Structural Requirements for Activity of Mind bomb1 in Notch Signaling.

Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2~Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region of MIB1 (ccRING3) drives MIB1 dimerization and that ubiquitin transfer activity of MIB1 relies solely on RING3. We report x-ray crystal structures of a UbcH5B-ccRING3 complex as a fusion protein and of the ANK region. Directly tethering the N-terminal region to ccRING3 forms a minimal MIB1 protein, which is sufficient to induce a Notch response in receiver cells. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.

Endothelial Notch1 signaling in white adipose tissue promotes cancer cachexia.

Cachexia is a major cause of morbidity and mortality in individuals with cancer and is characterized by weight loss due to adipose and muscle tissue wasting. Hallmarks of white adipose tissue (WAT) remodeling, which often precedes weight loss, are impaired lipid storage, inflammation and eventually fibrosis. Tissue wasting occurs in response to tumor-secreted factors. Considering that the continuous endothelium in WAT is the first line of contact with circulating factors, we postulated whether the endothelium itself may orchestrate tissue remodeling. Here, we show using human and mouse cancer models that during precachexia, tumors overactivate Notch1 signaling in distant WAT endothelium. Sustained endothelial Notch1 signaling induces a WAT wasting phenotype in male mice through excessive retinoic acid production. Pharmacological blockade of retinoic acid signaling was sufficient to inhibit WAT wasting in a mouse cancer cachexia model. This demonstrates that cancer manipulates the endothelium at distant sites to mediate WAT wasting by altering angiocrine signals.

Novel Association of the NOTCH Pathway Regulator MIB1 Gene With the Development of Bicuspid Aortic Valve.

Importance: Nonsyndromic bicuspid aortic valve (nsBAV) is the most common congenital heart valve malformation. BAV has a heritable component, yet only a few causative genes have been identified; understanding BAV genetics is a key point in developing personalized medicine. Objective: To identify a new gene for nsBAV. Design, Setting, and Participants: This was a comprehensive, multicenter, genetic association study based on candidate gene prioritization in a familial cohort followed by rare and common association studies in replication cohorts. Further validation was done using in vivo mice models. Study data were analyzed from October 2019 to October 2022. Three cohorts of patients with BAV were included in the study: (1) the discovery cohort was a large cohort of inherited cases from 29 pedigrees of French and Israeli origin; (2) the replication cohort 1 for rare variants included unrelated sporadic cases from various European ancestries; and (3) replication cohort 2 was a second validation cohort for common variants in unrelated sporadic cases from Europe and the US. Main Outcomes and Measures: To identify a candidate gene for nsBAV through analysis of familial cases exome sequencing and gene prioritization tools. Replication cohort 1 was searched for rare and predicted deleterious variants and genetic association. Replication cohort 2 was used to investigate the association of common variants with BAV. Results: A total of 938 patients with BAV were included in this study: 69 (7.4%) in the discovery cohort, 417 (44.5%) in replication cohort 1, and 452 (48.2%) in replication cohort 2. A novel human nsBAV gene, MINDBOMB1 homologue MIB1, was identified. MINDBOMB1 homologue (MIB1) is an E3-ubiquitin ligase essential for NOTCH-signal activation during heart development. In approximately 2% of nsBAV index cases from the discovery and replication 1 cohorts, rare MIB1 variants were detected, predicted to be damaging, and were significantly enriched compared with population-based controls (2% cases vs 0.9% controls; P = .03). In replication cohort 2, MIB1 risk haplotypes significantly associated with nsBAV were identified (permutation test, 1000 repeats; P = .02). Two genetically modified mice models carrying Mib1 variants identified in our cohort showed BAV on a NOTCH1-sensitized genetic background. Conclusions and Relevance: This genetic association study identified the MIB1 gene as associated with nsBAV. This underscores the crucial role of the NOTCH pathway in the pathophysiology of BAV and its potential as a target for future diagnostic and therapeutic intervention.

Cis-inhibition suppresses basal Notch signaling during sensory organ precursor selection.

