Wnt-Notch signalling: an integrated mechanism regulating transitions between cell states

The activity of Wnt and Notch signalling is central to many cell fate decisions during development and to the maintenance and differentiation of stem cell populations in homeostasis. While classical views refer to these pathways as independent signal transduction devices that co-operate in different systems, recent work has revealed intricate connections between their components. These observations suggest that rather than operating as two separate pathways, elements of Wnt and Notch signalling configure an integrated molecular device whose main function is to regulate transitions between cell states in development and homeostasis. Here, we propose a general framework for the structure and function of the interactions between these signalling systems that is focused on the notion of 'transition states', i.e. intermediates that arise during cell fate decision processes. These intermediates act as checkpoints in cell fate decision processes and are characterised by the mixed molecular identities of the states involved in these processes.     

Phase Separation in Biology and Disease; Current Perspectives and Open Questions

In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1, 2, 3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4, 5, 6, 7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, ～25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.     

Biomolecular condensates in neurodegeneration and cancer

The intracellular environment is partitioned into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates, also referred to as membrane‐less organelles, are diverse and abundant cellular compartments that lack membranous enclosures. Molecules assemble into condensates by phase separation; multivalent weak interactions drive molecules to separate from their surroundings and concentrate in discrete locations. Biomolecular condensates exist in all eukaryotes and in some prokaryotes, and participate in various essential house‐keeping, stress‐response and cell type‐specific processes. An increasing number of recent studies link abnormal condensate formation, composition and material properties to a number of disease states. In this review, we discuss current knowledge and models describing the regulation of condensates and how they become dysregulated in neurodegeneration and cancer. Further research on the regulation of biomolecular phase separation will help us to better understand their role in cell physiology and disease.     

Liquid-liquid phase separation as a common organizing principle of intracellular space and biomembranes providing dynamic adaptive responses

This work is devoted to the phenomenon of liquid-liquid phase separation (LLPS), which has come to be recognized as fundamental organizing principle of living cells. We distinguish separation processes with different dimensions. Well-known 3D-condensation occurs in aqueous solution and leads to membraneless organelle (MLOs) formation. 2D-films may be formed near membrane surfaces and lateral phase separation (membrane rafts) occurs within the membranes themselves. LLPS may also occur on 1D structures like DNA and the cyto- and nucleoskeleton. Phase separation provides efficient transport and sorting of proteins and metabolites, accelerates the assembly of metabolic and signaling complexes, and mediates stress responses. In this work, we propose a model in which the processes of polymerization (1D structures), phase separation in membranes (2D structures), and LLPS in the volume (3D structures) influence each other. Disordered proteins and whole condensates may provide membrane raft separation or polymerization of specific proteins. On the other hand, 1D and 2D structures with special composition or embedded IDRs can nucleate condensates. We hypothesized that environmental change may trigger a LLPS which can propagate within the cell interior moving along the cytoskeleton or as an autowave. New phase propagation quickly and using a low amount of energy adjusts cell signaling and metabolic systems to new demands. Cumulatively, the interconnected phase separation phenomena in different dimensions represent a previously unexplored system of intracellular communication and regulation which cannot be ignored when considering both physiological and pathological cell processes.     

Unravelling the evolution of neural stem cells in arthropods: Notch signalling in neural stem cell development in the crustacean Daphnia magna

The genetic regulatory networks controlling major developmental processes seem to be conserved in bilaterians regardless of an independent or a common origin of the structures. This has been explained by the employment of a genetic toolkit that was repeatedly used during bilaterian evolution to build the various forms and body plans. However, it is not clear how genetic networks were incorporated into the formation of novel structures and how homologous genes can regulate the disparate morphological processes. Here we address this question by analysing the role of Notch signalling, which is part of the bilaterian toolkit, in neural stem cell evolution in arthropods. Within arthropods neural stem cells have evolved in the last common ancestor of insects and crustaceans (Tetraconata). We analyse here for the first time the role of Notch signalling in a crustacean, the branchiopod Daphnia magna, and show that it is required in neural stem cells for regulating the time of neural precursor production and for binary cell fate decisions in the ventral neuroectoderm. The function of Notch signalling has diverged in the ventral neuroectoderm of insects and crustaceans accompanied by changes in the morphogenetic processes. In the crustacean, Notch controlled mechanisms of neuroblast regulation have evolved that are surprisingly similar to vertebrates and thus present a remarkable case of parallel evolution. These new data on a representative of crustaceans complete the arthropod data set on Notch signalling in the nervous system and allow for reconstructing how the Notch signalling pathway has been co-opted from pre-existing structures to the development of the evolving neural stem cells in the Tetraconata ancestor.     

