Sarcomere formation occurs by the assembly of multiple latent protein complexes.

The stereotyped striation of myofibrils is a conserved feature of muscle organization that is critical to its function. Although most components that constitute the basic myofibrils are well-characterized biochemically and are conserved across the animal kingdom, the mechanisms leading to the precise assembly of sarcomeres, the basic units of myofibrils, are poorly understood. To gain insights into this process, we investigated the functional relationships of sarcomeric protein complexes. Specifically, we systematically analyzed, using either RNAi in primary muscle cells or available genetic mutations, the organization of myofibrils in Drosophila muscles that lack one or more sarcomeric proteins. Our study reveals that the thin and thick filaments are mutually dependent on each other for striation. Further, the tension sensor complex comprised of zipper/Zasp/α-actinin is involved in stabilizing the sarcomere but not in its initial formation. Finally, integrins appear essential for the interdigitation of thin and thick filaments that occurs prior to striation. Thus, sarcomere formation occurs by the coordinated assembly of multiple latent protein complexes, as opposed to sequential assembly.

SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth.

Organization of actin filaments into a well-organized sarcomere structure is critical for muscle development and function. However, it is not completely understood how sarcomeric actin/thin filaments attain their stereotyped lengths. In an RNAi screen in Drosophila primary muscle cells, we identified a gene, sarcomere length short (sals), which encodes an actin-binding, WH2 domain-containing protein, required for proper sarcomere size. When sals is knocked down by RNAi, primary muscles display thin myofibrils with shortened sarcomeres and increased sarcomere number. Both loss- and gain-of-function analyses indicate that SALS may influence sarcomere lengths by promoting thin-filament lengthening from pointed ends. Furthermore, the complex localization of SALS and other sarcomeric proteins in myofibrils reveals that the full length of thin filaments is achieved in a two-step process, and that SALS is required for the second elongation phase, most likely because it antagonizes the pointed-end capping protein Tropomodulin.

Discovery of Orally Bioavailable and Liver-Targeted Hypoxia-Inducible Factor Prolyl Hydroxylase (HIF-PHD) Inhibitors for the Treatment of Anemia.

We report herein the design and synthesis of a series of orally active, liver-targeted hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitors for the treatment of anemia. In order to mitigate the concerns for potential systemic side effects, we pursued liver-targeted HIF-PHD inhibitors relying on uptake via organic anion transporting polypeptides (OATPs). Starting from a systemic HIF-PHD inhibitor (1), medicinal chemistry efforts directed toward reducing permeability and, at the same time, maintaining oral absorption led to the synthesis of an array of structurally diverse hydroxypyridone analogues. Compound 28a was chosen for further profiling, because of its excellent in vitro profile and liver selectivity. This compound significantly increased hemoglobin levels in rats, following chronic QD oral administration, and displayed selectivity over systemic effects.

Phenotypic responses of differentiated asthmatic human airway epithelial cultures to rhinovirus.

OBJECTIVES: Human airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model. METHODS: Air-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively. MAIN RESULTS: ALI cultures were readily infected by HRV. RNA-seq analysis of HRV infected ALI cultures identified sets of genes associated with asthma specific viral responses. These genes are related to inflammatory pathways, epithelial structure and remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1, MUC5AC, CDHR3), and novel ones that were identified for the first time in this study (e.g. CCRL1). CONCLUSIONS: ALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.

Primary cell cultures from Drosophila gastrula embryos.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.

We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.

Border-cell migration requires integration of spatial and temporal signals by the BTB protein Abrupt.

During development, elaborate patterns of cell differentiation and movement must occur in the correct locations and at the proper times. Developmental timing has been studied less than spatial pattern formation, and the mechanisms integrating the two are poorly understood. Border-cell migration in the Drosophila ovary occurs specifically at stage 9. Timing of the migration is regulated by the steroid hormone ecdysone, whereas spatial patterning of the migratory population requires localized activity of the JAK-STAT pathway. Ecdysone signalling is patterned spatially as well as temporally, although the mechanisms are not well understood. In stage 9 egg chambers, ecdysone signalling is highest in anterior follicle cells including the border cells. We identify the gene abrupt as a repressor of ecdysone signalling and border-cell migration. Abrupt protein is normally lost from border-cell nuclei during stage 9, in response to JAK-STAT activity. This contributes to the spatial pattern of the ecdysone response. Abrupt attenuates ecdysone signalling by means of a direct interaction with the basic helix-loop-helix (bHLH) domain of the P160 ecdysone receptor coactivator Taiman (Tai). Taken together, these findings provide a molecular mechanism by which spatial and temporal cues are integrated.

RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance.

To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.

Eyes absent, a key repressor of polar cell fate during Drosophila oogenesis.

Throughout Drosophila oogenesis, specialized somatic follicle cells perform crucial functions in egg chamber formation and in signaling between somatic and germline cells. In the ovary, at least three types of somatic follicle cells, polar cells, stalk cells and main body epithelial follicle cells, can be distinguished when egg chambers bud from the germarium. Although specification of these three somatic cell types is important for normal oogenesis and subsequent embryogenesis, the molecular basis for establishment of their cell fates is not completely understood. Our studies reveal the gene eyes absent (eya) to be a key repressor of polar cell fate. EYA is a nuclear protein that is normally excluded from polar and stalk cells, and the absence of EYA is sufficient to cause epithelial follicle cells to develop as polar cells. Furthermore, ectopic expression of EYA is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration. Finally, we show that ectopic Hedgehog signaling, which is known to cause ectopic polar cell formation, does so by repressing eya expression in epithelial follicle cells.