RESUMEN
Phase transitions involving biomolecular liquids are a fundamental mechanism underlying intracellular organization. In the cell nucleus, liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is implicated in assembly of the nucleolus, as well as transcriptional clusters, and other nuclear bodies. However, it remains unclear whether and how physical forces associated with nucleation, growth, and wetting of liquid condensates can directly restructure chromatin. Here, we use CasDrop, a novel CRISPR-Cas9-based optogenetic technology, to show that various IDPs phase separate into liquid condensates that mechanically exclude chromatin as they grow and preferentially form in low-density, largely euchromatic regions. A minimal physical model explains how this stiffness sensitivity arises from lower mechanical energy associated with deforming softer genomic regions. Targeted genomic loci can nonetheless be mechanically pulled together through surface tension-driven coalescence. Nuclear condensates may thus function as mechano-active chromatin filters, physically pulling in targeted genomic loci while pushing out non-targeted regions of the neighboring genome. VIDEO ABSTRACT.
Asunto(s)
Nucléolo Celular/metabolismo , Cromatina/metabolismo , Citoplasma/metabolismo , Genoma Humano , Proteínas Intrínsecamente Desordenadas/metabolismo , Transición de Fase , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Células 3T3 NIHRESUMEN
Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions but also their link to pathological aggregates.
Asunto(s)
Imagen Molecular/métodos , Transición de Fase , Proteínas/química , Animales , Proteínas de Arabidopsis , Criptocromos , Proteínas Intrínsecamente Desordenadas , Cinética , Luz , Ratones , Modelos Químicos , Células 3T3 NIH , Optogenética , Mapas de Interacción de Proteínas , Proteínas/metabolismoRESUMEN
ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of â¼1-4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multidomain substrates and ClpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the ClpX ring.
Asunto(s)
Endopeptidasa Clp/química , Endopeptidasa Clp/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Estructura Terciaria de Proteína , Desplegamiento Proteico , ProteolisisRESUMEN
Phase separation drives compartmentalization of intracellular contents into various biomolecular condensates. Individual condensate components are thought to differentially contribute to the organization and function of condensates. However, how intermolecular interactions among constituent biomolecules modulate the phase behaviors of multicomponent condensates remains unclear. Here, we used core components of the inhibitory postsynaptic density (iPSD) as a model system to quantitatively probe how the network of intra- and intermolecular interactions defines the composition and cellular distribution of biomolecular condensates. We found that oligomerization-driven phase separation of gephyrin, an iPSD-specific scaffold, is critically modulated by an intrinsically disordered linker region exhibiting minimal homotypic attractions. Other iPSD components, such as neurotransmitter receptors, differentially promote gephyrin condensation through distinct binding modes and affinities. We further demonstrated that the local accumulation of scaffold-binding proteins at the cell membrane promotes the nucleation of gephyrin condensates in neurons. These results suggest that in multicomponent systems, the extent of scaffold condensation can be fine-tuned by scaffold-binding factors, a potential regulatory mechanism for self-organized compartmentalization in cells.
Asunto(s)
Proteínas Portadoras , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/metabolismo , Sinapsis/metabolismo , TermodinámicaRESUMEN
Dynamic interaction between BRCA2 and telomeric G-quadruplexes (G4) is crucial for maintaining telomere replication homeostasis. Cells lacking BRCA2 display telomeric damage with a subset of these cells bypassing senescence to initiate break-induced replication (BIR) for telomere synthesis. Here we show that the abnormal stabilization of telomeric G4 following BRCA2 depletion leads to telomeric repeat-containing RNA (TERRA)-R-loop accumulation, triggering liquid-liquid phase separation (LLPS) and the assembly of Alternative Lengthening of Telomeres (ALT)-associated promyelocytic leukemia (PML) bodies (APBs). Disruption of R-loops abolishes LLPS and impairs telomere synthesis. Artificial engineering of telomeric LLPS restores telomere synthesis, underscoring the critical role of LLPS in ALT. TERRA-R-loops also recruit Polycomb Repressive Complex 2 (PRC2), leading to tri-methylation of Lys27 on histone H3 (H3K27me3) at telomeres. Half of paraffin-embedded tissue sections from human breast cancers exhibit APBs and telomere length heterogeneity, suggesting that BRCA2 mutations can predispose individuals to ALT-type tumorigenesis. Overall, BRCA2 abrogation disrupts the dynamicity of telomeric G4, producing TERRA-R-loops, finally leading to the assembly of telomeric liquid condensates crucial for ALT. We propose that modulating the dynamicity of telomeric G4 and targeting TERRA-R-loops in telomeric LLPS maintenance may represent effective therapeutic strategies for treating ALT-like cancers with APBs, including those with BRCA2 disruptions.
Asunto(s)
Proteína BRCA2 , Replicación del ADN , G-Cuádruplex , Homeostasis del Telómero , Telómero , Humanos , Telómero/metabolismo , Telómero/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Homeostasis del Telómero/genética , Replicación del ADN/genética , Histonas/metabolismo , Histonas/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Estructuras R-Loop , Complejo Represivo Polycomb 2/metabolismo , Complejo Represivo Polycomb 2/genética , Línea Celular Tumoral , Femenino , Separación de FasesRESUMEN
Pioneer transcription factors (TFs) like SOX2 are vital for stemness and cancer through enhancing gene expression within transcriptional condensates formed with coactivators, RNAs and mediators on super-enhancers (SEs). Despite their importance, how these factors work together for transcriptional condensation and activation remains unclear. SOX2, a pioneer TF found in SEs of pluripotent and cancer stem cells, initiates SE-mediated transcription by binding to nucleosomes, though the mechanism isn't fully understood. To address SOX2's role in SEs, we identified mSE078 as a model SOX2-enriched SE and p300 as a coactivator through bioinformatic analysis. In vitro and cell assays showed SOX2 forms condensates with p300 and SOX2-binding motifs in mSE078. We further proved that SOX2 condensation is highly correlated with mSE078's enhancer activity in cells. Moreover, we successfully demonstrated that p300 not only elevated transcriptional activity but also triggered chromatin acetylation via its direct interaction with SOX2 within these transcriptional condensates. Finally, our validation of SOX2-enriched SEs showcased their contribution to target gene expression in both stem cells and cancer cells. In its entirety, this study imparts valuable mechanistic insights into the collaborative interplay of SOX2 and its coactivator p300, shedding light on the regulation of transcriptional condensation and activation within SOX2-enriched SEs.
Asunto(s)
Células Madre Pluripotentes , Factores de Transcripción , Cromatina/genética , Elementos de Facilitación Genéticos , Nucleosomas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Humanos , Células Madre Pluripotentes/metabolismoRESUMEN
Inter-chromosomal interactions play a crucial role in genome organization, yet the organizational principles remain elusive. Here, we introduce a novel computational method to systematically characterize inter-chromosomal interactions using in situ Hi-C results from various cell types. Our method successfully identifies two apparently hub-like inter-chromosomal contacts associated with nuclear speckles and nucleoli, respectively. Interestingly, we discover that nuclear speckle-associated inter-chromosomal interactions are highly cell-type invariant with a marked enrichment of cell-type common super-enhancers (CSEs). Validation using DNA Oligopaint fluorescence in situ hybridization (FISH) shows a strong but probabilistic interaction behavior between nuclear speckles and CSE-harboring genomic regions. Strikingly, we find that the likelihood of speckle-CSE associations can accurately predict two experimentally measured inter-chromosomal contacts from Hi-C and Oligopaint DNA FISH. Our probabilistic establishment model well describes the hub-like structure observed at the population level as a cumulative effect of summing individual stochastic chromatin-speckle interactions. Lastly, we observe that CSEs are highly co-occupied by MAZ binding and MAZ depletion leads to significant disorganization of speckle-associated inter-chromosomal contacts. Taken together, our results propose a simple organizational principle of inter-chromosomal interactions mediated by MAZ-occupied CSEs.
Asunto(s)
Cromatina , Cromosomas , Humanos , Hibridación Fluorescente in Situ , Cromatina/genética , Cromatina/metabolismo , Núcleo Celular/metabolismo , ADN/genética , ADN/metabolismoRESUMEN
Programmability of DNA sequences enables the formation of synthetic DNA nanostructures and their macromolecular assemblies such as DNA hydrogels. The base pair-level interaction of DNA is a foundational and powerful mechanism to build DNA structures at the nanoscale; however, its temperature sensitivity and weak interaction force remain a barrier for the facile and scalable assembly of DNA structures toward higher-order structures. We conducted this study to provide an alternative, non-base-pairing approach to connect nanoscale DNA units to yield micrometer-sized gels based on the sequential phase transition of amphiphilic unit structures. Strong electrostatic interactions between DNA nanostructures and polyelectrolyte spermines led to the formation of giant phase-separated aggregates of monomer units. Gelation could be initiated by the addition of NaCl, which weakened the electrostatic DNA-spermine interaction while attractive interactions between cholesterols created stable networks by crosslinking DNA monomers. In contrast to the conventional DNA gelation techniques, our system used solid aggregates as a precursor for DNA microgels. Therefore, in situ gelation could be achieved by depositing aggregates on the desired substrate and subsequently initiating a phase transition. Our approach can expand the utility and functionality of DNA hydrogels by using more complex nucleic acid assemblies as unit structures and combining the technique with top-down microfabrication methods.
