Biology subjects

Sprinzak, D.

Publications and source records attributed to Sprinzak, D..

15 recordsLinked to original sources

bioRxiv2026

Notch mediated lateral inhibition is shaped by morphological differences to reinforce bias toward signal-sending or receiving roles

During neurogenesis, neuroblasts are selected from proneural-competent cells through lateral inhibition, a process controlled by the evolutionarily conserved Notch signalling pathway. By tracking transcription from Notch-target genes and cell morphologies in real time, we discovered that the presumptive neuroblast never initiates target-gene transcription. This implies a pre-existing bias directs Notch signalling. The bias correlates with a heterogeneity in apical cell areas which is further reinforced during neuroblast selection. Additionally, the length and duration of neuroblast-neighbour cell contacts predict the likelihood of transcription. Using mathematical modelling we show that lateral inhibition seeded with subtle morphological differences can bias cells toward signal-sending or receiving roles before transcriptional feedback occurs. Notch activation further alters apical cell area, reinforcing the initial bias. We propose that signalling and cell mechanics work together to ensure the robust selection of a single neural precursor.

developmental biology↗

bioRxiv2025

A model of transcriptional bursting dynamics based on coupling between chromatin and enhancer states

In recent years, it has become evident that transcription is not always a continuous process. Rather, many genes exhibit bursting behavior characterized by discrete periods of transcriptional activity and inactivity. While transcriptional bursting has been broadly observed across different organisms, from bacteria to mammals, we lack a mechanistic understanding of the molecular events that regulate this widespread process. Specifically, how the expression of a bursty gene is quantitatively determined by different molecular factors such as the concentration of transcription factors (TF) and architecture of the enhancers is not well understood. Here, we introduce a model based on the interplay between chromatin state and TF binding in order to describe bursting dynamics. We leverage the widespread Monod-Wyman-Changeux two-state model to predict the dependence of transcriptional bursting dynamics on TF concentration, binding affinity, and number of TF binding sites. We use the model to qualitatively reproduce the behavior of bursting dynamics observed in the C. elegans gonad. Overall, we provide a tractable model for transcriptional bursting that offers mechanistic insights into the factors regulating transcriptional bursting and generates experimentally testable predictions capable of uncovering the molecular basis of this widespread process.

biophysics↗

bioRxiv2025

Multimodal profiling reveals a Notch-responsive regenerative subpopulation of cochlear supporting cells

Regeneration enables organisms to repair damaged tissues, yet this capacity is strikingly limited in the cochlear sensory epithelium, essential for sound detection. A major cause of hearing loss arises from the irreversible loss of sensory hair cells (HCs) in the cochlea. While supporting cells (SCs) have a latent ability to transdifferentiate into HCs, this regenerative potential is rapidly lost after development. Using live imaging and single-cell multi-omics of cochlear explants, we uncovered the cellular and molecular heterogeneity underlying the limited regenerative capacity of the neonatal mouse cochlea. Notch repression broadly silenced key SC genes, yet only a rare subpopulation of Deiters cells (DC), termed transdifferentiating DCs (tDCs), initiated the transdifferentiation into HC fate. These cells underwent coordinated transcriptional and enhancer remodeling, linking epigenetic priming with morphological plasticity, while other SCs remained refractory despite robust Notch targets downregulation. Our study provides a molecular definition of an early induced transitional DC to HC state, revealing Notch inhibition as a selective trigger that unmasks rare regenerative competence.

developmental biology↗

bioRxiv2024

Neuralized-like proteins differentially activate Notch ligands

Notch signalling is a major signalling pathway coordinating cellular processes between neighbouring animal cells. In Drosophila, two ubiquitin ligases, Neuralized (Neur) and Mindbomb1 (Mib1), regulate Notch ligand activation and are essential for development. However, the mammalian orthologs of Neur, Neuralized-like (NEURL) 1 and 2, do not appear to be crucial for development, as double knock-out mice show no developmental defects. Thus, it is unclear if and how NEURL proteins regulate the four mammalian Notch ligands. To address these questions, we examined NEURL proteins ability to activate Notch ligands in humanized Drosophila and mammalian cell culture. We found that, unlike MIB1, NEURL proteins activate Notch only with a subset of mammalian ligands, which contain a Neuralized binding motif. This motif has the consensus sequence NxxN, present only in Notch ligands DLL1 and JAG1, but not in DLL4 and JAG2. Overall, we show that NEURL proteins activate specific Notch-ligands, suggesting a differential regulatory mechanism of Notch activation in mammals, which can potentially explain the limited role of NEURL proteins in mammalian development and homeostasis.

molecular biology↗

bioRxiv2024

The Fat4 intracellular domain controls turnover of Fat4/Dchs1 planar polarity membrane complexes