WNT signalling in the immune system: WNT is spreading its wings

WNT proteins are secreted morphogens that are required for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. In blood and immune cells, WNT signalling controls the proliferation of progenitor cells and might also affect the cell-fate decisions of stem cells. Recent studies indicate that WNT proteins also regulate effector T-cell development, regulatory T-cell activation and dendritic-cell maturation. WNT signalling seems to function as a universal mechanism in leukocytes to establish a pool of undifferentiated cells for further selection, effector-cell maturation and terminal differentiation. WNT signalling is therefore subject to strict molecular control, and dysregulated WNT signalling is implicated in the development of haematological malignancies.     

Signal transduction by tumor necrosis factor receptors

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.     

TRAIL signalling: decisions between life and death

The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumour cells. This property has made TRAIL and agonistic antibodies against its death inducing receptors (TRAIL-R1 and TRAIL-R2) to some of the most promising novel biotherapeutic agents for cancer therapy. Here we review the signalling pathways initiated by the apoptosis- as well as the non-apoptosis-inducing receptors, TRAIL-R3 and TRAIL-R4. The TRAIL "death-inducing signalling complex" (DISC) transmits the apoptotic signal. DISC formation leads to activation of a protease cascade, finally resulting in cell death. The TRAIL death receptor-mediated "extrinsic" pathway and the "intrinsic" pathway, which is controlled by the interaction of members of the Bcl-2 family, interact with each other in the decision about life or death of a cell. Apoptotic and non-apoptotic signalling is influenced by the NF-kappaB, PKB/Akt and the MAPK signalling pathways. In this review we intend to summarise the most important findings on the TRAIL signalling network and the interplay in the decisions between life and death of a tumor cell.     

A possible role for the canonical Wnt pathway in endocrine cell development in chicks

Wnt signalling is involved in many developmental processes such as proliferation, differentiation, cell fate decisions, and morphogenesis. However, little is known about Wnt signalling during pancreas development. Multiple Wnt ligands and Frizzled receptors are expressed in the embryonic mouse pancreas, the surrounding mesenchyme, and have also been detected in the chicken endoderm during development. The aim of this study was to investigate the role of canonical Wnt signalling on endocrine cell development by use of the in ovo electroporation of the chicken endoderm. Overexpression with a constitutive active form of beta-catenin in combination with Ngn3 resulted in reduced numbers of glucagon cells. dnLEF-1 or naked-1 did not alter endocrine cell differentiation when co-expressed with Ngn3, but dnLEF-1 appeared to have some potential for inhibiting delamination of Ngn3 cells. In addition, neuronal beta-III-tubulin, which had previously been considered a specific marker for neuronal cells, was observed in the pancreas and was upregulated in the electroporated Ngn3 cells and thus may be a new endocrine marker in the chicken.     

A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signalling Pathways during Early Xenopus Embryogenesis

Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions.     

WNTs in the vertebrate nervous system: from patterning to neuronal connectivity

WNT signalling has a key role in early embryonic patterning through the regulation of cell fate decisions, tissue polarity and cell movements. In the nervous system, WNT signalling also regulates neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation. Studies, from invertebrates to mammals, have led to a considerable understanding of WNT signal transduction pathways. This knowledge provides a framework for the study of the mechanisms by which WNTs regulate diverse neuronal functions. Manipulation of the WNT pathways could provide new strategies for nerve regeneration and neuronal circuit modulation.     

The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has emerged as a pervasive principle in cell biology, allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiological conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dilute aqueous solution. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liquid-liquid phase-separated droplets to provide experimental evidence for the key role of the solvent as a thermodynamic driving force. The FUS concentration inside LLPS droplets was determined to be increased to 2.0 mM independent of the initial protein concentration (5 or 10 μM solutions) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-separated FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodynamic driving force in phase separation.     

Phosphoinositide 3‐kinases in cell migration

Cell migration is essential for many biological processes in animals and is a complex highly co‐ordinated process that involves cell polarization, actin‐driven protrusion and formation and turnover of cell adhesions. The PI3K (phosphoinositide 3‐kinase) family of lipid kinases regulate cell migration in many different cell types, both through direct binding of proteins to their lipid products and indirectly through crosstalk with other pathways, such as Rho GTPase signalling. Emerging evidence suggests that the involvement of PI3Ks at different stages of migration varies even within one cell type, and is dependent on the combination of external stimuli, as well as on the signalling status of the cell. In addition, it appears that different PI3K isoforms have distinct roles in cell polarization and migration. This review describes how PI3K signalling is regulated by pro‐migratory stimuli, and the diverse ways in which PI3K‐mediated signal transduction contributes to different aspects of cell migration.     

