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Gene expression memory-based lineage inference (GEMLI) is a computational tool allowing to predict cell lineages solely from single-cell RNA-sequencing (scRNA-seq) datasets and is publicly available as an R package on GitHub. GEMLI is based on the occurrence of gene expression memory, i.e., the gene-specific maintenance of expression levels through cell divisions. This represents a shift away from experimental lineage tracing techniques based on genetic marks or physical cell lineage separation and greatly eases and expands lineage annotation. GEMLI allows to study cell lineages during differentiation in development, homeostasis, and regeneration, as well as disease onset and progression in various physiological and pathological contexts. This makes it possible to dissect cell type-specific gene expression memory, to discriminate symmetric and asymmetric cell fate decisions, and to reconstruct individual multicellular structures from pooled scRNA-seq datasets. GEMLI is particularly promising for its ability to identify small lineages in human samples, a context in which no other lineage tracing methods are applicable. In this chapter, we provide a detailed protocol of the GEMLI R package usage on gene expression matrices derived from standard scRNA-seq on various platforms. We cover the use of the main function to predict cell lineages and how to adjust its parameters to different tasks. We also show how lineage information is extracted, visualized, and fine-tuned. Finally, we describe the use of the package's functions for the detailed analysis of the predicted cell lineages. This includes the analysis of gene expression memory, cell type composition of individual large lineages, and identification of lineages at the transition point between two cell types., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Zebrafish are vertebrates with a high potential of regeneration after injury in the central nervous system. Therefore, they have emerged as a useful model system for studying traumatic spinal cord injuries., New Method: Using larval zebrafish, we have developed a robust paradigm to model the effects of anterior spinal cord injury, which correspond to the debilitating injuries of the cervical and thoracic regions in humans. Our new paradigm consists of a more anterior injury location compared to previous studies, a modified behavioral assessment using the visual motor response, and a new data analysis code., Results: Our approach enables a spinal cord injury closer to the hindbrain with more functional impact compared to previous studies using a more posterior injury location. Results reported in this work reveal recovery over seven days following spinal cord injury., Comparing With Existing Methods: The present work describes a modified paradigm for the in vivo study of spinal cord regeneration after injury using larval zebrafish, including an anterior injury location, a robust behavioral assessment, and a new data analysis software., Conclusions: Our findings lay the foundation for applying this paradigm to study the effects of drugs, nutrition, and other treatments to improve the regeneration process., Competing Interests: Declaration of Competing Interest The authors do not have any conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate how a single signalling pathway can both initiate and secure transition between cell states., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization., Competing Interests: Competing interests statement:The authors declare no competing interest.

Neuronal growth cones sense a variety of cues including chemical and mechanical ones to establish functional connections during nervous system development. Substrate-cytoskeletal coupling is an established model for adhesion-mediated growth cone advance; however, the detailed molecular and biophysical mechanisms underlying the mechanosensing and mechanotransduction process remain unclear. Here, we adapted a motor-clutch model to better understand the changes in clutch and cytoskeletal dynamics, traction forces, and substrate deformation when a growth cone interacts with adhesive substrates of different stiffnesses. Model parameters were optimized using experimental data from Aplysia growth cones probed with force-calibrated glass microneedles. We included a reinforcement mechanism at both motor and clutch level. Furthermore, we added a threshold for retrograde F-actin flow that indicates when the growth cone is strongly coupled to the substrate. Our modeling results are in strong agreement with experimental data with respect to the substrate deformation and the latency time after which substrate-cytoskeletal coupling is strong enough for the growth cone to advance. Our simulations show that it takes the shortest time to achieve strong coupling when substrate stiffness was low at 4 pN/nm. Taken together, these results suggest that Aplysia growth cones respond faster and more efficiently to soft than stiff substrates.

Assigning single cell transcriptomes to cellular lineage trees by lineage tracing has transformed our understanding of differentiation during development, regeneration, and disease. However, lineage tracing is technically demanding, often restricted in time-resolution, and most scRNA-seq datasets are devoid of lineage information. Here we introduce Gene Expression Memory-based Lineage Inference (GEMLI), a computational tool allowing to robustly identify small to medium-sized cell lineages solely from scRNA-seq datasets. GEMLI allows to study heritable gene expression, to discriminate symmetric and asymmetric cell fate decisions and to reconstruct individual multicellular structures from pooled scRNA-seq datasets. In human breast cancer biopsies, GEMLI reveals previously unknown gene expression changes at the onset of cancer invasiveness. The universal applicability of GEMLI allows studying the role of small cell lineages in a wide range of physiological and pathological contexts, notably in vivo. GEMLI is available as an R package on GitHub ( https://github.com/UPSUTER/GEMLI )., (© 2024. The Author(s).)