The Fat/Dachsous (Ft/Ds) pathway is a highly conserved pathway regulating planar cell polarity (PCP) across different animal species. Proteins from the Ft and Ds family are large transmembrane protocadherins that form heterophilic complexes on the boundaries between cells. Fat4 and Dchs1, the main mammalian homologues of this pathway, have been implicated in PCP in various epithelial tissues and were shown to form extremely stable complexes at the boundaries between cells. It is unclear however, what are the dynamics controlling such stable boundary complexes, and how the formation and turnover of these complexes is regulated. Here, we use quantitative live imaging to elucidate the role of the intracellular domains (ICD) of Fat4 and Dchs1 in regulating Fat4/Dchs1 complex dynamics. We show that removing the ICD of Fat4 results in a reduction of both Trans-endocytosis (TEC) of Dchs1 into the Fat4 cells and boundary accumulation, but does not affect the diffusion of the complexes at the boundary. We further show that the ICD of Fat4 controls the turnover rate of Fat4/Dchs1 complexes. Finally, we find that while actin polymerization is required for maintaining the boundary accumulation of Fat4/Dchs1 complexes, we do not identify correlations between Fat4/Dchs1 complexes and local actin accumulation. Overall, we suggest that the Fat4 ICD is important for the turnover and plasticity of the highly stable Fat4/Dchs1 complexes associated with PCP. Statement of SignificanceThe purpose of this study is to elucidate the dynamics leading to the formation and maintenance of Fat4/Dchs1 complexes at the cell boundary and how it is affected by the ICD of Fat4 in mammals. The insights from this work are important for obtaining a mechanistic molecular framework for understanding formation of planar cell polarity as well as for highlighting how cell boundary membrane complexes are regulated.

cell biology↗

bioRxiv2024

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.

biochemistry↗

bioRxiv2022

Cis-inhibition suppresses basal Notch signalling during sensory organ precursor selection

The emergence of the sensory organ precursor (SOP) from the proneural equivalence group in Drosophila melanogaster is a paradigm for studying single cell fate specification through the process of lateral inhibition. Classical lateral inhibition models describing this selection process are based on a transcriptional feedback mechanism where inhibitory signals between neighbouring cells, mediated by Notch pathway, are coupled to an intracellular circuit regulating the expression of the Notch ligand Delta (Dl). It was previously shown that in addition to its ability to trans-activate Notch in neighbouring cells, Dl can also cis-inhibit Notch in the same cell. However, it remains unclear what role does cis-inhibition play during SOP selection, and how it contributes to the selection of only one SOP. Here we address these questions using the unexpected observation that the mammalian ligand Delta-like 1 (Dll1) can trans-activate but not cis-inhibit Notch in Drosophila. We develop a mathematical model for SOP selection, termed the two-channel SOP (TCS) model, where Dl activity, but not its expression, is regulated by two channels associated with the two E3 ubiquitin ligases Neuralized (Neur) and Mindbomb1 (Mib1). While the Neur-dependent channel is regulated by Notch signalling, the Mib1-dependent channel is not, leading to tissue-wide basal inhibitory activity. We show theoretically and experimentally that cis-inhibition is required for suppressing Mib1-dependent basal Notch activity. Thus, our results highlight the trade-off between basal Notch activity and cis-inhibition as a mechanism for singling out an SOP from the proneural equivalence group.

developmental biology↗

bioRxiv2022

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. However, whereas 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 Notch 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 induced FKBP-FRB dimerization with rapamycin (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 EGF repeats cannot explain the differences in ligand ICD requirement that distinguishes aa-synNotch from endogenous Notch or ff-synNotch. 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.

molecular biology↗

bioRxiv2022

Matrix stiffness regulates Notch signaling activity in endothelial cells

The Notch signaling pathway plays a critical role in many developmental and disease related processes. It is widely accepted that Notch has a mechano-transduction module that regulates cleavage of the receptor. However, the role of biomechanical properties of the cellular environment on this module and on Notch signaling in general is still poorly understood. During angiogenesis, differentiation into tip and stalk cells is regulated by Notch. The endothelial cells in this process respond to biochemical and mechanical cues triggered by local stiffening of the ECM. Here, we investigated the influence of substrate stiffness on the Notch signaling pathway in endothelial cells. Using stiffness tuned PDMS substrates we show that Notch signaling pathway activity inversely correlates with the physiologically relevant substrate stiffness, with increased Notch activity on softer substrates. We show that trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness. Furthermore, we could show that integrin cell-matrix connections are both stiffness-dependent and influenced by Notch. Cadherin mediated cell-cell adhesion and Notch, however, influence each other in that basal Notch signaling is cell-cell contact dependent, but inhibition of the Notch signaling pathway also results in a reduction of VE-cadherin levels. We conclude that mechano-transduction of Notch activation depends on substrate stiffness highlighting the role of substrate rigidity as a modulator of Notch signaling. This may have important implications in pathological situations, such as tumor growth, associated with stiffening of the extracellular matrix.

