RESUMO
Dissection of cell signaling requires tools that can mimic spatiotemporal dynamics of individual pathways in living cells. Optogenetic methods enable manipulation of signaling processes with precise timing and local control. In this review, we describe recent optogenetic approaches for regulation of cell signaling, highlight their advantages and limitations, and discuss examples of their application.
Assuntos
Optogenética , Transdução de SinaisRESUMO
Increased endothelial permeability leads to excessive exudation of plasma proteins and leukocytes in the interstitium, which characterizes several vascular diseases including acute lung injury. The myosin light chain kinase long (MYLK-L) isoform is canonically known to regulate the endothelial permeability by phosphorylating myosin light chain (MLC-P). Compared to the short MYLK isoform, MYLK-L contains an additional stretch of ~919 amino acid at the N-terminus of unknown function. We show that thapsigargin and thrombin-induced SOCE was markedly reduced in Mylk-L-/- endothelial cells (EC) or MYLK-L-depleted human EC. These agonists also failed to increase endothelial permeability in MYLK-L-depleted EC and Mylk-L-/- lungs, thus demonstrating the novel role of MYLK-L-induced SOCE in increasing vascular permeability. MYLK-L augmented SOCE by increasing endoplasmic reticulum (ER)-plasma membrane (PM) junctions and STIM1 translocation to these junctions. Transduction of N-MYLK domain (amino acids 1-919 devoid of catalytic activity) into Mylk-L-/- EC rescued SOCE to the level seen in control EC in a STIM1-dependent manner. N-MYLK-induced SOCE augmented endothelial permeability without MLC-P via an actin-binding motif, DVRGLL. Liposomal-mediated delivery of N-MYLK mutant but not ∆DVRGLL-N-MYLK mutant in Mylk-L-/- mice rescued vascular permeability increase in response to endotoxin, indicating that targeting of DVRGLL motif within MYLK-L may limit SOCE-induced vascular hyperpermeability.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade Capilar , Membrana Celular/enzimologia , Retículo Endoplasmático/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Lesão Pulmonar Aguda/metabolismo , Animais , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoenzimas/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
Membrane protrusions are a critical facet of cell function. Mediating fundamental processes such as cell migration, cell-cell interactions, phagocytosis, as well as assessment and remodeling of the cell environment. Different protrusion types and morphologies can promote different cellular functions and occur downstream of distinct signaling pathways. As such, techniques to quantify and understand the inner workings of protrusion dynamics are critical for a comprehensive understanding of cell biology. In this chapter, we describe approaches to analyze cellular protrusions and correlate physical changes in cell morphology with biochemical signaling processes. We address methods to quantify and characterize protrusion types and velocity, mathematical approaches to predictive models of cytoskeletal changes, and implementation of protein engineering and biosensor design to dissect cell signaling driving protrusive activity. Combining these approaches allows cell biologists to develop a comprehensive understanding of the dynamics of membrane protrusions.
Assuntos
Extensões da Superfície Celular , Pseudópodes , Actinas , Movimento Celular , Citoesqueleto , EndocitoseRESUMO
Angiogenesis is initiated in response to a variety of external cues, including mechanical and biochemical stimuli; however, the underlying signaling mechanisms remain unclear. Here, we investigated the proangiogenic role of the endothelial mechanosensor Piezo1. Genetic deletion and pharmacological inhibition of Piezo1 reduced endothelial sprouting and lumen formation induced by wall shear stress and proangiogenic mediator sphingosine 1-phosphate, whereas Piezo1 activation by selective Piezo1 activator Yoda1 enhanced sprouting angiogenesis. Similarly to wall shear stress, sphingosine 1-phosphate functioned by activating the Ca2+ gating function of Piezo1, which in turn signaled the activation of the matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase during sprouting angiogenesis. Studies in mice in which Piezo1 was conditionally deleted in endothelial cells demonstrated the requisite role of sphingosine 1-phosphate-dependent activation of Piezo1 in mediating angiogenesis in vivo. These results taken together suggest that both mechanical and biochemical stimuli trigger Piezo1-mediated Ca2+ influx and thereby activate matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase and synergistically facilitate sprouting angiogenesis.
Assuntos
Canais Iônicos/deficiência , Metaloproteinase 14 da Matriz/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Canais Iônicos/genética , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two protein-engineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1- and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.
