Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.

Excitable Signal Transduction Networks in Directed Cell Migration.

Although directed migration of eukaryotic cells may have evolved to escape nutrient depletion, it has been adopted for an extensive range of physiological events during development and in the adult organism. The subversion of these movements results in disease, such as cancer. Mechanisms of propulsion and sensing are extremely diverse, but most eukaryotic cells move by extending actin-filled protrusions termed macropinosomes, pseudopodia, or lamellipodia or by extension of blebs. In addition to motility, directed migration involves polarity and directional sensing. The hundreds of gene products involved in these processes are organized into networks of parallel and interconnected pathways. Many of these components are activated or inhibited coordinately with stimulation and on each spontaneously extended protrusion. Moreover, these networks display hallmarks of excitability, including all-or-nothing responsiveness and wave propagation. Cellular protrusions result from signal transduction waves that propagate outwardly from an origin and drive cytoskeletal activity. The range of the propagating waves and hence the size of the protrusions can be altered by lowering or raising the threshold for network activation, with larger and wider protrusions favoring gliding or oscillatory behavior over amoeboid migration. Here, we evaluate the variety of models of excitable networks controlling directed migration and outline critical tests. We also discuss the utility of this emerging view in producing cell migration and in integrating the various extrinsic cues that direct migration.

Nucleating new branches from old.

Centrosome- and chromatin-based microtubule nucleation pathways have been implicated in spindle assembly. Using total internal reflection fluorescent microscopy and Xenopus egg extracts, Petry et al. demonstrate that new microtubules can also nucleate and branch out from existing ones in animal cells.

Nanotopography modulates intracellular excitable systems through cytoskeleton actuation.

Cellular sensing of most environmental cues involves receptors that affect a signal-transduction excitable network (STEN), which is coupled to a cytoskeletal excitable network (CEN). We show that the mechanism of sensing of nanoridges is fundamentally different. CEN activity occurs preferentially on nanoridges, whereas STEN activity is constrained between nanoridges. In the absence of STEN, waves disappear, but long-lasting F-actin puncta persist along the ridges. When CEN is suppressed, wave propagation is no longer constrained by nanoridges. A computational model reproduces these experimental observations. Our findings indicate that nanotopography is sensed directly by CEN, whereas STEN is only indirectly affected due to a CEN-STEN feedback loop. These results explain why texture sensing is robust and acts cooperatively with multiple other guidance cues in complex, in vivo microenvironments.

Reverse fountain flow of phosphatidylinositol-3,4-bisphosphate polarizes migrating cells.

The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and of the phospholipid phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Here, we show that lagging-edge component PI(3,4)P2 also localizes to retracting leading-edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Once internalized, macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which fuse with the plasma membrane at the rear of the cell. Subsequently, the phosphoinositide diffuses toward the front of the cell, where it is degraded. Computational modeling confirms that this cycle gives rise to stable back-to-front gradient. These results uncover a surprising "reverse-fountain flow" of PI(3,4)P2 that regulates polarity.

Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function.

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

Particle-based model of mechanosensory contractility kit assembly.

Cell shape change processes, such as proliferation, polarization, migration, and cancer metastasis, rely on a dynamic network of macromolecules. The proper function of this network enables mechanosensation, the ability of cells to sense and respond to mechanical cues. Myosin II and cortexillin I, critical elements of the cellular mechanosensory machinery, preassemble in the cytoplasm of Dictyostelium cells into complexes that we have termed contractility kits (CKs). Two IQGAP proteins then differentially regulate the mechanoresponsiveness of the cortexillin I-myosin II elements within CKs. To investigate the mechanism of CK self-assembly and gain insight into possible molecular means for IQGAP regulation, we developed a coarse-grained excluded volume molecular model in which all protein polymers are represented by nm-sized spheres connected by spring-like links. The model is parameterized using experimentally measured parameters acquired through fluorescence cross-correlation spectroscopy and fluorescence correlation spectroscopy, which describe the interaction affinities and diffusion coefficients for individual molecular components, and which have also been validated via several orthogonal methods. Simulations of wild-type and null-mutant conditions implied that the temporal order of assembly of these kits is dominated by myosin II dimer formation and that IQGAP proteins mediate cluster growth. In addition, our simulations predicted the existence of "ambiguous" CKs that incorporate both classes of IQGAPs, and we confirmed this experimentally using fluorescence cross-correlation spectroscopy. The model serves to describe the formation of the CKs and how their assembly enables and regulates mechanosensation at the molecular level.

Three-dimensional stochastic simulation of chemoattractant-mediated excitability in cells.

During the last decade, a consensus has emerged that the stochastic triggering of an excitable system drives pseudopod formation and subsequent migration of amoeboid cells. The presence of chemoattractant stimuli alters the threshold for triggering this activity and can bias the direction of migration. Though noise plays an important role in these behaviors, mathematical models have typically ignored its origin and merely introduced it as an external signal into a series of reaction-diffusion equations. Here we consider a more realistic description based on a reaction-diffusion master equation formalism to implement these networks. In this scheme, noise arises naturally from a stochastic description of the various reaction and diffusion terms. Working on a three-dimensional geometry in which separate compartments are divided into a tetrahedral mesh, we implement a modular description of the system, consisting of G-protein coupled receptor signaling (GPCR), a local excitation-global inhibition mechanism (LEGI), and signal transduction excitable network (STEN). Our models implement detailed biochemical descriptions whenever this information is available, such as in the GPCR and G-protein interactions. In contrast, where the biochemical entities are less certain, such as the LEGI mechanism, we consider various possible schemes and highlight the differences between them. Our simulations show that even when the LEGI mechanism displays perfect adaptation in terms of the mean level of proteins, the variance shows a dose-dependence. This differs between the various models considered, suggesting a possible means for determining experimentally among the various potential networks. Overall, our simulations recreate temporal and spatial patterns observed experimentally in both wild-type and perturbed cells, providing further evidence for the excitable system paradigm. Moreover, because of the overall importance and ubiquity of the modules we consider, including GPCR signaling and adaptation, our results will be of interest beyond the field of directed migration.

A mesoscale mechanical model of cellular interactions.

Computational models of cell mechanics allow the precise interrogation of cell shape change. These morphological changes are required for cells to survive in diverse tissue environments. Here, we present a mesoscale mechanical model of cell-substrate interactions using the level set method based on experimentally measured parameters. By implementing a viscoelastic mechanical equivalent circuit, we accurately model whole-cell deformations that are important for a variety of cellular processes. To effectively model shape changes as a cell interacts with a substrate, we have included receptor-mediated adhesion, which is governed by catch-slip bond behavior. The effect of adhesion was explored by subjecting cells to a variety of different substrates including flat, curved, and deformable surfaces. Finally, we increased the accuracy of our simulations by including a deformable nucleus in our cells. This model sets the foundation for further exploration into computational analyses of multicellular interactions.

How the mechanobiome drives cell behavior, viewed through the lens of control theory.

Cells have evolved sophisticated systems that integrate internal and external inputs to coordinate cell shape changes during processes, such as development, cell identity determination, and cell and tissue homeostasis. Cellular shape-change events are driven by the mechanobiome, the network of macromolecules that allows cells to generate, sense and respond to externally imposed and internally generated forces. Together, these components build the cellular contractility network, which is governed by a control system. Proteins, such as non-muscle myosin II, function as both sensors and actuators, which then link to scaffolding proteins, transcription factors and metabolic proteins to create feedback loops that generate the foundational mechanical properties of the cell and modulate cellular behaviors. In this Review, we highlight proteins that establish and maintain the setpoint, or baseline, for the control system and explore the feedback loops that integrate different cellular processes with cell mechanics. Uncovering the genetic, biophysical and biochemical interactions between these molecular components allows us to apply concepts from control theory to provide a systems-level understanding of cellular processes. Importantly, the actomyosin network has emerged as more than simply a 'downstream' effector of linear signaling pathways. Instead, it is also a significant driver of cellular processes traditionally considered to be 'upstream'.

Sensitivity minimization, biological homeostasis and information theory.