Actin flow refers to the motion of the F-actin cytoskeleton and has been observed in many different cell types, especially in motile cells including neuronal growth cones. The direction of the actin flow is generally retrograde from the periphery toward the center of the cell. Actin flow can be harnessed for forward movement of the cell through substrate-cytoskeletal coupling; thus, a key function of actin flow is in cell locomotion. In this chapter, we illustrate three different methods of quantifying retrograde F-actin flow in growth cones derived from cultured Aplysia bag cell neurons. These methods include tracking the movement of surface marker beads as well as kymograph analysis of time-lapse sequences acquired by differential interference contrast (DIC) imaging or fluorescent speckle microscopy (FSM). Due to their large size, Aplysia neuronal growth cones are uniquely suited for these methods; however, they can also be applied to any other growth cones with clear F-actin-rich peripheral domains., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

NADPH oxidase (Nox), a major source of reactive oxygen species (ROS), is involved in neurodegeneration after injury and disease. Nox is expressed in both neuronal and non-neuronal cells and contributes to an elevated ROS level after injury. Contrary to the well-known damaging effect of Nox-derived ROS in neurodegeneration, recently a physiological role of Nox in nervous system development including neurogenesis, neuronal polarity, and axonal growth has been revealed. Here, we tested a role for neuronal Nox in neurite regeneration following mechanical transection in cultured Aplysia bag cell neurons. Using a novel hydrogen peroxide (H 2 O 2 )-sensing dye, 5'-(p-borophenyl)-2'-pyridylthiazole pinacol ester (BPPT), we found that H 2 O 2 levels are elevated in regenerating growth cones following injury. Redistribution of Nox2 and p40 phox in the growth cone central domain suggests Nox2 activation after injury. Inhibiting Nox with the pan-Nox inhibitor celastrol reduced neurite regeneration rate. Pharmacological inhibition of Nox is correlated with reduced activation of Src2 tyrosine kinase and F-actin content in the growth cone. Taken together, these findings suggest that Nox-derived ROS regulate neurite regeneration following injury through Src2-mediated regulation of actin organization in Aplysia growth cones., (© 2023 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophil-like cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After initial spreading, the wild-type cells polarize and migrate, whereas the Mfn2 -/- cells maintain a circular shape. Increased cytosolic Ca 2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and endoplasmic reticulum to close vicinity. Elevated cytosolic Ca 2+ activates Ca 2+ /calmodulin-dependent protein kinase II, RhoA, and myosin light-chain kinase, causing an overactivation of nonmuscle myosin II, leading to a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases., Competing Interests: YW, LT, FX, AC, CD, HZ, LC, RW, TW, ST, JC, LL, DU, SK, DS, CY, DC, FH, PO, QD No competing interests declared, (© 2023, Wang et al.)

The level of a given protein is determined by the synthesis and degradation rates of its mRNA and protein. While several studies have quantified the contribution of different gene expression steps in regulating protein levels, these are limited by using equilibrium approximations in out-of-equilibrium biological systems. Here, we introduce gene expression flux analysis to quantitatively dissect the dynamics of the expression level for specific proteins and use it to analyze published transcriptomics and proteomics datasets. Our analysis reveals distinct regulatory modalities shared by sets of genes with clear functional signatures. We also find that protein degradation plays a stronger role than expected in the adaptation of protein levels. These findings suggest that shared regulatory strategies can lead to versatile responses at the protein level and highlight the importance of going beyond equilibrium approximations to dissect the quantitative contribution of different steps of gene expression to protein dynamics., Competing Interests: Authors declare no competing interests., (© 2023 The Author(s).)

Protein turnover (PT) has been formally defined only in equilibrium conditions, which is ill-suited to quantify PT during dynamic processes that occur during embryogenesis or (extra) cellular signaling. In this Hypothesis, we propose a definition of PT in an out-of-equilibrium regime that allows the quantification of PT in virtually any biological context. We propose a simple mathematical and conceptual framework applicable to a broad range of available data, such as RNA sequencing coupled with pulsed-SILAC datasets. We apply our framework to a published dataset and show that stimulation of mouse dendritic cells with LPS leads to a proteome-wide change in PT. This is the first quantification of PT out-of-equilibrium, paving the way for the analysis of biological systems in other contexts., (© 2023 The Authors. BioEssays published by Wiley Periodicals LLC.)

Recent developments such as multi-harmonic Atomic Force Microscopy (AFM) techniques have enabled fast, quantitative mapping of nanomechanical properties of living cells. Due to their high spatiotemporal resolution, these methods provide new insights into changes of mechanical properties of subcellular structures due to disease or drug response. Here, we propose three new improvements to significantly improve the resolution, identification, and mechanical property quantification of sub-cellular and sub-nuclear structures using multi-harmonic AFM on living cells. First, microcantilever tips are streamlined using long-carbon tips to minimize long-range hydrodynamic interactions with the cell surface, to enhance the spatial resolution of nanomechanical maps and minimize hydrodynamic artifacts. Second, simultaneous Spinning Disk Confocal Microscopy (SDC) with live-cell fluorescent markers enables the unambiguous correlation between observed heterogeneities in nanomechanical maps with subcellular structures. Third, computational approaches are then used to estimate the mechanical properties of sub-nuclear structures. Results are demonstrated on living NIH 3T3 fibroblasts and breast cancer MDA-MB-231 cells, where properties of nucleoli, a deep intracellular structure, were assessed. The integrated approach opens the door to study the mechanobiology of sub-cellular structures during disease or drug response., (© 2022. The Author(s).)

Cortactin is an actin-binding protein that regulates processes like cell migration, endocytosis, and tumor cell metastasis. Although cortactin is associated with actin-cytoskeletal dynamics in non-neuronal cells and cell-free systems, the exact mechanisms underlying its fundamental roles in neuronal growth cones are not fully explored. Recent reports show that Aplysia Src2 tyrosine kinase induces phosphorylation of cortactin as a mechanism to control lamellipodia protrusion and filopodia formation in cultured Aplysia bag cell neurons ( He et al. , 2015 ; Ren et al. , 2019 ). In order to provide in vitro evidence for Src2-mediated phosphorylation of cortactin, we developed a robust and cost-effective method for the efficient expression and purification of Aplysia cortactin and Src2 kinase that can be used for biochemical studies including phosphorylation assays. By co-purifying cortactin and Src kinase with a phosphatase (YopH) from Yersinia enterocolitica , we eliminated the problem of non-specific phosphorylation of induced proteins by bacterial kinases and also reduced costs by bypassing the need for commercial enzymatic treatments. This protocol is reproducible and can be modified to produce homogenous non-phosphorylated proteins during recombinant protein expression in Escherichia coli ., Competing Interests: Competing interestsThe authors declare that no competing interests exist., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

NADPH oxidases (Nox) are membrane-bound multi-subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox-derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, we cultured retinal ganglion cells (RGCs) derived from zebrafish embryos and exposed these neurons to netrin-1, slit2, and brain-derived neurotrophic factor (BDNF). To test the role of Nox in cue-mediated growth and guidance, we either pharmacologically inhibited Nox or investigated neurons from mutant fish that are deficient in Nox2. We found that slit2-mediated growth cone collapse, and axonal retraction were eliminated by Nox inhibition. Though we did not see an effect of either BDNF or netrin-1 on growth rates, growth in the presence of netrin-1 was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin-1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. We also investigated the possible relationship between Nox2 and slit2/Robo2 signaling in vivo. astray/nox2 double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. Our results provide evidence that Nox2 acts downstream of slit2/Robo2 by mediating growth and guidance of developing zebrafish RGC neurons., (© 2020 Wiley Periodicals LLC.)