Assuntos
Ativação Enzimática , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Células HeLa , Humanos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Engenharia de Proteínas , Transdução de Sinais , Biologia SintéticaRESUMO
Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury.
Assuntos
Endotélio Vascular/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais , Esfingolipídeos/metabolismo , Lesão Pulmonar Aguda/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Humanos , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pseudópodes/patologia , Espécies Reativas de Oxigênio/metabolismo , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases' homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.
Assuntos
Quinases da Família src/química , Quinases da Família src/metabolismo , Acilação , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Fenômenos Biofísicos , Células COS , Chlorocebus aethiops , Ativação Enzimática , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-fyn/química , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Domínios de Homologia de src , Quinases da Família src/genéticaRESUMO
We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed rapamycin-regulated targeted activation of pathways (RapRTAP), was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src-induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.
Assuntos
Movimento Celular/efeitos dos fármacos , Genes src/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Citoplasma/enzimologia , Citoplasma/ultraestrutura , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Microscopia de Fluorescência , Fenótipo , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.
Assuntos
Ativação Enzimática/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas/química , Animais , Biologia Computacional/métodos , Células HeLa , Humanos , Ligantes , Termodinâmica , Peixe-Zebra , Quinases da Família src/metabolismoRESUMO
Gastrin-releasing peptide receptor (GRPR) is ectopically expressed in over 60% of colon cancers. GRPR expression has been correlated with increased colon cancer cell migration. However, the signaling pathway by which GRPR activation leads to increased cancer cell migration is not well understood. We set out to molecularly dissect the GRPR signaling pathways that control colon cancer cell migration through regulation of small GTPase RhoA. Our results show that GRP stimulation activates RhoA predominantly through G13 heterotrimeric G-protein signaling. We also demonstrate that postsynaptic density 95/disk-large/ZO-1 (PDZ)-RhoGEF (PRG), a member of regulator of G-protein signaling (RGS)-homology domain (RH) containing guanine nucleotide exchange factors (RH-RhoGEFs), is the predominant activator of RhoA downstream of GRPR. We found that PRG is required for GRP-stimulated colon cancer cell migration, through activation of RhoA-Rho-associated kinase (ROCK) signaling axis. In addition, PRG-RhoA-ROCK pathway also contributes to cyclo-oxygenase isoform 2 (Cox-2) expression. Increased Cox-2 expression is correlated with increased production of prostaglandin-E2 (PGE2), and Cox-2-PGE2 signaling contributes to total GRPR-mediated cancer cell migration. Our analysis reveals that PRG is overexpressed in colon cancer cell lines. Overall, our results have uncovered a key mechanism for GRPR-regulated colon cancer cell migration through the Gα13-PRG-RhoA-ROCK pathway.
Assuntos
Neoplasias do Colo/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores da Bombesina/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células CACO-2 , Movimento Celular , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Proteína 4 Homóloga a Disks-Large , Células HT29 , Humanos , Estrutura Terciária de Proteína , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
Genetically encoded, Förster resonance energy transfer (FRET) biosensors enable live-cell optical imaging of signaling molecules. Small conformational changes often limit the dynamic range of biosensors that combine fluorescent proteins (FPs) and sensing domains into a single polypeptide. To address this, we developed FRET and lanthanide-based FRET (LRET) biosensors of Rac1 activation with two key features that enhance sensitivity and dynamic range. For one, alpha helical linker domains separate FRET partners and ensure a large conformational change and FRET increase when activated Rac1 at the biosensor C-terminus interacts with an amino-terminal Rac binding domain. Incorporation of a luminescent Tb(III) complex with long (~ ms) excited state lifetime as a LRET donor enabled time-gated luminescence measurements of Rac1 activity in cell lysates. The LRET dynamic range increased with ER/K linker length up to 1100% and enabled robust detection of Rac1 inhibition in 96-well plates. The ER/K linkers had a less pronounced, but still significant, effect on conventional FRET biosensors (with FP donors and acceptors), and we were able to dynamically image Rac1 activation at cell edges using fluorescence microscopy. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.