All organisms must be able to adapt to changes in the environment. To this end, they have developed sophisticated regulatory mechanisms to ensure homeostasis. Control engineers, who must design similar regulatory systems, have developed a number of general principles that govern feedback regulation. These lead to constraints which impose trade-offs that arise when developing controllers to minimize the effect of external disturbances on systems. Here, we review some of these trade-offs, particularly Bode's integral formula. We also highlight its connection to information theory, by showing that the constraints in sensitivity minimization can be cast as limitations on the information transmission through a system, and these have their root in causality. Finally, we look at how these constraints arise in two biological systems: glycolytic oscillations and the energy cost of perfect adaptation in a bacterial chemotactic pathway.

Mutually inhibitory Ras-PI(3,4)P2 feedback loops mediate cell migration.

Signal transduction and cytoskeleton networks in a wide variety of cells display excitability, but the mechanisms are poorly understood. Here, we show that during random migration and in response to chemoattractants, cells maintain complementary spatial and temporal distributions of Ras activity and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. In addition, depletion of PI(3,4)P2 by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading, and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2, and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down, causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive-feedback loop essential for excitability. Consistently, a computational model incorporating such a feedback loop in an excitable network model accurately simulates the dynamic distributions of active Ras and PI(3,4)P2 as well as cell migratory behavior. The mutually inhibitory Ras-PI(3,4)P2 mechanisms we uncovered here provide a framework for Ras regulation that may play a key role in many physiological processes.

Wave patterns organize cellular protrusions and control cortical dynamics.

Cellular protrusions are typically considered as distinct structures associated with specific regulators. However, we found that these regulators coordinately localize as propagating cortical waves, suggesting a common underlying mechanism. These molecular events fell into two excitable networks, the signal transduction network STEN and the cytoskeletal network CEN with different wave substructures. Computational studies using a coupled-network model reproduced these features and showed that the morphology and kinetics of the waves depended on strengths of feedback loops. Chemically induced dimerization at multiple nodes produced distinct, coordinated alterations in patterns of other network components. Taken together, these studies indicate: STEN positive feedback is mediated by mutual inhibition between Ras/Rap and PIP2, while negative feedback depends on delayed PKB activation; PKBs link STEN to CEN; CEN includes positive feedback between Rac and F-actin, and exerts fast positive and slow negative feedbacks to STEN The alterations produced protrusions resembling filopodia, ruffles, pseudopodia, or lamellipodia, suggesting that these structures arise from a common regulatory mechanism and that the overall state of the STEN-CEN system determines cellular morphology.

Controlling excitable wave behaviors through the tuning of three parameters.

Excitable systems are a class of dynamical systems that can generate self-sustaining waves of activity. These waves are known to manifest differently under diverse conditions, whereas some travel as planar or radial waves, and others evolve into rotating spirals. Excitable systems can also form stationary stable patterns through standing waves. Under certain conditions, these waves are also known to be reflected at no-flux boundaries. Here, we review the basic characteristics of these four entities: traveling, rotating, standing and reflected waves. By studying their mechanisms of formation, we show how through manipulation of three critical parameters: time-scale separation, space-scale separation and threshold, we can interchangeably control the formation of all the aforementioned wave types.

Control theory in biology and medicine : Introduction to the special issue.

From September-December 2017, the Mathematical Biosciences Institute at Ohio State University hosted a series of workshops on control theory in biology and medicine, including workshops on control and modulation of neuronal and motor systems, control of cellular and molecular systems, control of disease / personalized medicine across heterogeneous populations, and sensorimotor control of animals and robots. This special issue presents tutorials and research articles by several of the participants in the MBI workshops.

Chemical and mechanical stimuli act on common signal transduction and cytoskeletal networks.

Signal transduction pathways activated by chemoattractants have been extensively studied, but little is known about the events mediating responses to mechanical stimuli. We discovered that acute mechanical perturbation of cells triggered transient activation of all tested components of the chemotactic signal transduction network, as well as actin polymerization. Similarly to chemoattractants, the shear flow-induced signal transduction events displayed features of excitability, including the ability to mount a full response irrespective of the length of the stimulation and a refractory period that is shared with that generated by chemoattractants. Loss of G protein subunits, inhibition of multiple signal transduction events, or disruption of calcium signaling attenuated the response to acute mechanical stimulation. Unlike the response to chemoattractants, an intact actin cytoskeleton was essential for reacting to mechanical perturbation. These results taken together suggest that chemotactic and mechanical stimuli trigger activation of a common signal transduction network that integrates external cues to regulate cytoskeletal activity and drive cell migration.

