzyme-like activities and the actions of small toxic peptides. This review focuses on two of the most important enzymatic activities: snake venom phospholipases (svPLA2) and snake venom metalloproteases (svMP). These two enzyme classes are adept at enabling venom to recruit homologous endogenous signaling systems with sufficient magnitude and duration to produce and amplify cell injury beyond what would be expected from the direct impact of a whole venom dose. This magnification produces many of the most acutely important consequences of envenoming as well as chronic sequelae. Snake venom PLA2s and MPs enzymes recruit prey analogs of similar activity. The transduction mechanisms that recruit endogenous responses include arachidonic acid, intracellular calcium, cytokines, bioactive peptides, and possibly dimerization of venom and prey protein homologs. Despite years of investigation, the precise mechanism of svPLA2-induced neuromuscular paralysis remains incomplete. Based on recent studies, paralysis results from a self-amplifying cycle of endogenous PLA2 activation, arachidonic acid, increases in intracellular Ca2+ and nicotinic receptor deactivation. When prolonged, synaptic suppression supports the degeneration of the synapse. Interaction between endothelium-damaging MPs, sPLA2s and hyaluronidases enhance venom spread, accentuating venom-induced neurotoxicity, inflammation, coagulopathy and tissue injury. Improving snakebite treatment requires new tools to understand direct and indirect effects of envenoming. Homologous PLA2 and MP activities in both venoms and prey/snakebite victim provide molecular targets for non-antibody, small molecule agents for dissecting mechanisms of venom toxicity. Importantly, these tools enable the separation of venom-specific and prey-specific pathological responses to venom.

Predatory dipteran larva contributes to nutrient sequestration in a carnivorous pitcher plant

The fluids of Nepenthes pitcher plants are habitats to many specialized animals known as inquilines, which facilitate the conversion of prey protein into pitcher-absorbable nitrogen forms such as ammonium. Xenoplatyura beaveri (Diptera: Mycetophilidae) is a predatory dipteran inquiline that inhabits the pitchers of Nepenthes ampullaria . Larvae of X. beaveri construct sticky webs over the fluid surface of N. ampullaria to ensnare emerging adult dipteran inquilines. However, the interaction between X. beaveri and its host has never been examined before, and it is not known if X. beaveri can contribute to nutrient sequestration in N. ampullaria. Xenoplatyura beaveri individuals were reared in artificial pitchers in the laboratory on a diet of emergent Tripteroides tenax mosquitoes, and the ammonium concentration of the pitcher fluids was measured over time. Fluid ammonium concentration in tubes containing X. beaveri was significantly greater than those of the controls. Furthermore, fluid ammonium concentrations increased greatly after X. beaveri larvae metamorphosed, although the cause of this increase could not be identified. Our results show that a terrestrial, inquiline predator can contribute significantly to nutrient sequestration in the phytotelma it inhabits, and suggest that this interaction has a net mutualistic outcome for both species.

Dipteran larvae and microbes facilitate nutrient sequestration in the Nepenthes gracilis pitcher plant host

The fluid-containing traps of Nepenthes carnivorous pitcher plants (Nepenthaceae) are often inhabited by organisms known as inquilines. Dipteran larvae are key components of such communities and are thought to facilitate pitcher nitrogen sequestration by converting prey protein into inorganic nitrogen, although this has never been demonstrated in Nepenthes . Pitcher fluids are also inhabited by microbes, although the relationship(s) between these and the plant is still unclear. In this study, we examined the hypothesis of digestive mutualism between N. gracilis pitchers and both dipteran larvae and fluid microbes. Using dipteran larvae, prey and fluid volumes mimicking in situ pitcher conditions, we conducted in vitro experiments and measured changes in available fluid nitrogen in response to dipteran larvae and microbe presence. We showed that the presence of dipteran larvae resulted in significantly higher and faster releases of ammonium and soluble protein into fluids in artificial pitchers, and that the presence of fluid microbes did likewise for ammonium. We showed also that niche segregation occurs between phorid and culicid larvae, with the former fragmenting prey carcasses and the latter suppressing fluid microbe levels. These results clarify the relationships between several key pitcher-dwelling organisms, and show that pitcher communities facilitate nutrient sequestration in their host.

Feeding preference of adult females of ribbonfish Trichiurus lepturus through prey proximate-composition and caloric values

In the present study we analysed the proximate-composition and caloric values of the preferred prey consumed by ribbonfish, Trichiurus lepturus L. 1758 (adult females), that are distributed in the inner continental shelf from northern Rio de Janeiro State, southeastern Brazil (~22ºS), assessing the potential of nutritional and energetic approach as a tool to understand the feeding selective pattern of this marine top carnivore. The preferred prey of this predator composed of fish co-specifics, Pellona harroweri, Chirocentrodon bleekerianus, Lycengraulis grossidens, Peprilus paru, squid Doryteuthis plei, and shrimp Xiphopenaeus kroyeri were collected from 2007 to 2010 for proximate-composition (water, protein, lipid, ash, and carbohydrate) and caloric value analyses. The correspondence analysis showed that protein is the main component in the prey species (61.32% of variance explained), standing out from the other nutrients. Lipid has the highest percentage related to L. grossidens, ash to X. kroyeri and carbohydrate to D. plei. The strong correlations between protein and caloric value (positive) and lipid and caloric value (negative) indicated that T. lepturus is attending its energy demand through the prey protein content. This work elucidated the feeding preference of adult females of T. lepturus in relation to nutritional and caloric content of their preferred prey. The species showed food selectivity to prey that provide more energy per ingested biomass, so that the feeding events can maximize the predator's caloric gain, which is obtained by a protein-based diet.