Reactive oxygen species (ROS) are critical for maintaining cellular homeostasis and function when produced in physiological ranges. Important sources of cellular ROS include NADPH oxidases (Nox), which are evolutionary conserved multi-subunit transmembrane proteins. Nox-mediated ROS regulate variety of biological processes including hormone synthesis, calcium signaling, cell migration, and immunity. ROS participate in intracellular signaling by introducing post-translational modifications to proteins and thereby altering their functions. The central nervous system (CNS) expresses different Nox isoforms during both development and adulthood. Here, we review the role of Nox-mediated ROS during CNS development. Specifically, we focus on how individual Nox isoforms contribute to signaling in neural stem cell maintenance and neuronal differentiation, as well as neurite outgrowth and guidance. We also discuss how ROS regulates the organization and dynamics of the actin cytoskeleton in the neuronal growth cone. Finally, we review recent evidence that Nox-derived ROS modulate axonal regeneration upon nervous system injury., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Transcription factors (TFs) bind to specific DNA motifs to regulate the expression of target genes. To reach their binding sites, TFs diffuse in 3D and perform local motions such as 1D sliding, hopping, or intersegmental transfer. TF-DNA interactions depend on multiple parameters, such as the chromatin environment, TF partitioning into distinct subcellular regions, and cooperativity with other DNA-binding proteins. In this review, how current understanding of the search process has initially been shaped by prokaryotic studies is discussed, as well as what is known about the parameters regulating TF search efficiency in the context of the complex eukaryotic chromatin landscape., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state., Competing Interests: EF, CD, AM, SG, VG, BD, DS No competing interests declared, (© 2019, Friman et al.)

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here, we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates in undirected differentiation. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Cortactin is a Src tyrosine phosphorylation substrate that regulates multiple actin-related cellular processes. While frequently studied in nonneuronal cells, the functions of cortactin in neuronal growth cones are not well understood. We recently reported that cortactin mediates the effects of Src tyrosine kinase in regulating actin organization and dynamics in both lamellipodia and filopodia of Aplysia growth cones. Here, we identified a single cortactin tyrosine phosphorylation site (Y499) to be important for the formation of filopodia. Overexpression of a 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of a 499E phospho-mimetic mutant increased filopodia length. Using an antibody against cortactin pY499, we showed that tyrosine-phosphorylated cortactin is enriched along the leading edge. The leading edge localization of phosphorylated cortactin is Src2-dependent, F-actin-independent, and important for filopodia formation. In vitro kinase assays revealed that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro. Finally, we provide evidence that Arp2/3 complex acts downstream of phosphorylated cortactin to regulate density but not length of filopodia. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation.

The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models - transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) - to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells.

Protein expression levels depend on the balance between their synthesis and degradation rates. Even quiescent (G 0 ) cells display a continuous turnover of proteins, despite protein levels remaining largely constant over time. In cycling cells, global protein levels need to be precisely doubled at each cell division in order to maintain cellular homeostasis, but we still lack a quantitative understanding of how this is achieved. Recent studies have shed light on cell cycle-dependent changes in protein synthesis and degradation rates. Here we discuss current population-based and single cell approaches used to assess protein synthesis and degradation, and review the insights they have provided into the dynamics of protein turnover in different cell cycle phases.

The transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-β stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.

Phenotypically identical mammalian cells often display considerable variability in transcript levels of individual genes. How transcriptional activity propagates in cell lineages, and how this varies across genes is poorly understood. Here we combine live-cell imaging of short-lived transcriptional reporters in mouse embryonic stem cells with mathematical modelling to quantify the propagation of transcriptional activity over time and across cell generations in phenotypically homogenous cells. In sister cells we find mean transcriptional activity to be strongly correlated and transcriptional dynamics tend to be synchronous; both features control how quickly transcriptional levels in sister cells diverge in a gene-specific manner. Moreover, mean transcriptional activity is transmitted from mother to daughter cells, leading to multi-generational transcriptional memory and causing inter-family heterogeneity in gene expression.

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measurements of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an "enhanceosome," nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription., Competing Interests: The authors have declared that no competing interests exist.

Neurite outgrowth underlies the wiring of the nervous system during development and regeneration. Despite a significant body of research, the underlying cytoskeletal mechanics of growth and guidance are not fully understood, and the relative contributions of individual cytoskeletal processes to neurite growth are controversial. Here, we review the structural organization and biophysical properties of neurons to make a semi-quantitative comparison of the relative contributions of different processes to neurite growth. From this, we develop the idea that neurons are active fluids, which generate strong contractile forces in the growth cone and weaker contractile forces along the axon. As a result of subcellular gradients in forces and material properties, actin flows rapidly rearward in the growth cone periphery, and microtubules flow forward in bulk along the axon. With this framework, an integrated model of neurite outgrowth is proposed that hopefully will guide new approaches to stimulate neuronal growth.

The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer-range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs.