Cytokinesis: Robust cell shape regulation.

Cytokinesis, the final step of cell division, is a great example of robust cell shape regulation. A wide variety of cells ranging from the unicellular Dictyostelium to human cells in tissues proceed through highly similar, stereotypical cell shape changes during cell division. Typically, cells first round up forming a cleavage furrow in the middle, which constricts resulting in the formation of two daughter cells. Tight control of cytokinesis is essential for proper segregation of genetic and cellular materials, and its failure is deleterious to cell viability. Thus, biological systems have developed elaborate mechanisms to ensure high fidelity of cytokinesis, including the existence of multiple biochemical and mechanical pathways regulated through feedback. In this review, we focus on the built-in redundancy of the cytoskeletal machinery that allows cells to divide successfully in a variety of biological and mechanical contexts. Using Dictyostelium cytokinesis as an example, we demonstrate that the crosstalk between biochemical and mechanical signaling through feedback ensures correct assembly and function of the cell division machinery.

Novel protein Callipygian defines the back of migrating cells.

Asymmetric protein localization is essential for cell polarity and migration. We report a novel protein, Callipygian (CynA), which localizes to the lagging edge before other proteins and becomes more tightly restricted as cells polarize; additionally, it accumulates in the cleavage furrow during cytokinesis. CynA protein that is tightly localized, or "clustered," to the cell rear is immobile, but when polarity is disrupted, it disperses throughout the membrane and responds to uniform chemoattractant stimulation by transiently localizing to the cytosol. These behaviors require a pleckstrin homology-domain membrane tether and a WD40 clustering domain, which can also direct other membrane proteins to the back. Fragments of CynA lacking the pleckstrin homology domain, which are normally found in the cytosol, localize to the lagging edge membrane when coexpressed with full-length protein, showing that CynA clustering is mediated by oligomerization. Cells lacking CynA have aberrant lateral protrusions, altered leading-edge morphology, and decreased directional persistence, whereas those overexpressing the protein display exaggerated features of polarity. Consistently, actin polymerization is inhibited at sites of CynA accumulation, thereby restricting protrusions to the opposite edge. We suggest that the mutual antagonism between CynA and regions of responsiveness creates a positive feedback loop that restricts CynA to the rear and contributes to the establishment of the cell axis.

Endoplasmic Reticulum Lipid Flux Influences Enterocyte Nuclear Morphology and Lipid-dependent Transcriptional Responses.

Responding to a high-fat meal requires an interplay between multiple digestive tissues, sympathetic response pathways, and the gut microbiome. The epithelial enterocytes of the intestine are responsible for absorbing dietary nutrients and preparing them for circulation to distal tissues, which requires significant changes in cellular activity, including both morphological and transcriptional responses. Following a high-fat meal, we observe morphological changes in the enterocytes of larval zebrafish, including elongation of mitochondria, formation and expansion of lipid droplets, and the rapid and transient ruffling of the nuclear periphery. Dietary and pharmacological manipulation of zebrafish larvae demonstrated that these subcellular changes are specific to triglyceride absorption. The transcriptional changes that occur simultaneously with these morphological changes were determined using RNA sequencing, revealing a cohort of up-regulated genes associated with lipid droplet formation and lipid transport via lipoprotein particles. Using a microsomal triglyceride transfer protein (MTP) inhibitor to block ß-lipoprotein particle formation, we demonstrate that the transcriptional response to a high-fat meal is associated with the transfer of ER triglyceride to nascent ß-lipoproteins, possibly through the activation of Creb3l3/cyclic AMP-responsive element-binding protein. These data suggest that a transient increase in ER lipids is the likely mediator of the initial physiological response of intestinal enterocytes to dietary lipid.

Navigating through models of chemotaxis.

Chemotaxis in eukaryotic cells involves the coordination of several related but separable processes: motility, polarization, and gradient sensing. Mathematical models that have been proposed to explain chemotaxis typically focus on only one of these processes. We summarize the strengths and weaknesses of the models and point out the need for an integrated model.