Signaling from the living plasma membrane.

Our understanding of the plasma membrane, once viewed simply as a static barrier, has been revolutionized to encompass a complex, dynamic organelle that integrates the cell with its extracellular environment. Here, we discuss how bidirectional signaling across the plasma membrane is achieved by striking a delicate balance between restriction and propagation of information over different scales of time and space and how underlying dynamic mechanisms give rise to rich, context-dependent signaling responses. In this Review, we show how computer simulations can generate counterintuitive predictions about the spatial organization of these complex processes.

Smaug1 membrane-less organelles respond to AMPK and mTOR and affect mitochondrial function.

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.

Nfinder: automatic inference of cell neighborhood in 2D and 3D using nuclear markers.

BACKGROUND: In tissues and organisms, the coordination of neighboring cells is essential to maintain their properties and functions. Therefore, knowing which cells are adjacent is crucial to understand biological processes that involve physical interactions among them, e.g. cell migration and proliferation. In addition, some signaling pathways, such as Notch or extrinsic apoptosis, are highly dependent on cell-cell communication. While this is straightforward to obtain from membrane images, nuclei labelling is much more ubiquitous for technical reasons. However, there are no automatic and robust methods to find neighboring cells based only on nuclear markers. RESULTS: In this work, we describe Nfinder, a method to assess the cell's local neighborhood from images with nuclei labeling. To achieve this goal, we approximate the cell-cell interaction graph by the Delaunay triangulation of nuclei centroids. Then, links are filtered by automatic thresholding in cell-cell distance (pairwise interaction) and the maximum angle that a pair of cells subtends with shared neighbors (non-pairwise interaction). We systematically characterized the detection performance by applying Nfinder to publicly available datasets from Drosophila melanogaster, Tribolium castaneum, Arabidopsis thaliana and C. elegans. In each case, the result of the algorithm was compared to a cell neighbor graph generated by manually annotating the original dataset. On average, our method detected 95% of true neighbors, with only 6% of false discoveries. Remarkably, our findings indicate that taking into account non-pairwise interactions might increase the Positive Predictive Value up to + 11.5%. CONCLUSION: Nfinder is the first robust and automatic method for estimating neighboring cells in 2D and 3D based only on nuclear markers and without any free parameters. Using this tool, we found that taking non-pairwise interactions into account improves the detection performance significantly. We believe that using our method might improve the effectiveness of other workflows to study cell-cell interactions from microscopy images. Finally, we also provide a reference implementation in Python and an easy-to-use napari plugin.

Robust and unbiased estimation of the background distribution for automated quantitative imaging.

Background estimation is the first step in quantitative analysis of images. It has an impact on all subsequent analyses, in particular for segmentation and calculation of ratiometric quantities. Most methods recover only a single value such as the median or yield a biased estimation in non-trivial cases. We introduce, to our knowledge, the first method to recover an unbiased estimation of background distribution. It leverages the lack of local spatial correlation in background pixels to robustly select a subset that accurately represents the background. The resulting background distribution can be used to test for foreground membership of individual pixels or estimate confidence intervals in derived quantities.

Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains in cells.

Ubiquitin chains modify a major subset of the proteome, but detection of ubiquitin signaling dynamics and localization is limited due to a lack of appropriate tools. Here, we employ ubiquitin-binding domain (UBD)-based fluorescent sensors to monitor linear and K63-linked chains in vitro and in vivo. We utilize the UBD in NEMO and ABIN (UBAN) for detection of linear chains, and RAP80 ubiquitin-interacting motif (UIM) and TAB2 Npl4 zinc finger (NZF) domains to detect K63 chains. Linear and K63 sensors decorated the ubiquitin coat surrounding cytosolic Salmonella during bacterial autophagy, whereas K63 sensors selectively monitored Parkin-induced mitophagy and DNA damage responses in fixed and living cells. In addition, linear and K63 sensors could be used to monitor endogenous signaling pathways, as demonstrated by their ability to differentially interfere with TNF- and IL-1-induced NF-κB pathway. We propose that UBD-based biosensors could serve as prototypes to track and trace other chain types and ubiquitin-like signals in vivo.

Compact and reflective light-sheet microscopy for long-term imaging of living embryos.

The development of light-sheet fluorescence microscopy has been a revolution for developmental biology as it allows long-term imaging during embryonic development. An important reason behind the quick adoption has been the availability of open hardware alternatives. In this work, we present a robust and compact version of a light-sheet fluorescence microscope that is easy to assemble and requires little to no maintenance. An important aspect of the design is that the illumination unit consists of reflective elements, thereby reducing chromatic aberrations an order of magnitude as compared to refractive counterparts.

