Acting on identity: Myoblast fusion and the formation of the syncytial muscle fiber.

The study of Drosophila muscle development dates back to the middle of the last century. Since that time, Drosophila has proved to be an ideal system for studying muscle development, differentiation, function, and disease. As in humans, Drosophila muscle forms via a series of conserved steps, starting with muscle specification, myoblast fusion, attachment to tendon cells, interactions with motorneurons, and sarcomere and myofibril formation. The genes and mechanisms required for these processes share striking similarities to those found in humans. The highly tractable genetic system and imaging approaches available in Drosophila allow for an efficient interrogation of muscle biology and for application of what we learn to other systems. In this article, we review our current understanding of muscle development in Drosophila, with a focus on myoblast fusion, the process responsible for the generation of syncytial muscle cells. We also compare and contrast those genes required for fusion in Drosophila and vertebrates.

Gastric Cancer Extracellular Vesicles Tune the Migration and Invasion of Epithelial and Mesenchymal Cells in a Histotype-Dependent Manner.

Extracellular vesicles (EVs) secreted by tumor cells modulate recipient cells' behavior, but their effects in normal cells from the tumor microenvironment remain poorly known. In this study, we dissected the functional impact of gastric cancer cell-derived EVs (GC-EVs), representative of distinct GC histotypes, on the behavior of normal isogenic epithelial and mesenchymal cells. GC-EVs were isolated by differential centrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and imaging flow-cytometry. Epithelial and mesenchymal cells were challenged with GC-EVs and submitted to proliferation, migration, and invasion assays. Expression of epithelial and mesenchymal markers was followed by immunofluorescence and flow-cytometry. Our results indicated that GC-EVs secreted by diffuse-type cancer cells decrease the migration of recipient cells. This effect was more prominent and persistent for mesenchymal recipient cells, which also increased Fibronectin expression in response to EVs. GC-EVs secreted by cancer cells derived from tumors with an intestinal component increased invasion of recipient epithelial cells, without changes in EMT markers. In summary, this study demonstrated that GC-EVs modulate the migration and invasion of epithelial and mesenchymal cells from the tumor microenvironment, in a histotype-dependent manner, highlighting new features of intestinal and diffuse-type GC cells, which may help explaining differential metastasis patterns and aggressiveness of GC histotypes.

Codon misreading tRNAs promote tumor growth in mice.

Deregulation of tRNAs, aminoacyl-tRNA synthetases and tRNA modifying enzymes are common in cancer, raising the hypothesis that protein synthesis efficiency and accuracy (mistranslation) are compromised in tumors. We show here that human colon tumors and xenograft tumors produced in mice by two epithelial cancer cell lines mistranslate 2- to 4-fold more frequently than normal tissue. To clarify if protein mistranslation plays a role in tumor biology, we expressed mutant Ser-tRNAs that misincorporate Ser-at-Ala (frequent error) and Ser-at-Leu (infrequent error) in NIH3T3 cells and investigated how they responded to the proteome instability generated by the amino acid misincorporations. There was high tolerance to both misreading tRNAs, but the Ser-to-Ala misreading tRNA was a more potent inducer of cell transformation, stimulated angiogenesis and produced faster growing tumors in mice than the Ser-to-Leu misincorporating tRNA. Upregulation of the Akt pathway and the UPR were also observed. Most surprisingly, the relative expression of both misreading tRNAs increased during tumor growth, suggesting that protein mistranslation is advantageous in cancer contexts. These data highlight new features of protein synthesis deregulation in tumor biology.

Epithelial-Mesenchymal Plasticity Induced by Discontinuous Exposure to TGFß1 Promotes Tumour Growth.

Transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression. We hypothesize that EMT followed by MET promotes cell population heterogeneity, favouring tumour growth. We developed an EMT model by on and off exposure of epithelial EpH4 cells (E-cells) to TGFß1 that mimics phenotypic EMT (M-cells) and MET. We aimed at understanding whether phenotypic MET is accompanied by molecular and functional reversion back to epithelia by using RNA sequencing, immunofluorescence (IF), proliferation, wound healing, focus formation and mamosphere formation assays as well as cell xenografts in nude mice. Phenotypic reverted epithelial cells (RE-cells) obtained after MET induction presented epithelial morphologies and proliferation rates resembling E cells. However, the RE transcriptomic profile and IF staining of epithelial and mesenchymal markers revealed a uniquely heterogeneous mixture of cell subpopulations with a high self-renewal ability. RE cell heterogeneity was stably maintained for long periods after TGFß1 removal both in vitro and in large tumours derived from the nude mice. Overall, we show that phenotypic reverted epithelial cells (RE cells) do not return to the molecular and functional epithelial state and present mesenchymal features related to aggressiveness and cellular heterogeneity that favour tumour growth in vivo. This work strengthens epithelial cell reprogramming and cellular heterogeneity fostered by inflammatory cues as a tumour growth-promoting factor in vivo.

Getting into Position: Nuclear Movement in Muscle Cells.

