CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism

The tetraspanin CD53 protects stressed hematopoietic stem cells via promotion of DREAM complex-mediated quiescence

The hematopoietic stem cell (HSC) cycle responds to inflammatory and other proliferative stressors; however, these cells must quickly return to quiescence to avoid exhaustion and maintain their functional integrity. The mechanisms that regulate this return to quiescence are not well understood. Here, we show that tetraspanin CD53 is markedly upregulated in HSCs in response to a variety of inflammatory and proliferative stimuli and that the loss of CD53 is associated with prolonged cycling and reduced HSC function in the context of inflammatory stress. Mechanistically, CD53 promotes the activity of the dimerization partner, RB-like, E2F, and multi-vulva class B (DREAM) transcriptional repressor complex, which downregulates genes associated with cycling and division. Proximity labeling and confocal fluorescence microscopy studies showed that CD53 interacts with DREAM-associated proteins, specifically promoting the interaction between Rbl2/p130 and its phosphatase protein phosphatase 2A (PP2A), effectively stabilizing p130 protein availability for DREAM binding. Together, these data identified a novel mechanism by which stressed HSCs resist cycling

Staphylococcus aureus α-toxin impairs early neutrophil localization via electrogenic disruption of store-operated calcium entry

The pore-forming S. aureus α-toxin (Hla) contributes to virulence and disease pathogenesis. While high concentrations of toxin induce cell death, neutrophils exhibit relative resistance to lysis, suggesting that the action of Hla may not be solely conferred by lytic susceptibility. Using intravital microscopy, we observed that Hla disrupts neutrophil localization and clustering early in infection. Hla forms a narrow, ion-selective pore, suggesting that Hla may dysregulate calcium or other ions to impair neutrophil function. We found that sub-lytic Hla did not permit calcium influx but caused rapid membrane depolarization. Depolarization decreases the electrogenic driving force for calcium, and concordantly, Hla suppressed calcium signaling in vitro and in vivo and calcium-dependent leukotriene B4 (LTB4) production, a key mediator of neutrophil clustering. Thus, Hla disrupts the early patterning of the neutrophil response to infection, in part through direct impairment of neutrophil calcium signaling. This early mis-localization of neutrophils may contribute to establishment of infection

A Small Conductance Calcium-Activated K⁺ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al

SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers

Eco-evolutionary dynamics on deformable fitness landscapes

Conventional approaches to modelling ecological dynamics often do not include evolutionary changes in the genetic makeup of component species and, conversely, conventional approaches to modelling evolutionary changes in the genetic makeup of a population often do not include ecological dynamics. But recently there has been considerable interest in understanding the interaction of evolutionary and ecological dynamics as coupled processes. However, in the context of complex multi-species ecosytems, especially where ecological and evolutionary timescales are similar, it is difficult to identify general organising principles that help us understand the structure and behaviour of complex ecosystems. Here we introduce a simple abstraction of coevolutionary interactions in a multi-species ecosystem. We model non-trophic ecological interactions based on a continuous but low-dimensional trait/niche space, where the location of each species in trait space affects the overlap of its resource utilisation with that of other species. The local depletion of available resources creates, in effect, a deformable fitness landscape that governs how the evolution of one species affects the selective pressures on other species. This enables us to study the coevolution of ecological interactions in an intuitive and easily visualisable manner. We observe that this model can exhibit either of the two behavioural modes discussed in the literature; namely, evolutionary stasis or Red Queen dynamics, i.e., continued evolutionary change. We find that which of these modes is observed depends on the lag or latency between the movement of a species in trait space and its effect on available resources. Specifically, if ecological change is nearly instantaneous compared to evolutionary change, stasis results; but conversely, if evolutionary timescales are closer to ecological timescales, such that resource depletion is not instantaneous on evolutionary timescales, then Red Queen dynamics result. We also observe that in the stasis mode, the overall utilisation of resources by the ecosystem is relatively efficient, with diverse species utilising different niches, whereas in the Red Queen mode the organisation of the ecosystem is such that species tend to clump together competing for overlapping resources. These models thereby suggest some basic conditions that influence the organisation of inter-species interactions and the balance of individual and collective adaptation in ecosystems, and likewise they also suggest factors that might be useful in engineering artificial coevolution

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease