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.

It's not all that bad: associations among pain characteristics and sexual well-being in people living with chronic pain.

BACKGROUND: Individuals experiencing chronic pain often report adverse effects on their sexual functioning. However, other important aspects of sexual well-being (SWB), such as sexual distress and sexual self-esteem, have received little attention. This is an important omission because a SWB involves more than just good sexual function. Similarly, past research has not examined how chronic pain characteristics affect the different aspects of SWB. AIM: The goal of this cross-sectional study was to examine the SWB of individuals living with chronic pain and to examine the extent to which SWB is associated with different chronic pain characteristics. METHODS: A total of 310 individuals (28.1% men, 70.6% women, 1.3% transgender men) with ages between 21 and 50 (M = 31.96, SD = 6.13) who were in a romantic relationship and with self-reported chronic pain for three months or longer completed an online survey. OUTCOMES: The following indicators of SWB were included in the study: frequency of genital sexual activity, sexual satisfaction, sexual self-esteem, sexual desire, sexual function, genital pain, and sexual distress. RESULTS: The results show that most individuals with chronic pain maintain an active and satisfying sexual life and feel positive about themselves as a sexual partner. Slightly more than a fourth reported experiencing at least one sexual functioning difficulty and almost three-fourths of them found those difficulties sexually distressing. A first canonical correlation showed that more negative pain characteristics were associated with poorer SWB. The second canonical correlation showed that greater perceived partner support can offset the negative relationship between pain and some aspects of SWB. CLINICAL IMPLICATIONS: These findings show that individuals living with chronic pain can experience positive SWB. Furthermore, the buffering effect of partner support suggests it is important to involve romantic partners in interventions aimed at improving the SWB of people living with chronic pain. STRENGTHS AND LIMITATIONS: The study examined a large number of indicators of SWB using a sample of individuals with different types of chronic pain. Limitations include potential self-selection bias and a sample that was predominantly white and highly educated. CONCLUSIONS: The results paint a more positive picture of the SWB of individuals living with pain and show that individuals living with chronic pain can experience positive SWB. These findings can help for researchers, educators, and clinicians about how to conceptualize, understand, and improve the SWB of individuals living with chronic pain.

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.

A New Opportunity for "Old" Molecules: Targeting PARP1 Activity through a Non-Enzymatic Mechanism.

In recent years, new therapies have been developed based on molecules that target molecular mechanisms involved in both the initiation and maintenance of the oncogenic process. Among these molecules are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. PARP1 has emerged as a target with great therapeutic potential for some tumor types, drawing attention to this enzyme and resulting in many small molecule inhibitors of its enzymatic activity. Therefore, many PARP inhibitors are currently in clinical trials for the treatment of homologous recombination (HR)-deficient tumors, BRCA-related cancers, taking advantage of synthetic lethality. In addition, several novel cellular functions unrelated to its role in DNA repair have been described, including post-translational modification of transcription factors, or acting through protein-protein interactions as a co-activator or co-repressor of transcription. Previously, we reported that this enzyme may play a key role as a transcriptional co-activator of an important component of cell cycle regulation, the transcription factor E2F1. Here, we show that PARP inhibitors, which interfere with its activity in cell cycle regulation, perform this without affecting its enzymatic function.

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.

Psychometric Validation of the Sexual Distress Scale in Male and Female Portuguese Samples.

BACKGROUND: The Female Sexual Distress Scale and the Female Sexual Distress Scale-Revised-herein called the Sexual Distress Scale (SDS and SDS-R)-are among the most widely used self-report instruments to assess sexual distress, but no version for use in the Portuguese population is available to date. AIM: The current study aimed to validate the Portuguese version of the SDS/SDS-R in samples of women and men with and without distressing sexual problems. METHODS: A sample of 1,109 participants without distressing sexual problems (761 women) and 382 participants with distressing sexual problems (283 women), ages ranging from 18 to 72 years, were used to examine the psychometric properties of the Portuguese SDS and SDS-R. OUTCOMES: Participants completed a survey that included a sociodemographic and health questionnaire, the Portuguese version of the SDS and SDS-R, and measures of sexual satisfaction, sexual quality of life, sexual function, dyadic adjustment, and psychological distress. RESULTS: Results indicated that the Portuguese SDS and the SDS-R assess 1 general domain of sexual distress and showed good evidences of validity based on content and on relations with other variables. Sexual distress was associated with poorer sexual function, satisfaction, and quality of life, with higher psychological distress, and lower dyadic adjustment. Internal consistency and test-retest (1 month) reliabilities were excellent. Tests of differential functioning of items indicated that the SDS and SDS-R scores can be used to compare women and men on sexual distress, but the SDS/SDS-R scores flagged differential functioning of items and test (DFIT) between participants with and without distressing sexual problems. CLINICAL TRANSLATION: Clinicians and researchers can now make use of the SDS and of the SDS-R in the Portuguese population, facilitating the assessment of sexual distress in clinical settings. STRENGTHS & LIMITATIONS: The Portuguese SDS/SDS-R scores can be compared between women and men, providing information on sexual distress independently of sexual function. With the current evidence, comparisons between individuals with and without distressing sexual problems should be made with caution, as the scores may be biased against the former. CONCLUSION: This study provides a validation of the Portuguese version of the SDS/SDS-R that can be used to assess sexual distress in Portuguese women and men and can be used to compare between these 2 groups. Tavares IM, Santos-Iglesias P, Nobre PJ. Psychometric Validation of the Sexual Distress Scale in Male and Female Portuguese Samples. J Sex Med 2022;19:834-845.