Asunto(s)
Microgeles , Nanoestructuras , Emparejamiento Base , ADN/química , Hidrogeles/química , Nanoestructuras/químicaRESUMEN
Kinesin-8s are plus-end-directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are "ultraprocessive," a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end-directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.
Asunto(s)
Fenómenos Biofísicos , Cinesinas/metabolismo , Microtúbulos/metabolismo , Movimiento (Física) , Rastreo Celular , Difusión , Células HeLa , Humanos , Cinesinas/química , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Grabación en VideoRESUMEN
Biomolecular condensates, often assembled through phase transition mechanisms, play key roles in organizing diverse cellular activities. The material properties of condensates, ranging from liquid droplets to solid-like glasses or gels, are key features impacting the way resident components associate with one another. However, it remains unclear whether and how different material properties would influence specific cellular functions of condensates. Here, we combine optogenetic control of phase separation with single-molecule mRNA imaging to study relations between phase behaviors and functional performance of condensates. Using light-activated condensation, we show that sequestering target mRNAs into condensates causes translation inhibition. Orthogonal mRNA imaging reveals highly transient nature of interactions between individual mRNAs and condensates. Tuning condensate composition and material property towards more solid-like states leads to stronger translational repression, concomitant with a decrease in molecular mobility. We further demonstrate that ß-actin mRNA sequestration in neurons suppresses spine enlargement during chemically induced long-term potentiation. Our work highlights how the material properties of condensates can modulate functions, a mechanism that may play a role in fine-tuning the output of condensate-driven cellular activities.
Asunto(s)
Actinas , Optogenética , Humanos , Actinas/genética , Condensados Biomoleculares , Hipertrofia , Potenciación a Largo PlazoRESUMEN
Liquid-liquid phase separation (LLPS) facilitates the formation of membraneless organelles within cells, with implications in various biological processes and disease states. AT-rich interactive domain-containing protein 1A (ARID1A) is a chromatin remodeling factor frequently associated with cancer mutations, yet its functional mechanism remains largely unknown. Here, we find that ARID1A harbors a prion-like domain (PrLD), which facilitates the formation of liquid condensates through PrLD-mediated LLPS. The nuclear condensates formed by ARID1A LLPS are significantly elevated in Ewing's sarcoma patient specimen. Disruption of ARID1A LLPS results in diminished proliferative and invasive abilities in Ewing's sarcoma cells. Through genome-wide chromatin structure and transcription profiling, we identify that the ARID1A condensate localizes to EWS/FLI1 target enhancers and induces long-range chromatin architectural changes by forming functional chromatin remodeling hubs at oncogenic target genes. Collectively, our findings demonstrate that ARID1A promotes oncogenic potential through PrLD-mediated LLPS, offering a potential therapeutic approach for treating Ewing's sarcoma.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Factores de Transcripción , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Línea Celular Tumoral , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genética , Regulación Neoplásica de la Expresión Génica , Proliferación Celular , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Cromatina/metabolismo , Carcinogénesis/genética , Animales , Ratones , Dominios Proteicos , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Separación de FasesRESUMEN
Phase separation is a key mechanism for intracellular organization, driving the segregation of biomolecules into distinct condensates. Intracellular condensates play diverse functional roles including gene expression, stress response, and cell signaling. Technologies that enable the control of intracellular phase separation can be highly useful not only for a better understanding of the biophysical principles of phase separation processes but also for engineering novel condensates. Here, we describe an optogenetic approach for spatiotemporal control of phase separation in living cells.
Asunto(s)
Optogenética , Biofisica , Citoplasma/metabolismoRESUMEN
Biomolecular condensates play a key role in organizing cellular reactions by concentrating a specific set of biomolecules. However, whether condensate formation is accompanied by an increase in the total mass concentration within condensates or by the demixing of already highly crowded intracellular components remains elusive. Here, using refractive index imaging, we quantify the mass density of several condensates, including nucleoli, heterochromatin, nuclear speckles, and stress granules. Surprisingly, the latter two condensates exhibit low densities with a total mass concentration similar to the surrounding cyto- or nucleoplasm. Low-density condensates display higher permeability to cellular protein probes. We find that RNA tunes the biomolecular density of condensates. Moreover, intracellular structures such as mitochondria heavily influence the way phase separation proceeds, impacting the localization, morphology, and growth of condensates. These findings favor a model where segregative phase separation driven by non-associative or repulsive molecular interactions together with RNA-mediated selective association of specific components can give rise to low-density condensates in the crowded cellular environment.