The emergence of the sensory organ precursor (SOP) from an equivalence group in Drosophila is a paradigm for studying single-cell fate specification through Notch-mediated lateral inhibition. Yet, it remains unclear how only a single SOP is selected from a relatively large group of cells. We show here that a critical aspect of SOP selection is controlled by cis-inhibition (CI), whereby the Notch ligands, Delta (Dl), cis-inhibit Notch receptors in the same cell. Based on the observation that the mammalian ligand Dl-like 1 cannot cis-inhibit Notch in Drosophila, we probe the role of CI in vivo. We develop a mathematical model for SOP selection where Dl activity is independently regulated by the ubiquitin ligases Neuralized and Mindbomb1. We show theoretically and experimentally that Mindbomb1 induces basal Notch activity, which is suppressed by CI. Our results highlight the trade-off between basal Notch activity and CI as a mechanism for singling out a SOP from a large equivalence group.

Therapeutic gene silencing of CKAP5 leads to lethality in genetically unstable cancer cells.

The potential of microtubule-associated protein targets for cancer therapeutics remains largely unexplored due to the lack of target-specific agents. Here, we explored the therapeutic potential of targeting cytoskeleton-associated protein 5 (CKAP5), an important microtubule-associated protein, with CKAP5-targeting siRNAs encapsulated in lipid nanoparticles (LNPs). Our screening of 20 solid cancer cell lines demonstrated selective vulnerability of genetically unstable cancer cell lines in response to CKAP5 silencing. We identified a highly responsive chemo-resistant ovarian cancer cell line, in which CKAP5 silencing led to significant loss in EB1 dynamics during mitosis. Last, we demonstrated the therapeutic potential in an in vivo ovarian cancer model, showing 80% survival rate of siCKAP5 LNPs-treated animals. Together, our results highlight the importance of CKAP5 as a therapeutic target for genetically unstable ovarian cancer and warrants further investigation into its mechanistic aspects.

Precise alternating cellular pattern in the inner ear by coordinated hopping intercalations and delaminations.

The mammalian hearing organ, the organ of Corti, is one of the most organized tissues in mammals. It contains a precisely positioned array of alternating sensory hair cells (HCs) and nonsensory supporting cells. How such precise alternating patterns emerge during embryonic development is not well understood. Here, we combine live imaging of mouse inner ear explants with hybrid mechano-regulatory models to identify the processes that underlie the formation of a single row of inner hair cells (IHCs). First, we identify a previously unobserved morphological transition, termed "hopping intercalation," that allows cells differentiating toward IHC fate to "hop" under the apical plane into their final position. Second, we show that out-of-row cells with low levels of the HC marker Atoh1 delaminate. Last, we show that differential adhesion between cell types contributes to straightening of the IHC row. Our results support a mechanism for precise patterning based on coordination between signaling and mechanical forces that is likely relevant for many developmental processes.

Structure of the planar cell polarity cadherins Fat4 and Dachsous1.

The atypical cadherins Fat and Dachsous are key regulators of cell growth and animal development. In contrast to classical cadherins, which form homophilic interactions to segregate cells, Fat and Dachsous cadherins form heterophilic interactions to induce cell polarity within tissues. Here, we determine the co-crystal structure of the human homologs Fat4 and Dachsous1 (Dchs1) to establish the molecular basis for Fat-Dachsous interactions. The binding domains of Fat4 and Dchs1 form an extended interface along extracellular cadherin (EC) domains 1-4 of each protein. Biophysical measurements indicate that Fat4-Dchs1 affinity is among the highest reported for cadherin superfamily members, which is attributed to an extensive network of salt bridges not present in structurally similar protocadherin homodimers. Furthermore, modeling suggests that unusual extracellular phosphorylation modifications directly modulate Fat-Dachsous binding by introducing charged contacts across the interface. Collectively, our analyses reveal how the molecular architecture of Fat4-Dchs1 enables them to form long-range, high-affinity interactions to maintain planar cell polarity.

Notch signaling in development and homeostasis.