Cells need to reliably control their proteome composition to maintain homeostasis and regulate growth. How protein synthesis and degradation interplay to control protein expression levels remains unclear. Here, we combined a tandem fluorescent timer and pulse-chase protein labeling to disentangle how protein synthesis and degradation control protein homeostasis in single live mouse embryonic stem cells. We discovered substantial cell-cycle dependence in protein synthesis rates and stabilization of a large number of proteins around cytokinesis. Protein degradation rates were highly variable between cells, co-varied within individual cells for different proteins, and were positively correlated with synthesis rates. This suggests variability in proteasome activity as an important source of global extrinsic noise in gene expression. Our approach paves the way toward understanding the complex interplay of synthesis and degradation processes in determining protein levels of individual mammalian cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Many mammalian genes are transcribed during short bursts of variable frequencies and sizes that substantially contribute to cell-to-cell variability. However, which molecular mechanisms determine bursting properties remains unclear. To probe putative mechanisms, we combined temporal analysis of transcription along the circadian cycle with multiple genomic reporter integrations, using both short-lived luciferase live microscopy and single-molecule RNA-FISH. Using the Bmal1 circadian promoter as our model, we observed that rhythmic transcription resulted predominantly from variations in burst frequency, while the genomic position changed the burst size. Thus, burst frequency and size independently modulated Bmal1 transcription. We then found that promoter histone-acetylation level covaried with burst frequency, being greatest at peak expression and lowest at trough expression, while remaining unaffected by the genomic location. In addition, specific deletions of ROR-responsive elements led to constitutively elevated histone acetylation and burst frequency. We then investigated the suggested link between histone acetylation and burst frequency by dCas9p300-targeted modulation of histone acetylation, revealing that acetylation levels influence burst frequency more than burst size. The correlation between acetylation levels at the promoter and burst frequency was also observed in endogenous circadian genes and in embryonic stem cell fate genes. Thus, our data suggest that histone acetylation-mediated control of transcription burst frequency is a common mechanism to control mammalian gene expression., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) have been linked to neuronal polarity, axonal outgrowth, cerebellar development, regeneration of sensory axons, and neuroplasticity. However, the specific roles that individual Nox isoforms play during nervous system development in vivo remain unclear. To address this problem, we investigated the role of Nox activity in the development of retinotectal connections in zebrafish embryos. Zebrafish broadly express four nox genes ( nox1 , nox2/cybb , nox5 , and duox ) throughout the CNS during early development. Application of a pan-Nox inhibitor, celastrol, during the time of optic nerve (ON) outgrowth resulted in significant expansion of the ganglion cell layer (GCL), thinning of the ON, and a decrease in retinal axons reaching the optic tectum (OT). With the exception of GCL expansion, these effects were partially ameliorated by the addition of H 2 O 2 , a key ROS involved in Nox signaling. To address isoform-specific Nox functions, we used CRISPR/Cas9 to generate mutations in each zebrafish nox gene. We found that nox2/cybb chimeric mutants displayed ON thinning and decreased OT innervation. Furthermore, nox2/cybb homozygous mutants ( nox2/cybb -/- ) showed significant GCL expansion and mistargeted retinal axons in the OT. Neurite outgrowth from cultured zebrafish retinal ganglion cells was reduced by Nox inhibitors, suggesting a cell-autonomous role for Nox in these neurons. Collectively, our results show that Nox2/Cybb is important for retinotectal development in zebrafish. SIGNIFICANCE STATEMENT Most isoforms of NADPH oxidase (Nox) only produce reactive oxygen species (ROS) when activated by an upstream signal, making them ideal candidates for ROS signaling. Nox enzymes are present in neurons and their activity has been shown to be important for neuronal development and function largely by in vitro studies. However, whether Nox is involved in the development of axons and formation of neuronal connections in vivo has remained unclear. Using mutant zebrafish embryos, this study shows that a specific Nox isoform, Nox2/Cybb, is important for the establishment of axonal connections between retinal ganglion cells and the optic tectum., (Copyright © 2018 the authors 0270-6474/18/385854-18$15.00/0.)

