The regular spacing of somites during vertebrate embryogenesis involves a dynamic gradient of FGF signaling that controls the timing of maturation of cells in the presomitic mesoderm (PSM). How the ...FGF signal is transduced by PSM cells is unclear. Here, we first show that the FGF gradient is translated into graded activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway along the PSM in the chicken embryo. Using in ovo electroporation of PSM cells, we demonstrate that constitutive activation of ERK signaling in the PSM blocks segmentation by preventing maturation of PSM cells, thus phenocopying the overexpression of FGF8. Conversely, inhibition of ERK phosphorylation mimics a loss of function of FGF signaling in the PSM. Interestingly, video microscopy analysis of cell movements shows that ERK regulates the motility of PSM cells, suggesting that the decrease of cell movements along the PSM enables mesenchymal PSM cells to undergo proper segmentation. Together, our data demonstrate that ERK is the effector of the gradient of FGF in the PSM that controls the segmentation process.
Background: Cisplatin (CDDP) is commonly utilized in the treatment of advanced solid tumors including head and neck squamous cell carcinoma (HNSCC). Cisplatin response remains highly variable among ...individual tumors and development of cisplatin resistance is common. We hypothesized that development of cisplatin resistance is partially driven by metabolic reprogramming. Methods: Using a pre-clinical HNSCC model and an integrated approach to steady state metabolomics, metabolic flux and gene expression data we characterized the interaction between cisplatin resistance and metabolic reprogramming. Results: Cisplatin toxicity in HNSCC was driven by generation of intra-cellular oxidative stress. This was validated by demonstrating that acquisition of cisplatin resistance generates cross-resistance to ferroptosis agonists despite the fact that cisplatin itself does not trigger ferroptosis. Acquisition of cisplatin resistance dysregulated the expression of genes involved in amino acid, fatty acid metabolism and central carbon catabolic pathways, enhanced glucose catabolism and serine synthesis. Acute cisplatin exposure increased intra-tumoral levels of S-methyl-5-thiadenosine (MTA) precursors and metabotoxins indicative of generalized oxidative stress. Conclusions: Acquisition of cisplatin resistance is linked to metabolic recovery from oxidative stress. Although this portends poor effectiveness for directed metabolic targeting, it supports the potential for biomarker development of cisplatin effectiveness using an integrated approach.
Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains ...unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells.
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For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.isci.2023.105965.
•The cytoplasm is a two-phase system consisting of viscous fluid and elastic solid•The elastic solid can be separated, from the viscous fluid, as the cytomatrix•The cytomatrix immobilizes catalytic complexes for efficient biocatalysis•Immobilization and segregation of catalytic complexes and proteome can form organelles
Molecular biology; Cell biology; Proteomics; Transcriptomics
Formation and patterning of the vertebrate embryo occur in a head-to-tail sequence. This progressive mode of body formation from the posterior end of the embryo requires a strict temporal ...coordination of tissue differentiation--a process involving fibroblast growth factor (FGF) signalling. Here we show that transcription of fgf8 messenger RNA is restricted to the growing posterior tip of the embryo. fgf8 mRNA is progressively degraded in the newly formed tissues, resulting in the formation of an mRNA gradient in the posterior part of the embryo. This fgf8 mRNA gradient is translated into a gradient of FGF8 protein, which correlates with graded phosphorylation of the kinase Akt, a downstream effector of FGF signalling. Such a mechanism provides an efficient means to monitor the timing of FGF signalling, coupling the differentiation of embryonic tissues to the posterior elongation of the embryo. In addition, this mechanism provides a novel model for morphogen gradient formation.
The rate of mRNA degradation plays an important role in the control of gene expression. The mRNA stability is mainly dependent on
cis-regulatory elements contained in the 3′ or 5′ untranslated region ...(UTR) of the mature mRNAs, and its regulation is an efficient way to adapt the level of a given transcript in the cell. Although this process has been well studied in cell culture, little is known about mRNA stability during embryonic development. Here, we describe an assay that combines the tetracyclin-dependent inducible system Tet-Off with in ovo electroporation to monitor mRNA stability in the chick neural tube. We show, by using the GFP intensity as an indirect reporter system, that the 3′UTR of
Lunatic Fringe strongly destabilizes transcripts, while transcripts bearing the 3′UTR of
Fgf8 are much more stable. This simple assay provides a powerful tool to study mRNA dynamics in vivo.