Notch signaling is a highly conserved signaling pathway that coordinates cellular differentiation during the development and homeostasis in numerous organs and tissues across metazoans. Activation of Notch signaling relies on direct contact between neighboring cells and mechanical pulling of the Notch receptors by the Notch ligands. Notch signaling is commonly used in developmental processes to coordinate the differentiation into distinct cell fates of neighboring cells. In this Development at a Glance article, we describe the current understanding of the Notch pathway activation and the different regulatory levels that control the pathway. We then describe several developmental processes where Notch is crucial for coordinating differentiation. These examples include processes that are largely based on lateral inhibition mechanisms giving rise to alternating patterns (e.g. SOP selection, hair cell in the inner ear and neural stem cell maintenance), as well as processes where Notch activity is oscillatory (e.g. somitogenesis and neurogenesis in mammals).

Matrix stiffness regulates Notch signaling activity in endothelial cells.

Notch signaling is critical for many developmental and disease-related processes. It is widely accepted that Notch has a mechanotransduction module that regulates receptor cleavage. However, the role of biomechanical properties of the cellular environment in Notch signaling in general is still poorly understood. During angiogenesis, differentiation of endothelial cells into tip and stalk cells is regulated by Notch signaling, and remodeling of the extracellular matrix occurs. We investigated the influence of substrate stiffness on the Notch signaling pathway in endothelial cells. Using stiffness-tuned polydimethylsiloxane (PDMS) substrates, we show that activity of the Notch signaling pathway inversely correlates with a physiologically relevant range of substrate stiffness (i.e. increased Notch signaling activity on softer substrates). Trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness, and integrin cell-matrix connections are both stiffness dependent and influenced by Notch signaling. We conclude that mechanotransduction of Notch activation is modulated by substrate stiffness, highlighting the role of substrate rigidity as an important cue for signaling. This might have implications in pathological situations associated with stiffening of the extracellular matrix, such as tumor growth.

A Nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells.

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Functional Comparison between Endogenous and Synthetic Notch Systems.

The Notch pathway converts receptor-ligand interactions at the cell surface into a transcriptional response in the receiver cell. In recent years, synthetic Notch systems (synNotch) that respond to different inputs and transduce different transcriptional responses have been engineered. One class of synNotch systems uses antibody-antigen interactions at the cell surface to induce the proteolytic cleavage cascade of the endogenous Notch autoregulatory core and the consequent release of a synNotch intracellular domain (ICD), converting surface antigen detection into a cellular response. While the activation of endogenous Notch requires ubiquitylation and subsequent endocytosis of the ligand ICD, these synNotch systems do not seem to have such a requirement because the synNotch ligands completely lack an ICD. This observation raises questions about existing models for the synNotch activation mechanism. Here, we test how different structural and biochemical factors affect the dependence of endogenous and synthetic Notch activation on ligand ICD. We compare the behavior of antibody-antigen synNotch (aa-synNotch) to that of endogenous Notch, and to a synNotch system that uses rapamycin induced dimerization of FK506 binding protein (FKBP) and FKBP rapamycin binding (FRB) domaindimerization domains (ff-synNotch), which still requires a ligand ICD. We found that differences in receptor-ligand affinity, in the identity of the transmembrane domain, or in the presence or absence of extracellular epidermal growth factor repeats cannot explain the differences in ligand ICD requirement that distinguishes aa-synNotch from endogenous Notch or ff-synNotch. We also found that unlike endogenous Notch and ff-synNotch, the aa-synNotch system does not exhibit trans-endocytosis of the receptor extracellular domain into the sender cell. These findings suggest that the aa-synNotch systems bypass the ligand ICD requirement because antigen-antibody pairs are able to promote other adhesive cell-cell interactions that provide the mechanical tension needed for ligand activation.

Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor.

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

Mechanical forces shaping the development of the inner ear.