Objective: Current neuronal cell culture is mostly performed on two-dimensional (2D) surfaces, which lack many of the important features of the native environment of neurons, including topographical cues, deformable extracellular matrix, and spatial isotropy or anisotropy in three dimensions. Although three-dimensional (3D) cell culture systems provide a more physiologically relevant environment than 2D systems, their popularity is greatly hampered by the lack of easy-to-make-and-use devices. We aim to develop a widely applicable 3D culture procedure to facilitate the transition of neuronal cultures from 2D to 3D., Approach: We made a simple microwell device for 3D neuronal cell culture that is inexpensive, easy to assemble, and fully compatible with commonly used imaging techniques, including super-resolution microscopy., Main Results: We developed a novel gel mixture to support 3D neurite regeneration of Aplysia bag cell neurons, a system that has been extensively used for quantitative analysis of growth cone dynamics in 2D. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo. We demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles., Significance: Neuronal cell culture has been a valuable tool for neuroscientists to study the behavior of neurons in a controlled environment. Compared to 2D, neurons cultured in 3D retain the majority of their native characteristics, while offering higher accessibility, control, and repeatability. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration.

During mitosis, gene transcription stops, and the bulk of DNA-binding proteins are excluded from condensed chromosomes. While most gene-specific transcription factors are largely evicted from mitotic chromosomes, a subset remains bound to specific and non-specific DNA sites. Here, we review the current knowledge on the mechanisms leading to the retention of a subset of transcription factors on mitotic chromosomes and discuss the implications in gene expression regulation and their potential as an epigenetic mechanism controlling stem cell self-renewal and differentiation., (© 2017 Federation of European Biochemical Societies.)

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

The regulation of fundamental processes such as gene expression or cell differentiation involves chromatin states, demarcated by combinatorial histone post-translational modification (PTM) patterns. The subnuclear organization and dynamics of chromatin states is not well understood, as tools for their detection and modulation in live cells are lacking. Here, we report the development of genetically encoded chromatin-sensing multivalent probes, cMAPs, selective for bivalent chromatin, a PTM pattern associated with pluripotency in embryonic stem cells (ESCs). cMAPs were engineered from a set of PTM-binding (reader) proteins and optimized using synthetic nucleosomes carrying defined PTMs. Applied in live ESCs, cMAPs formed discrete subnuclear foci, revealing the organization of bivalent chromatin into local clusters. Moreover, cMAPs enabled direct monitoring of the loss of bivalency upon treatment with small-molecule epigenetic modulators. cMAPs thus provide a versatile platform to monitor chromatin state dynamics in live cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

During the development of the nervous system and regeneration following injury, microtubules (MTs) are required for neurite elongation. Whether this elongation occurs primarily through tubulin assembly at the tip of the axon, the transport of individual MTs, or because MTs translocate forward in bulk is unclear. Using fluorescent speckle microscopy (FSM), differential interference contrast (DIC), and phase contrast microscopy, we tracked the movement of MTs, phase dense material, and docked mitochondria in chick sensory and Aplysia bag cell neurons growing rapidly on physiological substrates. In all cases, we find that MTs and other neuritic components move forward in bulk at a rate that on average matches the velocity of neurite elongation. To better understand whether and why MT assembly is required for bulk translocation, we disrupted it with nocodazole. We found this blocked the forward bulk advance of material along the neurite and was paired with a transient increase in axonal tension. This indicates that disruption of MT dynamics interferes with neurite outgrowth, not by disrupting the net assembly of MTs at the growth cone, but rather because it alters the balance of forces that power the bulk forward translocation of MTs.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells., (Copyright © 2017. Published by Elsevier Inc.)

The ability of some transcription factors to remain bound to specific genes on condensed mitotic chromosomes has been suggested to play a role in their rapid transcriptional reactivation upon mitotic exit. We have recently shown that SOX2 and OCT4 remain associated to mitotic chromosomes, and that depletion of SOX2 at the mitosis-G1 (M-G1) transition impairs its ability to maintain pluripotency and drive neuroectodermal commitment. Here we report on the role of SOX2 at the M-G1 transition in regulating transcriptional activity of embryonic stem cells. Using single cell time-lapse analysis of reporter constructs for STAT3 and SOX2/OCT4 activity, we show that SOX2/OCT4 do not lead to more rapid transcriptional reactivation in G1 than STAT3, a transcription factor that is excluded from mitotic chromosomes. We also report that only few endogenous target genes show decreased pre-mRNA levels after mitotic exit or in other cell cycle phases in the absence of SOX2 at the M-G1 transition. This suggests that bookmarked SOX2 target genes are not differently regulated than non-bookmarked target genes, and we discuss an alternative hypothesis on how mitotic bookmarking by SOX2 and other sequence-specific transcription factors could be involved in transcriptional regulation.

Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation., (© 2016 Deluz et al.; Published by Cold Spring Harbor Laboratory Press.)