Assuntos
Técnicas Biossensoriais , Elementos da Série dos Lantanídeos , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Luminescência , ProteínasRESUMO
Allosteric regulation of proteins has been utilized to study various aspects of cell signaling, from unicellular events to organism-wide phenotypes. However, traditional methods of allosteric regulation, such as constitutively active mutants and inhibitors, lack tight spatiotemporal control. This often leads to unintended signaling consequences that interfere with data interpretation. To overcome these obstacles, researchers employed protein engineering approaches that enable tight control of protein function through allosteric mechanisms. These methods provide high specificity as well as spatial and temporal precision in regulation of protein activity in vitro and in vivo. In this review, we focus on the recent advancements in engineered allosteric regulation and discuss the various bioengineered allosteric techniques available now, from chimeric GPCRs to chemogenetic and optogenetic switches. We highlight the benefits and pitfalls of each of these techniques as well as areas in which future improvements can be made. Additionally, we provide a brief discussion on implementation of engineered allosteric regulation approaches, demonstrating that these tools can shed light on elusive biological events and have the potential to be utilized in precision medicine.
Assuntos
Optogenética , Engenharia de Proteínas , Proteínas , Regulação Alostérica , Optogenética/métodos , Proteínas/química , Transdução de SinaisRESUMO
Protein tyrosine phosphatases (PTPases) are critical mediators of dynamic cell signaling. A tool capable of identifying transient signaling events downstream of PTPases is essential to understand phosphatase function on a physiological time scale. We report a broadly applicable protein engineering method for allosteric regulation of PTPases. This method enables dissection of transient events and reconstruction of individual signaling pathways. Implementation of this approach for Shp2 phosphatase revealed parallel MAPK and ROCK II dependent pathways downstream of Shp2, mediating transient cell spreading and migration. Furthermore, we show that the N-SH2 domain of Shp2 regulates MAPK-independent, ROCK II-dependent cell migration. Engineered targeting of Shp2 activity to different protein complexes revealed that Shp2-FAK signaling induces cell spreading whereas Shp2-Gab1 or Shp2-Gab2 mediates cell migration. We identified specific transient morphodynamic processes induced by Shp2 and determined the role of individual signaling pathways downstream of Shp2 in regulating these events. Broad application of this approach is demonstrated by regulating PTP1B and PTP-PEST phosphatases.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 11 , Transdução de Sinais , Regulação Alostérica , Movimento Celular , Quinase 1 de Adesão Focal/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The cytoskeleton and cell-matrix adhesions constitute a dynamic network that controls cellular behavior during development and cancer. The Focal Adhesion Kinase (FAK) is a central actor of these cell dynamics, promoting cell-matrix adhesion turnover and active membrane fluctuations. However, the initial steps leading to FAK activation and subsequent promotion of cell dynamics remain elusive. Here, we report that the serine/threonine kinase PKCθ participates in the initial steps of FAK activation. PKCθ, which is strongly expressed in aggressive human breast cancers, controls the dynamics of cell-matrix adhesions and active protrusions through direct FAK activation, thereby promoting cell invasion and lung metastases. Using various tools for in vitro and live cell studies, we precisely decipher the molecular mechanisms of FAK activation. PKCθ directly interacts with the FAK FERM domain to open FAK conformation through PKCθ's specific V3 domain, while phosphorylating FAK at newly identified serine/threonine residues within nascent adhesions, inducing cell dynamics and aggressive behavior. This study thus places PKCθ-directed FAK opening and phosphorylations as an original mechanism controlling dynamic, migratory, and invasive abilities of aggressive breast cancer cells, further strengthening the emerging oncogenic function of PKCθ.
Assuntos
Neoplasias da Mama/fisiopatologia , Citoesqueleto/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteína Quinase C-theta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pseudópodes/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , FosforilaçãoRESUMO
We developed a new system for light-induced protein dimerization in living cells using a photocaged analogue of rapamycin together with an engineered rapamycin binding domain. Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Luz , Sirolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Dimerização , Células HEK293 , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas de Ligação a Tacrolimo/químicaRESUMO
Cell surface G protein-coupled receptors (GPCRs), upon agonist binding, undergo serine-threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y143, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y143-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y143D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca2+ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y143 and its import into ER via BiP. BiP depletion restored Y143D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y143-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.