cell biology↗

bioRxiv2022

Mechano-signaling feedback underlies precise inner hair cell patterning in the organ of Corti

The mammalian hearing organ, the organ of Corti, is one of the most organized tissues in the mammalian body plan. It contains precisely positioned array of alternating sensory hair cells (HC) and non-sensory supporting cells that emerge during embryonic development from an initially disordered pro-sensory domain. While much is known on the genetics and biochemistry underlying this process, it is still unclear how such precise alternating patterns emerge during embryonic development. Here, we combine live imaging of mouse inner ear explants with hybrid mechano-regulatory models to elucidate the mechanisms underlying the formation of a single row of inner HC (IHC). We show that a narrow strip of initially disordered salt-and-pepper pattern, generated by Notch-mediated lateral inhibition, is dynamically refined by coordinated intercalations, delaminations, and differential adhesion. We identify a new morphological transition, termed hopping intercalation, that allows nascent IHC to hop under the apical plane into their final position. We further show that IHC patterning is associated with boundary localization of the cell adhesion molecules, Nectin-3 and Nectin-1. Our experimental results and modeling support a mechanism for precise patterning based on a feedback between Notch-mediated differentiation and mechanically driven cellular reorganization that is likely relevant for many developmental processes.

developmental biology↗

bioRxiv2021

A nesprin-4/kinesin-1 cargo model for nucleus 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.

cell biology↗

bioRxiv2020

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

Neural stem cell (NSC) populations persist in the adult vertebrate brain over a life time, and their homeostasis is controlled at the population level. The nature and properties of these coordination mechanisms remain unknown. Here we combine dynamic imaging of entire NSC populations in their in vivo niche over weeks, pharmacological manipulations, mathematical modeling and spatial statistics, and demonstrate that NSCs use spatiotemporally resolved local feedbacks to coordinate their decision to divide. These involve a Notch-mediated inhibition from transient neural progenitors, and a dispersion effect from dividing NSCs themselves, exerted with a delay of 9-12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that they 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 with specific spatiotemporal correlations.

neuroscience↗

bioRxiv2020

Exposure to Mites Sensitizes Intestinal Stem Cell Maintenance, Splenic Marginal Zone B Cell Homeostasis, And Heart Development to Notch Dosage and Cooperativity.

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. While 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, compromising viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% DSS. The most striking phenotypes - gender imbalance and splenic marginal zone B cell lymphoma - emerged in combination with 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 the dimer deficient animals. HighlightsO_LINotch dimerization has an in vivo role in contributing to intestinal homeostasis C_LIO_LILoss of cooperativity can manifest as Notch gain or loss of function phenotypes C_LIO_LIMite infestation exacerbates all phenotypes, triggers MZB hyperproliferation in mutant animals C_LIO_LIMite-infested mutant mice develop SMZL with age C_LI

developmental biology↗

bioRxiv2019

Enhancer architecture sensitizes cell specific responses to Notch gene dose via a bind and discard mechanism

Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we show that inserting a single enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can unexpectedly induce a subset of Drosophila Notch haploinsufficiency phenotypes in an animal with wild type Notch gene dose. Mechanistically, this enhancer couples Notch DNA binding to degradation in a Cdk8-dependent, transcription-independent manner. Using mathematical modeling combined with quantitative trait and expression analysis, we show that tissues requiring long duration Notch signals are more sensitive to perturbations in Notch degradation compared to tissues relying upon short duration processes. These findings support a novel \"bind and discard\" mechanism in which enhancers with specific binding sites promote rapid Notch turnover, reduce Notch-dependent transcription at other loci, and thereby sensitize tissues to gene dose based upon signal duration.

developmental biology↗

bioRxiv2019

Shear forces drive precise patterning of hair cells in the mammalian inner ear

Precise cellular organizations are required for the function of many organs and tissues. It is often unclear, however, how such precise patterns emerge during development. The mammalian hearing organ, the organ of Corti, consists of a remarkably organized pattern of four rows of hair cells (HCs) interspersed by non-sensory supporting cells (SCs). This checkerboard-like pattern of HCs and SCs emerges from a disordered epithelium over several days, yet the transition to an ordered cellular pattern is not well understood. Using a combination of quantitative morphological analysis and time-lapse imaging of mouse cochlear explants, we show here that patterning of the organ of Corti involves dynamic reorganizations that include lateral shear motion, cell intercalations, and delaminations. A mathematical model, where tissue morphology is described in terms of the mechanical forces that act on cells and cellular junctions, suggests that global shear on HCs and local repulsion between HCs are sufficient to drive the tissue into the final checkerboard-like pattern. Our findings suggest that precise patterns can emerge during development from reorganization processes, driven by a combination of global and local forces in a process analogous to shear-induced crystallization.

developmental biology↗