AGS3-based optogenetic GDI induces GPCR-independent Gßγ signaling and macrophage migration.

G protein-coupled receptors (GPCRs) are efficient Guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP, and free Gßγ, and are major disease drivers. Evidence shows that the ambient low threshold signaling required for cells is likely supplemented by signaling regulators such as non-GPCR GEFs and Guanine nucleotide Dissociation Inhibitors (GDIs). Activators of G protein Signaling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signaling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3's G protein regulatory (GPR) motif, to understand its GDI activity and induce standalone Gßγ signaling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gßγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signaling pathways and triggering GPCR-independent Gßγ signaling in cells and in vivo.

Spatiotemporal Optical Control of Gαq-PLCß Interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Spatiotemporal optical control of Gαq-PLCß interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Molecular regulation of PLCß signaling.

Phospholipase C (PLC) enzymes convert the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG regulate numerous downstream pathways, eliciting diverse and profound cellular changes and physiological responses. In the six PLC subfamilies in higher eukaryotes, PLCß is intensively studied due to its prominent role in regulating crucial cellular events underlying many processes including cardiovascular and neuronal signaling, and associated pathological conditions. In addition to GαqGTP, Gßγ generated upon G protein heterotrimer dissociation also regulates PLCß activity. Here, we not only review how Gßγ directly activates PLCß, and also extensively modulates Gαq-mediated PLCß activity, but also provide a structure-function overview of PLC family members. Given that Gαq and PLCß are oncogenes, and Gßγ shows unique cell-tissue-organ specific expression profiles, Gγ subtype-dependent signaling efficacies, and distinct subcellular activities, this review proposes that Gßγ is a major regulator of Gαq-dependent and independent PLCß signaling.

The spatial distribution of GPCR and Gßγ activity across a cell dictates PIP3 dynamics.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various diseases, including cancer, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.

G protein gamma subunit, a hidden master regulator of GPCR signaling.

Heterotrimeric G proteins (αßγ subunits) that are activated by G protein-coupled receptors (GPCRs) mediate the biological responses of eukaryotic cells to extracellular signals. The α subunits and the tightly bound ßγ subunit complex of G proteins have been extensively studied and shown to control the activity of effector molecules. In contrast, the potential roles of the large family of γ subunits have been less studied. In this review, we focus on present knowledge about these proteins. Induced loss of individual γ subunit types in animal and plant models result in strikingly distinct phenotypes indicating that γ subtypes play important and specific roles. Consistent with these findings, downregulation or upregulation of particular γ subunit types result in various types of cancers. Clues about the mechanistic basis of γ subunit function have emerged from imaging the dynamic behavior of G protein subunits in living cells. This shows that in the basal state, G proteins are not constrained to the plasma membrane but shuttle between membranes and on receptor activation ßγ complexes translocate reversibly to internal membranes. The translocation kinetics of ßγ complexes varies widely and is determined by the membrane affinity of the associated γ subtype. On translocating, some ßγ complexes act on effectors in internal membranes. The variation in translocation kinetics determines differential sensitivity and adaptation of cells to external signals. Membrane affinity of γ subunits is thus a parsimonious and elegant mechanism that controls information flow to internal cell membranes while modulating signaling responses.

A short C-terminal peptide in Gγ regulates Gßγ signaling efficacy.

G protein beta-gamma (Gßγ) subunits anchor to the plasma membrane (PM) through the carboxy-terminal (CT) prenyl group in Gγ. This interaction is crucial for the PM localization and functioning of Gßγ, allowing GPCR-G protein signaling to proceed. The diverse Gγ family has 12 members, and we have recently shown that the signaling efficacies of major Gßγ effectors are Gγ-type dependent. This dependency is due to the distinct series of membrane-interacting abilities of Gγ. However, the molecular process allowing for Gßγ subunits to exhibit a discrete and diverse range of Gγ-type-dependent membrane affinities is unclear and cannot be explained using only the type of prenylation. The present work explores the unique designs of membrane-interacting CT residues in Gγ as a major source for this Gγ-type-dependent Gßγ signaling. Despite the type of prenylation, the results show signaling efficacy at the PM, and associated cell behaviors of Gßγ are governed by crucially located specific amino acids in the five to six residue preprenylation region of Gγ. The provided molecular picture of Gγ-membrane interactions may explain how cells gain Gγ-type-dependent G protein-GPCR signaling as well as how Gßγ elicits selective signaling at various subcellular compartments.

Dissociation of the G protein ßγ from the Gq-PLCß complex partially attenuates PIP2 hydrolysis.