Multiplexed imaging of intracellular protein networks.

Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.

The autodepalmitoylating activity of APT maintains the spatial organization of palmitoylated membrane proteins.

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.

Multi-step loading of human minichromosome maintenance proteins in live human cells.

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.

Fluorescence fluctuations of quantum-dot sensors capture intracellular protein interaction dynamics.

We extend the in vitro principle of co-immunoprecipitation to quantify dynamic protein interactions in living cells. Using a multiresolution implementation of fluorescence correlation spectroscopy to achieve maximal temporal resolution, we monitored the interactions of endogenous bait proteins, recruited by quantum dots, with fluorescently tagged prey. With this approach, we analyzed the rapid physiological regulation of protein kinase A.

In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.

Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.

flatFLIM: enhancing the dynamic range of frequency domain FLIM.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a quantitative technique to probe the nanoenvironment of fluorescent molecules. It is the most robust way to quantify Förster Resonance Energy Transfer (FRET) as it allows reliable differentiation between concentration changes and quenching. In this way, molecular interactions can be imaged in single living cells. The most common wide-field implementation is homodyne Frequency Domain (FD) FLIM, which determines the fluorescence lifetime by measuring the phase and modulation changes of the fluorescence in each pixel upon excitation with a light source modulated at a high frequency. The fluorescence lifetimes are derived from a stack of images acquired at different phase shifts between excitation and detection. In this work we describe a simple method to enhance the dynamic range of FD-FLIM based on precompensating the expected fluorescence modulation by varying the laser power through the phase stack. We show theoretically and experimentally that most of the dynamic range of the camera can be recovered to quantify cells with different intensities. This improvement can be added to any FD-FLIM setup with minimal modifications, enhancing the throughput of information content.

FRET in cell biology: still shining in the age of super-resolution?

Interest in imaging of Förster resonance energy transfer (FRET) in biological systems has been steadily increasing in the last 30 years. The ability to transduce a near-field interaction into a far-field signal has provided a unique optical tool to assess biological phenomena well below the resolution of standard optical microscopy. In recent years, sub-diffraction microscopy techniques have achieved maturation and are increasingly used in biological applications. As the resolution of these methods increases they will slowly encroach on the domains where FRET is now dominant. Herein we review the major applications in biological FRET imaging and we discuss the possibilities and challenges in the super-resolution era.

CASPAM: A Triple-Modality Biosensor for Multiplexed Imaging of Caspase Network Activity.

Understanding signal propagation across biological networks requires to simultaneously monitor the dynamics of several nodes to uncover correlations masked by inherent intercellular variability. To monitor the enzymatic activity of more than two components over short time scales has proven challenging. Exploiting the narrow spectral width of homo-FRET-based biosensors, up to three activities can be imaged through fluorescence polarization anisotropy microscopy. We introduce Caspase Activity Sensor by Polarization Anisotropy Multiplexing (CASPAM) a single-plasmid triple-modality reporter of key nodes of the apoptotic network. Apoptosis provides an ideal molecular framework to study interactions between its three composing pathways (intrinsic, extrinsic, and effector). We characterized the biosensor performance and demonstrated the advantages that equimolar expression has in both simplifying experimental procedure and reducing observable variation, thus enabling robust data-driven modeling. Tools like CASPAM become essential to analyze molecular pathways where multiple nodes need to be simultaneously monitored.

Effective description of bistability and irreversibility in apoptosis.

Apoptosis is a mechanism of programmed cell death in which cells engage in a controlled demolition and prepare to be digested without damaging their environment. In normal conditions, apoptosis is repressed until it is irreversibly induced by an appropriate signal. In adult organisms, apoptosis is a natural way to dispose of damaged cells and its disruption or excess is associated with cancer and autoimmune diseases. Apoptosis is regulated by a complex signaling network controlled by caspases, specialized enzymes that digest essential cellular components and promote the degradation of genomic DNA. In this work, we propose an effective description of the signaling network focused on caspase-3 as a readout of cell fate. We integrate intermediate network interactions into a nonlinear feedback function acting on caspase-3 and introduce the effect of pro-apoptotic stimuli and regulatory elements as a saturating activation function. We show that activation dynamics in the theory is similar to previously reported experimental results. We compute bifurcation diagrams and obtain cell fate maps describing how stimulus intensity and feedback strength affect cell survival and death fates. These fates overlap within a bistable region that depends on total caspase concentration, regulatory elements, and feedback nonlinearity. We study a strongly nonlinear regime to obtain analytical expressions for bifurcation curves and fate map boundaries. For a broad range of parameters, strong stimuli can induce an irreversible switch to the death fate. We use the theory to explore dynamical stimulation conditions and determine how cell fate depends on stimulation temporal patterns. This analysis predicts a critical relation between transient stimuli intensity and duration to trigger irreversible apoptosis. We derive an analytical expression for this critical relation, valid for short stimuli. Our description provides distinct predictions and offers a framework to study how this signaling network processes different stimuli to make a cell fate decision.