The positioning of nuclei within the cell is a dynamic process that depends on the cell's fate and developmental stage and that is adjusted for optimal cell function. This is especially true in skeletal muscle cells, which contain hundreds of myonuclei distributed evenly along the periphery of the muscle cell. Mispositioned myonuclei are often associated with muscle dysfunction and disease. Different mechanisms governing myonuclear positioning are now emerging, with several of the new genes implicated in nuclear movement linked to human muscle disease. Here we discuss the recent advances in myonuclear positioning and its implications for muscle size and function from the view of Drosophila. Additionally, we highlight similarities and differences to mammalian systems and provide connections to human muscle disease.

Reverse-engineering forces responsible for dynamic clustering and spreading of multiple nuclei in developing muscle cells.

How cells position their organelles is a fundamental biological question. During Drosophila embryonic muscle development, multiple nuclei transition from being clustered together to splitting into two smaller clusters to spreading along the myotube's length. Perturbations of microtubules and motor proteins disrupt this sequence of events. These perturbations do not allow intuiting which molecular forces govern the nuclear positioning; we therefore used computational screening to reverse-engineer and identify these forces. The screen reveals three models. Two suggest that the initial clustering is due to nuclear repulsion from the cell poles, while the third, most robust, model poses that this clustering is due to a short-ranged internuclear attraction. All three models suggest that the nuclear spreading is due to long-ranged internuclear repulsion. We test the robust model quantitatively by comparing it with data from perturbed muscle cells. We also test the model using agent-based simulations with elastic dynamic microtubules and molecular motors. The model predicts that, in longer mammalian myotubes with a large number of nuclei, the spreading stage would be preceded by segregation of the nuclei into a large number of clusters, proportional to the myotube length, with a small average number of nuclei per cluster.

The Drosophila Ninein homologue Bsg25D cooperates with Ensconsin in myonuclear positioning.

Skeletal muscle consists of multinucleated cells in which the myonuclei are evenly spaced throughout the cell. In Drosophila, this pattern is established in embryonic myotubes, where myonuclei move via microtubules (MTs) and the MT-associated protein Ensconsin (Ens)/MAP7, to achieve their distribution. Ens regulates multiple aspects of MT biology, but little is known about how Ens itself is regulated. We find that Ens physically interacts and colocalizes with Bsg25D, the Drosophila homologue of the centrosomal protein Ninein. Bsg25D loss enhances myonuclear positioning defects in embryos sensitized by partial Ens loss. Bsg25D overexpression causes severe positioning defects in immature myotubes and fully differentiated myofibers, where it forms ectopic MT organizing centers, disrupts perinuclear MT arrays, reduces muscle stiffness, and decreases larval crawling velocity. These studies define a novel relationship between Ens and Bsg25D. At endogenous levels, Bsg25D positively regulates Ens activity during myonuclear positioning, but excess Bsg25D disrupts Ens localization and MT organization, with disastrous consequences for myonuclear positioning and muscle function.

Imaging Approaches to Investigate Myonuclear Positioning in Drosophila.

In the skeletal muscle, nuclei are positioned at the periphery of each myofiber and are evenly distributed along its length. Improper positioning of myonuclei has been correlated with muscle disease and decreased muscle function. Several mechanisms required for regulating nuclear position have been identified using the fruit fly, Drosophila melanogaster. The conservation of the myofiber between the fly and vertebrates, the availability of advanced genetic tools, and the ability to visualize dynamic processes using fluorescent proteins in vivo makes the fly an excellent system to study myonuclear positioning. This chapter describes time-lapse and fixed imaging methodologies using both the Drosophila embryo and the larva to investigate mechanisms of myonuclear positioning.

Dies1/VISTA expression loss is a recurrent event in gastric cancer due to epigenetic regulation.

Dies1/VISTA induces embryonic stem-cell differentiation, via BMP-pathway, but also acts as inflammation regulator and immune-response modulator. Dies1 inhibition in a melanoma-mouse model led to increased tumour-infiltrating T-cells and decreased tumour growth, emphasizing Dies1 relevance in tumour-microenvironment. Dies1 is involved in cell de/differentiation, inflammation and cancer processes, which mimic those associated with Epithelial-to-Mesenchymal-Transition (EMT). Despite this axis linking Dies1 with EMT and cancer, its expression, modulation and relevance in these contexts is unknown. To address this, we analysed Dies1 expression, its regulation by promoter-methylation and miR-125a-5p overexpression, and its association with BMP-pathway downstream-effectors, in a TGFß1-induced EMT-model, cancer cell-lines and primary samples. We detected promoter-methylation as a mechanism controlling Dies1 expression in our EMT-model and in several cancer cell-lines. We showed that the relationship between Dies1 expression and BMP-pathway effectors observed in the EMT-model, was not present in all cell-lines, suggesting that Dies1 has other cell-specific effectors, beyond the BMP-pathway. We further demonstrated that: Dies1 expression loss is a recurrent event in GC, caused by promoter methylation and/or miR-125a-5p overexpression and; GC-microenvironment myofibroblasts overexpress Dies1. Our findings highlight Dies1 as a novel player in GC, with distinct roles within tumour cells and in the tumour-microenvironment.