Phospholipase C ß (PLCß), which is activated by the Gq family of heterotrimeric G proteins, hydrolyzes the inner membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). Because Gq and PLCß regulate many crucial cellular processes and have been identified as major disease drivers, activation and termination of PLCß signaling by the Gαq subunit have been extensively studied. Gq-coupled receptor activation induces intense and transient PIP2 hydrolysis, which subsequently recovers to a low-intensity steady-state equilibrium. However, the molecular underpinnings of this equilibrium remain unclear. Here, we explored the influence of signaling crosstalk between Gq and Gi/o pathways on PIP2 metabolism in living cells using single-cell and optogenetic approaches to spatially and temporally constrain signaling. Our data suggest that the Gßγ complex is a component of the highly efficient lipase GαqGTP-PLCß-Gßγ. We found that over time, Gßγ dissociates from this lipase complex, leaving the less-efficient GαqGTP-PLCß lipase complex and allowing the significant partial recovery of PIP2 levels. Our findings also indicate that the subtype of the Gγ subunit in Gßγ fine-tunes the lipase activity of Gq-PLCß, in which cells expressing Gγ with higher plasma membrane interaction show lower PIP2 recovery. Given that Gγ shows cell- and tissue-specific subtype expression, our findings suggest the existence of tissue-specific distinct Gq-PLCß signaling paradigms. Furthermore, these results also outline a molecular process that likely safeguards cells from excessive Gq signaling.

Subtype-dependent regulation of Gßγ signalling.

G protein-coupled receptors (GPCRs) transmit information to the cell interior by transducing external signals to heterotrimeric G protein subunits, Gα and Gßγ subunits, localized on the inner leaflet of the plasma membrane. Though the initial focus was mainly on Gα-mediated events, Gßγ subunits were later identified as major contributors to GPCR-G protein signalling. A broad functional array of Gßγ signalling has recently been attributed to Gß and Gγ subtype diversity, comprising 5 Gß and 12 Gγ subtypes, respectively. In addition to displaying selectivity towards each other to form the Gßγ dimer, numerous studies have identified preferences of distinct Gßγ combinations for specific GPCRs, Gα subtypes and effector molecules. Importantly, Gß and Gγ subtype-dependent regulation of downstream effectors, representing a diverse range of signalling pathways and physiological functions have been found. Here, we review the literature on the repercussions of Gß and Gγ subtype diversity on direct and indirect regulation of GPCR/G protein signalling events and their physiological outcomes. Our discussion additionally provides perspective in understanding the intricacies underlying molecular regulation of subtype-specific roles of Gßγ signalling and associated diseases.

Optical approaches for single-cell and subcellular analysis of GPCR-G protein signaling.

G protein-coupled receptors (GPCRs), G proteins, and their signaling associates are major signal transducers that control the majority of cellular signaling and regulate key biological functions including immune, neurological, cardiovascular, and metabolic processes. These pathways are targeted by over one-third of drugs on the market; however, the current understanding of their function is limited and primarily derived from cell-destructive approaches providing an ensemble of static, multi-cell information about the status and composition of molecules. Spatiotemporal behavior of molecules involved is crucial to understanding in vivo cell behaviors both in health and disease, and the advent of genetically encoded fluorescence proteins and small fluorophore-based biosensors has facilitated the mapping of dynamic signaling in cells with subcellular acuity. Since we and others have developed optogenetic methods to regulate GPCR-G protein signaling in single cells and subcellular regions using dedicated wavelengths, the desire to develop and adopt optogenetically amenable assays to measure signaling has motivated us to take a broader look at the available optical tools and approaches compatible with measuring single-cell and subcellular GPCR-G protein signaling. Here we review such key optical approaches enabling the examination of GPCR, G protein, secondary messenger, and downstream molecules such as kinase and lipid signaling in living cells. The methods reviewed employ both fluorescence and bioluminescence detection. We not only further elaborate the underlying principles of these sensors but also discuss the experimental criteria and limitations to be considered during their use in single-cell and subcellular signal mapping.

G protein αq exerts expression level-dependent distinct signaling paradigms.

G protein αq-coupled receptors (Gq-GPCRs) primarily signal through GαqGTP mediated phospholipase Cß (PLCß) stimulation and the subsequent hydrolysis of phosphatidylinositol 4, 5 bisphosphate (PIP2). Though Gq-heterotrimer activation results in both GαqGTP and Gßγ, unlike Gi/o-receptors, it is unclear if Gq-coupled receptors employ Gßγ as a major signal transducer. Compared to Gi/o- and Gs-coupled receptors, we observed that most cell types exhibit a limited free Gßγ generation upon Gq-pathway and Gαq/11 heterotrimer activation. We show that cells transfected with Gαq or endogenously expressing more than average-levels of Gαq/11 compared to Gαs and Gαi exhibit a distinct signaling regime primarily characterized by recovery-resistant PIP2 hydrolysis. Interestingly, the elevated Gq-expression is also associated with enhanced free Gßγ generation and signaling. Furthermore, the gene GNAQ, which encodes for Gαq, has recently been identified as a cancer driver gene. We also show that GNAQ is overexpressed in tumor samples of patients with Kidney Chromophobe (KICH) and Kidney renal papillary (KIRP) cell carcinomas in a matched tumor-normal sample analysis, which demonstrates the clinical significance of Gαq expression. Overall, our data indicates that cells usually express low Gαq levels, likely safeguarding cells from excessive calcium as wells as from Gßγ signaling.

Statins Perturb Gßγ Signaling and Cell Behavior in a Gγ Subtype Dependent Manner.

Guanine nucleotide-binding proteins (G proteins) facilitate the transduction of external signals to the cell interior, regulate most eukaryotic signaling, and thus have become crucial disease drivers. G proteins largely function at the inner leaflet of the plasma membrane (PM) using covalently attached lipid anchors. Both small monomeric and heterotrimeric G proteins are primarily prenylated, either with a 15-carbon farnesyl or a 20-carbon geranylgeranyl polyunsaturated lipid. The mevalonate [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase] pathway synthesizes lipids for G-protein prenylation. It is also the source of the precursor lipids for many biomolecules, including cholesterol. Consequently, the rate-limiting enzymes of the mevalonate pathway are major targets for cholesterol-lowering medications and anticancer drug development. Although prenylated G protein γ (Gγ) is essential for G protein-coupled receptor (GPCR)-mediated signaling, how mevalonate pathway inhibitors, statins, influence subcellular distribution of Gßγ dimer and Gαßγ heterotrimer, as well as their signaling upon GPCR activation, is poorly understood. The present study shows that clinically used statins not only significantly disrupt PM localization of Gßγ but also perturb GPCR-G protein signaling and associated cell behaviors. The results also demonstrate that the efficiency of prenylation inhibition by statins is Gγ subtype-dependent and is more effective toward farnesylated Gγ types. Since Gγ is required for Gßγ signaling and shows a cell- and tissue-specific subtype distribution, the present study can help understand the mechanisms underlying clinical outcomes of statin use in patients. This work also reveals the potential of statins as clinically usable drugs to control selected GPCR-G protein signaling.

G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gßγ.

Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gßγ. Since we have shown that signaling efficacy of Gßγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gßγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gßγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization.

Regulation of G Protein ßγ Signaling.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) deliver external signals to the cell interior, upon activation by the external signal stimulated G protein-coupled receptors (GPCRs).While the activated GPCRs control several pathways independently, activated G proteins control the vast majority of cellular and physiological functions, ranging from vision to cardiovascular homeostasis. Activated GPCRs dissociate GαGDPßγ heterotrimer into GαGTP and free Gßγ. Earlier, GαGTP was recognized as the primary signal transducer of the pathway and Gßγ as a passive signaling modality that facilitates the activity of Gα. However, Gßγ later found to regulate more number of pathways than GαGTP does. Once liberated from the heterotrimer, free Gßγ interacts and activates a diverse range of signaling regulators including kinases, lipases, GTPases, and ion channels, and it does not require any posttranslation modifications. Gßγ family consists of 48 members, which show cell- and tissue-specific expressions, and recent reports show that cells employ the subtype diversity in Gßγ to achieve desired signaling outcomes. In addition to activated GPCRs, which induce free Gßγ generation and the rate of GTP hydrolysis in Gα, which sequester Gßγ in the heterotrimer, terminating Gßγ signaling, additional regulatory mechanisms exist to regulate Gßγ activity. In this chapter, we discuss structure and function, subtype diversity and its significance in signaling regulation, effector activation, regulatory mechanisms as well as the disease relevance of Gßγ in eukaryotes.

Melanopsin (Opn4) utilizes Gαi and Gßγ as major signal transducers.

Melanopsin (Opn4), a ubiquitously expressed photoreceptor in all classes of vertebrates, is crucial for both visual and non-visual signaling. Opn4 supports visual functions of the eye by sensing radiance levels and discriminating contrast and brightness. Non-image-forming functions of Opn4 not only regulate circadian behavior, but also control growth and development processes of the retina. It is unclear how a single photoreceptor could govern such a diverse range of physiological functions; a role in genetic hardwiring could be one explanation, but molecular and mechanistic evidence is lacking. In addition to its role in canonical Gq pathway activation, here we demonstrate that Opn4 efficiently activates Gi heterotrimers and signals through the G protein ßγ. Compared with the low levels of Gi pathway activation observed for several Gq-coupled receptors, the robust Gαi and Gßγ signaling of Opn4 led to both generation of PIP3 and directional migration of RAW264.7 macrophages. We propose that the ability of Opn4 to signal through Gαi and Gßγ subunits is a major contributor to its functional diversity.