A first approach to the use of upconversion nanoparticles to measure fluorescent tracers in water: a proof of concept.

In this work we use lanthanide based NaYF4:Er3+, Yb3+upconversion nanoparticles (UCNP) to detect ppb-level sensitibity of a xanthene dye, Rhodamine B (RB) dye, under NIR excitation. A static energy transfer was observed between the luminescent UCNP energy donors and RB acceptor in aqueous solution for three different sizes of UCNP. No specific covalent functionalization of the UCNPs was performed providing a direct method of detection, particularly promising in natural systems where the interfering fluorescence background is a detrimental limitation to the performance of the detection method. This procedure is a first approach to be applied in estuarine and coastal zone where the high content of suspended particulate matter prevents the detection of tracers.

Furry is required for cell movements during gastrulation and functionally interacts with NDR1.

Gastrulation is a key event in animal embryogenesis during which germ layer precursors are rearranged and the embryonic axes are established. Cell polarization is essential during gastrulation, driving asymmetric cell division, cell movements, and cell shape changes. The furry (fry) gene encodes an evolutionarily conserved protein with a wide variety of cellular functions, including cell polarization and morphogenesis in invertebrates. However, little is known about its function in vertebrate development. Here, we show that in Xenopus, Fry plays a role in morphogenetic processes during gastrulation, in addition to its previously described function in the regulation of dorsal mesoderm gene expression. Using morpholino knock-down, we demonstrate a distinct role for Fry in blastopore closure and dorsal axis elongation. Loss of Fry function drastically affects the movement and morphological polarization of cells during gastrulation and disrupts dorsal mesoderm convergent extension, responsible for head-to-tail elongation. Finally, we evaluate a functional interaction between Fry and NDR1 kinase, providing evidence of an evolutionarily conserved complex required for morphogenesis.

Interaction Between the Angiotensin-(1-7) Mas Receptor and the Dopamine D2 Receptor: Implications in Inflammation.

[Figure: see text].

An Integrative and Modular Framework to Recapitulate Emergent Behavior in Cell Migration.

Cell migration has been a subject of study in a broad variety of biological systems, from morphogenetic events during development to cancer progression. In this work, we describe single-cell movement in a modular framework from which we simulate the collective behavior of glioblastoma cells, the most prevalent and malignant primary brain tumor. We used the U87 cell line, which can be grown as a monolayer or spatially closely packed and organized in 3D structures called spheroids. Our integrative model considers the most relevant mechanisms involved in cell migration: chemotaxis of attractant factor, mechanical interactions and random movement. The effect of each mechanism is integrated into the overall probability of the cells to move in a particular direction, in an automaton-like approach. Our simulations fit and reproduced the emergent behavior of the spheroids in a set of migration assays where single-cell trajectories were tracked. We also predicted the effect of migration inhibition on the colonies from simple experimental characterization of single treated cell tracks. The development of tools that allow complementing molecular knowledge in migratory cell behavior is relevant for understanding essential cellular processes, both physiological (such as organ formation, tissue regeneration among others) and pathological perspectives. Overall, this is a versatile tool that has been proven to predict individual and collective behavior in U87 cells, but that can be applied to a broad variety of scenarios.

Robustness in spatially driven bistability in signaling systems.

Biological systems are spatially organized. This microscopic heterogeneity has been shown to produce emergent complex behaviors such as bistability. Even though the connection between spatiality and dynamic response is essential to understand biological output, its robustness and extent has not been sufficiently explored. This work focuses on a previously described system which is composed of two monostable modules acting on different cellular compartments and sharing species through linear shuttling reactions. One of the two main purposes of this paper is to quantify the frequency of occurrence of bistability throughout the parameter space and to identify which parameters and in which value ranges control the emergence and the properties of bistability. We found that a very small fraction of the sampled parameter space produced a bistable response. Most importantly, shuttling parameters were among the most influential ones to control this property. The other goal of this paper is to simplify the same system as much as possible without losing compartment-induced bistability. This procedure provided a simplified model that still connects two monostable systems by a reduced set of linear shuttling reactions that circulates all the species around the two compartments. Bistable systems are one of the main building blocks of more complex behaviors such as oscillations, memory, and digitalization. Therefore, we expect that the proposed minimal system provides insight into how these behaviors can arise from compartmentalization.