The GLI1, GLI2 and GLI3 transcription factors mediate Hedgehog (Hh) signaling, which is crucial for bone development. During intramembranous ossification, mesenchymal stem cells (MSCs) are directly differentiated into osteoblasts. Under basal and Hh pathway-stimulated conditions, primary cilia play essential roles in proteolytic processing of GLI3 to its repressor form (GLI3R) and in activation of GLI2. Although previous studies in mice have suggested that Gli1 expression depends on GLI2 and GLI3, coordinated roles of GLI1, GLI2 and GLI3 in osteogenic differentiation are not fully understood at the cellular level. From the MSC line C3H10T1/2, we established Gli2-knockout (KO) and Gli3-KO cells, as well as constitutively GLI3R-producing (cGLI3R) cells, and expressed GLI1, GLI2 and GLI3 constructs in these cell lines. The results demonstrate at the cellular level that GLI2 and GLI3R counterregulate osteogenic differentiation via activation and repression of Gli1 expression, respectively; GLI3R, which results from GLI3 processing requiring protein kinase A-mediated phosphorylation, downregulates expression of Gli2 as well as Gli1; and GLI1 upregulates expression of Gli1 itself and Gli2, constituting a GLI1-GLI2 positive feedback loop.

Summary: Journal of Cell Science's publisher, The Company of Biologists, is celebrating its 100th birthday in 2025. Here, we review JCS's history since being founded as the Quarterly Journal of Microscopical Sciences.

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Dharma Pally is first author on ‘ The novel ECM protein SNED1 mediates cell adhesion via the RGD-binding integrins α5β1 and αvβ3’, published in JCS. Dharma is a postdoctoral research associate in the lab of Alexandra Naba at University of Illinois Chicago, USA. His research interest in the Naba lab focuses on deciphering the molecular mechanisms by which SNED1 regulates breast cancer cell migration during metastasis and neural crest cell migration during craniofacial development.

Protein liquid-liquid phase separation (LLPS) is driven by intrinsically disordered regions and multivalent binding domains, both of which are common features of diverse microtubule (MT) regulators. Many in vitro studies have dissected the mechanisms by which MT-binding proteins (MBPs) regulate MT nucleation, stabilization and dynamics, and investigated whether LLPS plays a role in these processes. However, more recent in vivo studies have focused on how MBP LLPS affects biological functions throughout neuronal development. Dysregulation of MBP LLPS can lead to formation of aggregates - an underlying feature in many neurodegenerative diseases - such as the tau neurofibrillary tangles present in Alzheimer's disease. In this Review, we highlight progress towards understanding the regulation of MT dynamics through the lens of phase separation of MBPs and associated cytoskeletal regulators, from both in vitro and in vivo studies. We also discuss how LLPS of MBPs regulates neuronal development and maintains homeostasis in mature neurons.

Cells can communicate with neighboring and more distant cells by secretion of extracellular vesicles (EVs). EVs are lipid bilayer membrane-bound structures that can be packaged with proteins, nucleic acids and lipids that mediate cell-cell signaling. EVs are increasingly recognized to play numerous important roles in both normal physiological processes and pathological conditions. Steady progress in the field has uncovered a great diversity and heterogeneity of distinct vesicle types that appear to be secreted from most, if not all, cell types. Recently, it has become apparent that cells also release non-vesicular extracellular nanoparticles (NVEPs), including the newly discovered exomeres and supermeres. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of the diversity of EVs and nanoparticles that are released from cells into the extracellular space, highlighting recent advances in the field.

Biomolecular condensates have recently retained much attention given that they provide a fundamental mechanism of cellular organization. Among those, cytoplasmic ribonucleoprotein (RNP) granules selectively and reversibly concentrate RNA molecules and regulatory proteins, thus contributing to the spatiotemporal regulation of associated RNAs. Extensive in vitro work has unraveled the molecular and chemical bases of RNP granule assembly. The signaling pathways controlling this process in a cellular context are, however, still largely unknown. Here, we aimed at identifying regulators of cytoplasmic RNP granules characterized by the presence of the evolutionarily conserved Imp RNA-binding protein (a homolog of IGF2BP proteins). We performed a high-content image-based RNAi screen targeting all Drosophila genes encoding RNA-binding proteins, phosphatases and kinases. This led to the identification of dozens of genes regulating the number of Imp-positive RNP granules in S2R+ cells, among which were components of the MAPK pathway. Combining functional approaches, phospho-mapping and generation of phospho-variants, we further showed that EGFR signaling inhibits Imp-positive RNP granule assembly through activation of the MAPK-ERK pathway and downstream phosphorylation of Imp at the S15 residue. This work illustrates how signaling pathways can regulate cellular condensate assembly by post-translational modifications of specific components.

ABSTRACTThe Borg (or Cdc42EP) family consists of septin-binding proteins that are known to promote septin-dependent stress fibers and acto-myosin contractility. We show here that epithelial Borg5 (also known as Cdc42EP1) instead limits contractility, cell–cell adhesion tension and motility, as is required for the acquisition of columnar, isotropic cell morphology in mature MDCK monolayers. Borg5 depletion inhibited the development of the lateral F-actin cortex and stimulated microtubule-dependent leading-edge lamellae as well as radial stress fibers and, independently of the basal F-actin phenotype, caused anisotropy of apical surfaces within compacted monolayers. We determined that Borg5 limits colocalization of septin proteins with microtubules, and that like septin 2, Borg5 interacts with the rod-domain of myosin IIA (herein referring to the MYH9 heavy chain). The interaction of myosin IIA with Borg5 was reduced in the presence of septins. Because septins also mediate myosin activation, we propose that Borg5 limits contractility in MDCK cells in part by counteracting septin-associated myosin activity.