Cells are subjected to a barrage of daily insults that often lead to their cortices being ripped open and requiring immediate repair. An important component of the cell's repair response is the ...formation of an actomyosin ring at the wound periphery to mediate its closure. Here we show that inhibition of myosin or the linear actin nucleation factors Diaphanous and/or dishevelled associated activator of morphogenesis results in a disrupted contractile apparatus and delayed wound closure. We also show that the branched actin nucleators WASp and SCAR function nonredundantly as scaffolds to assemble and maintain this contractile actomyosin cable. Removing branched actin leads to the formation of smaller circular actin-myosin structures at the cell cortex and to slow wound closure. Removing linear and branched actin simultaneously results in failed wound closure. Surprisingly, removal of branched actin and myosin results in the formation of parallel linear F-actin filaments that undergo a chiral swirling movement to close the wound, uncovering a new mechanism of cell wound closure. Taken together, we demonstrate the roles of different actin substructures that are required for optimal actomyosin ring formation and the extraordinary resilience of the cell to undergo wound repair when it is unable to form different subsets of these substructures.
It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during ...zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mesendodermal cells during zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell movement, suggesting that Toddler is neither an attractant nor a repellent but acts globally as a motogen. Toddler drives internalization of G protein-coupled APJ/Apelin receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes gastrulation movements, and might be the first in a series of uncharacterized developmental signals.
Information flow through neural circuits often requires their organization into topographic maps in which the positions of cell bodies and synaptic targets correspond. To understand how topographic ...map development is controlled, we examine the mechanism underlying targeting of vagus motor axons to the pharyngeal arches in zebrafish. We reveal that retinoic acid organizes topography by specifying anterior-posterior identity in vagus motor neurons. We then show that chemoattractant signaling between Hgf and Met is required for vagus innervation of the pharyngeal arches. Finally, we find that retinoic acid controls the spatiotemporal dynamics of Hgf/Met signaling to coordinate axon targeting with the developmental progression of the pharyngeal arches and show that experimentally altering the timing of Hgf/Met signaling is sufficient to redirect axon targeting and disrupt the topographic map. These findings establish a mechanism of topographic map development in which the regulation of chemoattractant signaling in space and time guides axon targeting.
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•Differential retinoic acid signaling levels specify vagus motor axon target choice•Hgf-Met chemoattractant signaling is required for vagus motor axon guidance•Retinoic acid drives coordinated A-P waves of hgf and met expression•The timing of Hgf-Met signaling determines vagus motor axon target choice
Nervous system function requires that synaptic connections within neural circuits be precisely organized. Here, Isabella et al. examine the development of the intricately organized vagus nerve and find that chemoattractant signaling dynamics are spatiotemporally coordinated between neurons and their target tissues to guide complex axon targeting decisions.
Eukaryotic chromosome segregation requires the kinetochore, a megadalton‐sized machine that forms on specialized centromeric chromatin containing CENP‐A, a histone H3 variant. CENP‐A deposition ...requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP‐A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP‐A can arrive at centromeres without its dedicated centromere‐specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA‐binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP‐A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.
Synopsis
Deposition of centromeric histone H3 variant CENP‐A is essential for kinetochore formation, but has remained difficult to study in vivo. Here, a new method to visualize CENP‐A deposition at single molecule resolution reveals that stable nucleosome formation requires coordination between several protein cofactors and the AT run content of centromeric DNA.
Centromeric histone CENP‐A/Cse4 can associate with centromeric DNA in the absence of its conserved chaperone HJURP, but requires the chaperone for stable deposition.
Physical restriction of centromeric DNA to prevent wrapping prevents stable deposition of the centromeric nucleosome but it does not prevent transient association.
Association of constitutive centromere association network (CCAN) proteins is required for stable centromeric nucleosome formation.
The AT run content of yeast centromeres is correlated to the stability of the centromeric nucleosome.
A new method to visualize CENP‐A deposition at single molecule resolution reveals that stable nucleosome formation requires coordination between several protein cofactors and the AT run content of centromeric DNA.
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