Gγ identity dictates efficacy of Gßγ signaling and macrophage migration.

G protein ßγ subunit (Gßγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gßγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gßγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gßγ and thereby differentially modulate Gßγ effector signaling in a Gγ-specific manner. Both Gßγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-CaaX motif residues at the PM-interacting region of Gγ control the PM affinity of Gßγ. We further show that the overall PM affinity of the Gßγ pool of a cell type is a strong predictor of its Gßγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gßγ behaviors not only recapitulated experimentally observed signaling of Gßγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gßγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gßγ.

Measurement of GPCR-G protein activity in living cells.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic genomes. They control a variety of cellular and physiological processes such as hormone secretion and heart rate, and therefore are associated with a majority of pathological conditions including cancer and heart diseases. Currently established assays to measure ligand-induced activation of GPCRs and G proteins possess limitations such as being time consuming, indirect, and expensive. Thus, an efficient method to measure GPCR-G protein activation is required to identify novel pharmacological modulators to control them and gain insights about molecular underpinnings of the associated pathways. Activation of GPCRs induces dissociation of G protein heterotrimers to form GαGTP and free Gßγ. Free Gßγ subunits have been shown to translocate reversibly from the plasma membrane to internal membranes. Gßγ translocation therefore represents the GPCR-G protein activation, and thus, imaging of this process can be used to quantify the kinetics and magnitude of the pathway activation-deactivation in real time in living cells. The objective of this chapter is to elaborate the protocols of (i) generation and optimization of the required sensor constructs; (ii) development of cell culture, transient transfection, imaging, and optogenetic procedures; (iii) imaging and data analysis methods; and (iv) stable cell line generation, pertaining to this assay to measure GPCR-G protein activation.

Two independent but synchronized Gßγ subunit-controlled pathways are essential for trailing-edge retraction during macrophage migration.

Chemokine-induced directional cell migration is a universal cellular mechanism and plays crucial roles in numerous biological processes, including embryonic development, immune system function, and tissue remodeling and regeneration. During the migration of a stationary cell, the cell polarizes, forms lamellipodia at the leading edge (LE), and triggers the concurrent retraction of the trailing edge (TE). During cell migration governed by inhibitory G protein (Gi)-coupled receptors (GPCRs), G protein ßγ (Gßγ) subunits control the LE signaling. Interestingly, TE retraction has been linked to the activation of the small GTPase Ras homolog family member A (RhoA) by the Gα12/13 pathway. However, it is not clear how the activation of Gi-coupled GPCRs at the LE orchestrates the TE retraction in RAW264.7 macrophages. Here, using an optogenetic approach involving an opsin to activate the Gi pathway in defined subcellular regions of RAW cells, we show that in addition to their LE activities, free Gßγ subunits also govern TE retraction by operating two independent, yet synchronized, pathways. The first pathway involves RhoA activation, which prevents dephosphorylation of the myosin light chain, allowing actomyosin contractility to proceed. The second pathway activates phospholipase Cß and induces myosin light chain phosphorylation to enhance actomyosin contractility through increasing cytosolic calcium. We further show that both of these pathways are essential, and inhibition of either one is sufficient to abolish the Gi-coupled GPCR-governed TE retraction and subsequent migration of RAW cells.

Comparison of Calcium Dynamics and Specific Features for G Protein-Coupled Receptor-Targeting Drugs Using Live Cell Imaging and Automated Analysis.

G protein-coupled receptors (GPCRs) are targets for designing a large fraction of the drugs in the pharmaceutical industry. For GPCR-targeting drug screening using cell-based assays, measurement of cytosolic calcium has been widely used to obtain dose-response profiles. However, it remains challenging to obtain drug-specific features due to cell-to-cell heterogeneity in drug-cell responses obtained from live cell imaging. Here, we present a framework combining live cell imaging of a cell population and a feature extraction method for classification of responses of drugs targeting GPCRs CXCR4 and α2AR. We measured the calcium dynamics using confocal microscopy and compared the responses for SDF-1α and norepinephrine. The results clearly show that the clustering patterns of responses for the two GPCRs are significantly different. Additionally, we show that different drugs targeting the same GPCR induce different calcium response signatures. We also implemented principal component analysis and k means for feature extraction and used nondominated (ND) sorting for ranking a group of drugs at various doses. The presented approach can be used to model a cell population as a mixture of subpopulations. It also offers specific advantages, such as higher spatial resolution, classification of responses, and ranking of drugs, potentially providing a platform for high-content drug screening.