Asunto(s)
Núcleo Celular , ARN , ARN/metabolismo , Núcleo Celular/metabolismo , Nucléolo Celular/metabolismo , Heterocromatina/metabolismoRESUMEN
Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.
Asunto(s)
Retículo Endoplásmico , Proteínas de Transporte Vesicular , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Transporte de Proteínas/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismoRESUMEN
ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5'-[gamma-thio]triphosphate (ATPgammaS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas/metabolismo , Conectina , Endopeptidasa Clp/química , Proteínas de Escherichia coli/química , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas/química , ARN Bacteriano/química , ARN Bacteriano/metabolismoRESUMEN
Phase separation is a thermodynamic process leading to the formation of compositionally distinct phases. For the past few years, numerous works have shown that biomolecular phase separation serves as biogenesis mechanisms of diverse intracellular condensates, and aberrant phase transitions are associated with disease states such as neurodegenerative diseases and cancers. Condensates exhibit rich phase behaviors including multiphase internal structuring, noise buffering, and compositional tunability. Recent studies have begun to uncover how a network of intermolecular interactions can give rise to various biophysical features of condensates. Here, we review phase behaviors of biomolecules, particularly with regard to regular solution models of binary and ternary mixtures. We discuss how these theoretical frameworks explain many aspects of the assembly, composition, and miscibility of diverse biomolecular phases, and highlight how a model-based approach can help elucidate the detailed thermodynamic principle for multicomponent intracellular phase separation.
Asunto(s)
Enfermedades Neurodegenerativas , Humanos , TermodinámicaRESUMEN
Biomolecular condensates participate in diverse cellular processes, ranging from gene regulation to stress survival. Bottom-up engineering of synthetic condensates advances our understanding of the organizing principle of condensates. It also enables the synthesis of artificial systems with novel functions. However, building synthetic condensates with a predictable organization and function remains challenging. Here, we use DNA as a building block to create synthetic condensates that are assembled through phase separation. The programmability of intermolecular interactions between DNA molecules enables the control over various condensate properties including assembly, composition, and function. Similar to the way intracellular condensates are organized, DNA clients are selectively partitioned into cognate condensates. We demonstrate that the synthetic condensates can accelerate DNA strand displacement reactions and logic gate operation by concentrating specific reaction components. We envision that the DNA-based condensates could help the realization of the high-order functions required to build more life-like artificial systems.
RESUMEN
Phase separation of biomolecules plays key roles in physiological compartmentalization as well as pathological aggregation. A deeper understanding of biomolecular phase separation requires dissection of a relation between intermolecular interactions and resulting phase behaviors. DNA nanostars, multivalent DNA assemblies of which sticky ends define attractive interactions, represent an ideal system to probe this fundamental relation governing phase separation processes. Here, we use DNA nanostars to systematically study how structural flexibility exhibited by interacting species impacts their phase behaviors. We design multiple nanostars with a varying degree of flexibility using single-stranded gaps of different lengths in the arm of each nanostar unit. We find that structural flexibility drastically alters the phase diagram of DNA nanostars in such a way that the phase separation of more flexible structures is strongly inhibited. This result is not due to self-inhibition from the loss of valency but rather ascribed to a generic flexibility-driven change in the thermodynamics of the system. Our work provides not only potential regulatory mechanisms cells may exploit to dynamically control intracellular phase separation but also a route to build synthetic systems of which assembly can be controlled in a signal dependent manner.
Asunto(s)
ADN , TermodinámicaRESUMEN
Single-cell level analysis of various cellular behaviors has been aided by recent developments in microfluidic technology. Polydimethylsiloxane (PDMS)-based microfluidic devices have been widely used to elucidate cell differentiation and migration under spatiotemporal stimulation. However, microfluidic devices fabricated with PDMS have inherent limitations due to material issues and non-scalable fabrication process. In this study, we designed and fabricated an injection molded microfluidic device that enables real-time chemical profile control. This device is made of polystyrene (PS), engineered with channel dimensions optimized for injection molding to achieve functionality and compatibility with single cell observation. We demonstrated the spatiotemporal dynamics in the device with computational simulation and experiments. In temporal dynamics, we observed extracellular signal-regulated kinase (ERK) activation of PC12 cells by stimulating the cells with growth factors (GFs). Also, we confirmed yes-associated protein (YAP) phase separation of HEK293 cells under stimulation using sorbitol. In spatial dynamics, we observed the migration of NIH 3T3 cells (transfected with Lifeact-GFP) under different spatiotemporal stimulations of PDGF. Using the injection molded plastic devices, we obtained comprehensive data more easily than before while using less time compared to previous PDMS models. This easy-to-use plastic microfluidic device promises to open a new approach for investigating the mechanisms of cell behavior at the single-cell level.