ABSTRACT Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G-protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the medioapical region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data support a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.
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From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway
The morphogenetic process of apical constriction, which relies on non-muscle myosin II (NMII) generated constriction of apical domains of epithelial cells, is key to the development of complex cellular patterns. Apical constriction occurs in almost all multicellular organisms, but one of the most well-characterized systems is the Folded-gastrulation (Fog)-induced apical constriction that occurs in
- Award ID(s):
- 1716964
- NSF-PAR ID:
- 10468559
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- BMC Molecular and Cell Biology
- Volume:
- 24
- Issue:
- 1
- ISSN:
- 2661-8850
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Fehon, Richard (Ed.)To identify novel regulators of nonmuscle myosin II (NMII) we performed an image-based RNA interference screen using stable Drosophila melanogaster S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. We identified the Rab-specific GTPase-activating protein (GAP) RN-tre as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, our results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility.more » « less
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SUMMARY Extracellular ATP (eATP) is a key signaling molecule that plays a pivotal role in plant growth and defense responses. The receptor P2K1 is responsible for perceiving eATP and initiating its signaling cascade. However, the signal transduction mechanisms downstream of P2K1 activation remain incompletely understood. We conducted a comprehensive analysis of the P2K1 interactome using co‐immunoprecipitation‐coupled tandem mass spectrometry, leading to the identification of 121 candidate proteins interacting with P2K1. In silico analysis narrowed down the candidates to 47 proteins, including Ca2+‐binding proteins, ion transport‐related proteins, and receptor kinases. To investigate their involvement in eATP signaling, we employed a screening strategy based on changes in gene expression in response to eATP in mutants of the identified interactors. This screening revealed several Ca2+‐dependent protein kinases (CPKs) that significantly affected the expression of eATP‐responsive genes, suggesting their potential roles in eATP signaling. Notably, CPK28 and CPK6 showed physical interactions with P2K1 both in yeast and plant systems. Calcium influx and gene expression studies demonstrated that CPK28 perturbed eATP‐induced Ca2+mobilization and some early transcriptional responses. Overexpression of CPK28 resulted in an antagonistic physiological response to P2K1‐mediated eATP signaling during both plant growth and defense responses to the necrotrophic pathogen
Botrytis cinerea . Our findings highlight CPK28, among other CPKs, as a modulator of P2K1‐mediated eATP signaling, providing valuable insights into the coordination of eATP signaling in plant growth and immunity. -
Over the past three-decades, Janus kinase (Jak) and signal transducer and activator of transcription (STAT) signaling has emerged as a paradigm to understand the involvement of signal transduction in development and disease pathology. At the molecular level, cytokines and interleukins steer Jak/STAT signaling to transcriptional regulation of target genes, which are involved in cell differentiation, migration, and proliferation. Jak/STAT signaling is involved in various types of blood cell disorders and cancers in humans, and its activation is associated with carcinomas that are more invasive or likely to become metastatic. Despite immense information regarding Jak/STAT regulation, the signaling network has numerous missing links, which is slowing the progress towards developing drug therapies. In mammals, many components act in this cascade, with substantial cross-talk with other signaling pathways. In Drosophila, there are fewer pathway components, which has enabled significant discoveries regarding well-conserved regulatory mechanisms. Work across species illustrates the relevance of these regulators in humans. In this review, we showcase fundamental Jak/STAT regulation mechanisms in blood cells, stem cells, and cell motility. We examine the functional relevance of key conserved regulators from Drosophila to human cancer stem cells and metastasis. Finally, we spotlight less characterized regulators of Drosophila Jak/STAT signaling, which stand as promising candidates to be investigated in cancer biology. These comparisons illustrate the value of using Drosophila as a model for uncovering the roles of Jak/STAT signaling and the molecular means by which the pathway is controlled.more » « less
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Abstract Background Collective cell migration underlies many essential processes, including sculpting organs during embryogenesis, wound healing in the adult, and metastasis of cancer cells. At mid-oogenesis,
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