Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity. This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or ß-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

mRNA cycles through hypoxia-induced stress granules in live Drosophila embryonic muscles.

In some myopathies, hypoxia can be the result of pathologic effects like muscle necrosis and abnormal blood flow. At the molecular level, the consequence of hypoxic conditions is not yet fully understood. Under stress conditions, many housekeeping gene mRNAs are translationally silenced, while translation of other mRNAs increases. Alterations to the pool of mRNAs available for translation lead to the formation of so-called stress granules containing both mRNAs and proteins. Stress granule formation and dynamics have been investigated using cells in culture, but have not yet been examined in vivo. In Drosophila embryonic muscles, we found that hypoxia induces the formation of sarcoplasmic granules containing the established stress granule markers RIN and dFMR1. Upon restoration of normoxia, the observed granules were decreased in size, indicating that their formation might be reversible. Employing photobleaching approaches, we found that a cytoplasmic reporter mRNA rapidly shuttles in and out of the granules. Hence, stress granules are highly dynamic complexes and not simple temporary storage sites. Although mRNA rapidly cycles through the granules, its movement throughout the muscle is, remarkably, spatially restricted by the presence of yet undefined myofiber domains. Our results suggest that in hypoxic muscles mRNA remains highly mobile; however, its movement throughout the muscle is restricted by certain boundaries. The development of this Drosophila hypoxia model makes it possible to study the formation and dynamics of stress granules and their associated mRNAs and proteins in a living organism.

In vivo monitoring of mRNA movement in Drosophila body wall muscle cells reveals the presence of myofiber domains.

BACKGROUND: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. METHODOLOGY/PRINCIPAL FINDINGS: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. CONCLUSIONS/SIGNIFICANCE: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cell-based therapies.