The inner ear is one of the most complex structures in the mammalian body. Embedded within it are the hearing and balance sensory organs that contain arrays of hair cells that serve as sensors of sound and acceleration. Within the sensory organs, these hair cells are prototypically arranged in regular mosaic patterns. The development of such complex, yet precise, patterns require the coordination of differentiation, growth, and morphogenesis, both at the tissue and cellular scales. In recent years, there is accumulating evidence that mechanical forces at the tissue, the cellular, and the subcellular scales coordinate the development and organization of this remarkable organ. Here, we review recent works that reveal how such mechanical forces shape the inner ear, control its size, and establish regular cellular patterns. The insights learned from studying how mechanical forces drive the inner ear development are relevant for many other developmental systems in which precise cellular patterns are essential for their function.

Dynamic spatiotemporal coordination of neural stem cell fate decisions occurs through local feedback in the adult vertebrate brain.

Neural stem cell (NSC) populations persist in the adult vertebrate brain over a lifetime, and their homeostasis is controlled at the population level through unknown mechanisms. Here, we combine dynamic imaging of entire NSC populations in their in vivo niche over several weeks with pharmacological manipulations, mathematical modeling, and spatial statistics and demonstrate that NSCs use spatiotemporally resolved local feedback signals to coordinate their decision to divide in adult zebrafish brains. These involve Notch-mediated short-range inhibition from transient neural progenitors and a dispersion effect from the dividing NSCs themselves exerted with a delay of 9-12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that these signals are linked by lineage progression and control the spatiotemporal distribution of output neurons. These results highlight how local and temporally delayed interactions occurring between brain germinal cells generate self-propagating dynamics that maintain NSC population homeostasis and coordinate specific spatiotemporal correlations.

Biophysics of Notch Signaling.

Notch signaling is a conserved system of communication between adjacent cells, influencing numerous cell fate decisions in the development of multicellular organisms. Aberrant signaling is also implicated in many human pathologies. At its core, Notch has a mechanotransduction module that decodes receptor-ligand engagement at the cell surface under force to permit proteolytic cleavage of the receptor, leading to the release of the Notch intracellular domain (NICD). NICD enters the nucleus and acts as a transcriptional effector to regulate expression of Notch-responsive genes. In this article, we review and integrate current understanding of the detailed molecular basis for Notch signal transduction, highlighting quantitative, structural, and dynamic features of this developmentally central signaling mechanism. We discuss the implications of this mechanistic understanding for the functionality of the signaling pathway in different molecular and cellular contexts.

Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness.

Genetic variants account for approximately half the cases of congenital and early-onset deafness. Methods and technologies for viral delivery of genes into the inner ear have evolved over the past decade to render gene therapy a viable and attractive approach for treatment. Variants in SYNE4, encoding the protein nesprin-4, a member of the linker of nucleoskeleton and cytoskeleton (LINC), lead to DFNB76 human deafness. Syne4-/- mice have severe-to-profound progressive hearing loss and exhibit mislocalization of hair cell nuclei and hair cell degeneration. We used AAV9-PHP.B, a recently developed synthetic adeno-associated virus, to deliver the coding sequence of Syne4 into the inner ears of neonatal Syne4-/- mice. Here we report rescue of hair cell morphology and survival, nearly complete recovery of auditory function, and restoration of auditory-associated behaviors, without observed adverse effects. Uncertainties remain regarding the durability of the treatment and the time window for intervention in humans, but our results suggest that gene therapy has the potential to prevent hearing loss in humans with SYNE4 mutations.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Notch dimerization and gene dosage are important for normal heart development, intestinal stem cell maintenance, and splenic marginal zone B-cell homeostasis during mite infestation.

Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.

Mechanical forces drive ordered patterning of hair cells in the mammalian inner ear.

Periodic organization of cells is required for the function of many organs and tissues. The development of such periodic patterns is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here we show that the transition from disordered to ordered checkerboard-like pattern of hair cells and supporting cells in the mammalian hearing organ, the organ of Corti, is likely based on mechanical forces rather than signaling events. Using time-lapse imaging of mouse cochlear explants, we show that hair cells rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events. Using mechanical models of the tissue, we show that global shear and local repulsion forces on hair cells are sufficient to drive the transition from disordered to ordered cellular pattern. Our findings suggest that mechanical forces drive ordered hair cell patterning in a process strikingly analogous to the process of shear-induced crystallization in polymer and granular physics.