‘RNA modulation of transport properties and stability in phase-separated condensates

One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a chemically-accurate sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1, and TDP-43 proteins along with that of their corresponding prion-like domains and RNA recognition motifs from absence to moderately high RNA concentration. By characterizing the phase diagram, key molecular interactions, surface tension, and transport properties of the condensates, we report a dual RNA-induced behavior: on the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whereas at high concentration, it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity even at high concentration, but barely modify protein self-diffusion which mainly depends on RNA concentration and on the effect RNA has on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.     

Inhibition of gamma-secretases alters both proliferation and differentiation of mesenchymal stem cells

INTRODUCTION: Human mesenchymal stem cell (hMSC) proliferation and development is regulated by many signalling pathways. gamma-Secretases play an important role in Notch signalling as well as other processes that are involved in developmental decisions, but their role in hMSC proliferation and cell fate decisions has not been explored. OBJECTIVE: To investigate the role of gamma-secretases in hMSC proliferation and differentiation. MATERIALS AND METHODS: Using the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), we investigated their role in hMSC growth and differentiation to chondrogenic, osteogenic and adipogenic fates. RESULTS: We found that inhibiting gamma-secretases reduced the rate of hMSC proliferation, and altered hMSC differentiation in vitro. Addition of DAPT had an inhibitory effect on chondrogenesis resulting in impaired cartilage matrix production and altered chondrocyte morphology. DAPT treated chrodrocytic pellets had reduced levels of Hes1 and Hey1 suggesting that these effects are mediated via Notch signalling. Addition of the DAPT inhibitor to osteogenic cultures did not alter the appearance of bone markers, however, adipogenesis occurred in these cultures in a DAPT concentration-dependent manner. DAPT did not enhance adipogenesis in the presence of a potent adipogenic cocktail, but had an adipogenic effect when combined with dexamethasone only. CONCLUSION: We conclude that gamma-secretases play an important role in both hMSC proliferation and differentiation.     

Membrane transport of hydrogen peroxide

Hydrogen peroxide (H2O2) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work has demonstrated that H2O2 also functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H2O2 is likely to be subjected to tight regulation via processes involved in production, distribution and removal. Substantial progress has been made exploring the formation and scavenging of H2O2, whereas little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. From work in yeast and bacteria it is clear that the diffusion of H2O2 across membranes is limited. We have now obtained direct evidence that selected aquaporin homologues from plants and mammals have the capacity to channel H2O2 across membranes. The main focus of this review is (i) to summarize the most recent evidence for a signalling role of H2O2 in various pathways in plants and mammals and (ii) to discuss the relevance of specific transport of H2O2.     

From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamicity

Mitogen-activated protein kinases (MAPKs) are ubiquitous phosphorylation enzymes involved in signal transduction, gene expression and activation of diverse cytoskeletal proteins. MAPKs participate in the regulation of a broad range of crucial cellular processes including cell survival, division, polarization, stress responses, and metabolism. Phosphorylation of cytoskeletal proteins usually results in the rearrangement of cytoskeletal arrays leading to morphological changes and cell polarization. On the other hand, some cytoskeletal motor proteins, such as kinesins, could activate MAPK members and participate in signal delivery to the proper cellular destination (e.g. during cell division). Moreover, changes in the integrity of cytoskeletal elements have direct impacts on MAPK activity. Recent evidence suggests that there is bi-directional signalling between MAPK cascades and cytoskeleton. The focus here is on this cross-talk between MAPK signalling and the cytoskeleton in various eukaryotic systems including yeast, plants, and mammals and a role is proposed for MAPKs as sensors monitoring the cytoskeleton-dependent balance of forces within the cell.     

Grb2 regulates B-cell maturation, B-cell memory responses and inhibits B-cell Ca2+ signalling

Grb2 is a ubiquitously expressed adaptor protein, which activates Ras and MAP kinases in growth factor receptor signalling, while in B-cell receptor (BCR) signalling this role is controversial. In B cell lines it was shown that Grb2 can inhibit BCR-induced Ca(2+) signalling. Nonetheless, the physiological role of Grb2 in primary B cells is still unknown. We generated a B-cell-specific Grb2-deficient mouse line, which had a severe reduction of mature follicular B cells in the periphery due to a differentiation block and decreased B-cell survival. Moreover, we found several changes in important signalling pathways: enhanced BCR-induced Ca(2+) signalling, alterations in mitogen-activated protein kinase activation patterns and strongly impaired Akt activation, the latter pointing towards a defect in PI3K signalling. Interestingly, B-cell-specific Grb2-deficient mice showed impaired IgG and B-cell memory responses, and impaired germinal centre formation. Thus, Grb2-dependent signalling pathways are crucial for lymphocyte differentiation processes, as well as for control of secondary humoral immune responses.