Assuntos
Retículo Endoplasmático/genética , Inflamação/genética , Receptores de Esfingosina-1-Fosfato/genética , Fator de Necrose Tumoral alfa/genética , Citosol/metabolismo , Endocitose/genética , Chaperona BiP do Retículo Endoplasmático/química , Chaperona BiP do Retículo Endoplasmático/genética , Células Endoteliais/metabolismo , Humanos , Inflamação/patologia , Fosforilação/genética , Proteólise , Receptores Acoplados a Proteínas G/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Tirosina/genéticaRESUMO
Podosomes are compartmentalized actin-rich adhesions, defined by their ability to locally secrete proteases and remodel extracellular matrix. Matrix remodeling by endothelial podosomes facilitates invasion and thereby vessel formation. However, the mechanisms underlying endothelial podosome formation and function remain unclear. Here, we demonstrate that Septin2, Septin6, and Septin7 are required for maturation of nascent endothelial podosomes into matrix-degrading organelles. We show that podosome development occurs through initial mobilization of the scaffolding protein Tks5 and F-actin accumulation, followed by later recruitment of Septin2. Septin2 localizes around the perimeter of podosomes in close proximity to the basolateral plasma membrane, and phosphoinositide-binding residues of Septin2 are required for podosome function. Combined, our results suggest that the septin cytoskeleton forms a diffusive barrier around nascent podosomes to promote their maturation. Finally, we show that Septin2-mediated regulation of podosomes is critical for endothelial cell invasion associated with angiogenesis. Therefore, targeting of Septin2-mediated podosome formation is a potentially attractive anti-angiogenesis strategy.
Assuntos
Proteínas de Ciclo Celular/genética , Neovascularização Fisiológica/genética , Septinas/genética , Citoesqueleto de Actina/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Movimento Celular/genética , Células Cultivadas , Células Endoteliais/metabolismo , Matriz Extracelular/genética , Humanos , Morfogênese/genética , Podossomos/genéticaRESUMO
Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.
Cells need to sense and respond to their environment. To do this, they have dedicated proteins that interpret outside signals and convert them into appropriate responses that are only active at a specific time and location within the cell. However, in many diseases, including cancer, these signaling proteins are switched on for too long or are active in the wrong place. To better understand why this is the case, researchers manipulate proteins to identify the processes they regulate. One way to do this is to engineer proteins so that they can be controlled by light, turning them either on or off. Ideally, a light-controlled tool can activate proteins at defined times, control proteins in specific locations within the cell and regulate any protein of interest. However, current methods do not combine all of these requirements in one tool, and scientists often have to use different methods, depending on the topic they are researching. Now, Shaaya et al. set out to develop a single tool that combines all required features. The researchers engineered a light-sensitive 'switch' that allowed them to activate a specific protein by illuminating it with blue light and to deactivate it by turning the light off. Unlike other methods, the new tool uses a light-sensitive switch that works like a clamp. In the dark, the clamp is open, which 'stretches' and distorts the protein, rendering it inactive. In light, however, the clamp closes and the structure of the protein and its activity are restored. Moreover, it can activate proteins multiple times, control proteins in specific locations within the cell and it can be applied to a variety of proteins. This specific design makes it possible to combine multiple features in one tool that will both simplify and broaden its use to investigate specific proteins and signaling pathways in a broad range of diseases.
Assuntos
Optogenética/métodos , Quinases da Família src/química , Regulação Alostérica , Enzimas/química , LuzRESUMO
Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation-dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding-deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk-cortactin complex in actin polymerization downstream of tyrosine kinase signaling.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Células Epiteliais/microbiologia , Proteínas dos Microfilamentos/fisiologia , Shigella/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Extensões da Superfície Celular/microbiologia , Cortactina , Endocitose , Células Epiteliais/ultraestrutura , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-crk , RNA Interferente Pequeno/farmacologiaRESUMO
In the current model of endothelial barrier regulation, the tyrosine kinase SRC is purported to induce disassembly of endothelial adherens junctions (AJs) via phosphorylation of VE cadherin, and thereby increase junctional permeability. Here, using a chemical biology approach to temporally control SRC activation, we show that SRC exerts distinct time-variant effects on the endothelial barrier. We discovered that the immediate effect of SRC activation was to transiently enhance endothelial barrier function as the result of accumulation of VE cadherin at AJs and formation of morphologically distinct reticular AJs. Endothelial barrier enhancement via SRC required phosphorylation of VE cadherin at Y731. In contrast, prolonged SRC activation induced VE cadherin phosphorylation at Y685, resulting in increased endothelial permeability. Thus, time-variant SRC activation differentially phosphorylates VE cadherin and shapes AJs to fine-tune endothelial barrier function. Our work demonstrates important advantages of synthetic biology tools in dissecting complex signaling systems.