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'.

Hard Times: Prostate Cancer Patients' Experiences with Erectile Aids.

BACKGROUND: Prostate cancer (PCa) treatments commonly lead to erectile difficulties. While the mainstay treatment is erectile aids (EAs) to promote erectile recovery, some men never use these treatments and those whose do use EAs often abandon them in the long-term. AIM: The goal of this study was to examine PCa patients' experiences with EAs, to elucidate relationships between experiences with EAs on psychological and sexual well-being, and to explore benefits and drawbacks to EA use. METHODS: A self-report survey including validated questionnaires was administered to examine PCa patients' use and perceptions of helpfulness of EAs, and to characterize associations between use, perceived helpfulness, and psychological and sexual well-being. The survey was followed by an open-ended prompt to explore participants' experiences with EAs. OUTCOMES: We surveyed 260 North American men, up to 25 years after receiving treatment for PCa. Three groups of patients were observed, including those who used EAs and perceived them to be helpful, those who used EAs and perceived them to be unhelpful, as well as a smaller group of patients who never used EAs. RESULTS: Around 80% of the sample were using or had used EAs. Despite the high frequency of use, not all men found EAs helpful. Men who used EAs and found them unhelpful reported poorer psychological and sexual well-being compared to men who didn't use aids or who used EAs but found them helpful. Results indicated both benefits and drawbacks to the use of EAs. Benefits related largely to the efficacy of the aid in promoting erections. A wide range of drawbacks were also reported. CLINICAL IMPLICATIONS: Given the negative sexual and psychological impacts associated with using EAs and finding them unhelpful, we suggest that researchers and health care providers should take care to proactively address potential challenges that are common with EA use, and also to consider the risks of failed attempts with EAs. STRENGTHS & LIMITATIONS: By using both scaled and open-ended questions, a more nuanced picture of the relative benefits and limitations of EA use within the PCa population is presented. As responses were not mandatory, a subset of participants provided comments about the use of EAs. Additionally, the sample was quite homogenous, with mostly white, American and well-educated participants, so it therefore lacks generalizability to other populations. CONCLUSION: This paper illustrates several challenges to EA use, while providing insight into reasons for abandonment of use of EAs. Walker LM, Sears CS, Santos-Iglesias P, et al. Hard Times: Prostate Cancer Patients' Experiences with Erectile Aids. J Sex Med 2021;18:1775-1787.

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.

Preliminary validation of the Sexual Distress Scale-Short Form: Applications to Women, Men, and Prostate Cancer Survivors.

The Sexual Distress Scale (SDS) can be used to assess sexual distress in women, men, and prostate cancer (PCa) survivors. Despite its strong psychometric properties, researchers and clinicians could benefit from a short form of the scale. Two studies were conducted to develop (Study 1) and validate (Study 2) a short form of the SDS (SDS-SF) using samples of women, men, and PCa survivors from previous studies. Results of Study 1 suggested a 5-item SDS-SF. Study 2 showed that the SDS-SF items clustered in one factor with good fit across the three samples and excellent reliability. Sexual distress was associated with higher sexual bother, and poorer sexual satisfaction, sexual function, and relationship quality. The SDS-SF discriminated participants with and without distressing sexual problems. The SDS-SF facilitates the assessment of sexual distress in clinical settings by providing a quick way of screening patients with high levels of sexual distress.

On the Relationship Between Erectile Function and Sexual Distress in Men with Prostate Cancer.

Erectile difficulties are common after prostate cancer (PCa) treatment and are associated with sexual distress. However, the relationship between erectile function and sexual distress has yet to be carefully examined. This study had three goals: (1) examine the relationship between erectile function and sexual distress; (2) determine groups of men based on erectile function and sexual distress; and (3) examine the psychosexual characteristics of these groups. A cross section of 233 sexually active men after PCa treatment (age M = 64.90 years, SD = 7.50) completed an online survey containing demographic, health, and sexuality and relationship questionnaires. The relationship between erectile function and sexual distress was curvilinear. Four groups of men were found: good erectile function and low sexual distress, poor erectile function and high sexual distress, but also good erectile function yet high sexual distress, and poor erectile function and low sexual distress. Regardless of erectile function, men with greater sexual distress were more depressed, reported additional sexual concerns, placed less value on sex, were less sexually satisfied, and used protective buffering communication more frequently. They were also less likely to be satisfied with their adaptation to sexual changes and less likely to have found a solution to those changes. The relationship between erectile function and sexual distress is complex, characterized by a wide array of responses to erectile function (high and low distress) and multiple correlates of sexual distress. These results broaden the concept of sexual recovery after PCa treatment, which may assist clinicians and researchers to better address sexual problems after PCa treatment.

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.