Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.

Protein activities depend heavily on protein complex formation and dynamic posttranslational modifications, such as phosphorylation. The dynamic nature of protein complex formation and posttranslational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (separation of phases-based protein interaction reporter) and kinase activities (separation of phases-based activity reporter of kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary PPIs among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other posttranslational modifications with unprecedented ease and sensitivity. Vectors for adding homo-oligomerizing tags were developed and used for detecting dynamic protein-protein interactions and phosphorylation changes in plants.

Homeostasis disruption is visible at the molecular and cellular levels and may often lead to cell death. This vital process allows us to maintain the more extensive system's integrity by keeping the different features (genetic, metabolic, physiologic, and individual) intact. Interestingly, while cells can die in different manners, dying cells still communicate with their environment. This communication was, for a long time, perceived as only driven by the release of soluble factors. However, it has now been reconsidered with the increasing interest in extracellular vesicles (EVs), which are discovered to be released during different regulated cell death programs, with the observation of specific effects. EVs are game changers in the paradigm of cell–cell communication with tremendous implications in fundamental research with regard to noncell autonomous functions, as well as in biomarkers research, all of which are geared toward diagnostic and therapeutic purposes. This review is composed of two main parts. The first is a comprehensive presentation of the state of the art of the EV field at large. In the second part, we focus on EVs discovered to be released during different regulated cell death programs, also known as cell death EVs (cdEVs), and EV‐associated specific effects on recipient cells in the context of cell death and inflammation/inflammatory responses.

Abstract Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell‐to‐cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady‐state (ssEVs) and cell death‐associated EVs (cdEVs) following TNF‐induced necroptosis (necEVs), anti‐Fas‐induced apoptosis (apoEVs), and ML162‐induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death‐associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady‐state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.

Abstract Protein complex formation and dynamic post-translational modifications are notoriously difficult to monitor at cellular resolution. Here, we developed a versatile modular toolbox of fluorescently labelled, artificial homo-oligomerizing peptide-tags (HOTag) that install interaction-dependent liquid-liquid phase-separation upon interaction between two proteins of interest. We deployed our novel toolbox for the in planta visualization of inducible, binary and ternary protein-protein interactions (PPIs), as well as specific phosphorylation, showing its great potential to become a robust standard technique to study PPIs and phosphorylation in plants.

Protein activities depend heavily on protein complex formation and dynamic post-translational modifications, such as phosphorylation. Their dynamic nature is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization and high-end microscopy. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to study protein-protein interactions (PPIs) and kinase activities in planta based on phase separation. This technology enabled easy detection of inducible, binary and ternary protein-protein interactions among cytoplasmic, nuclear and plasma membrane proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SnRK1 kinase activity, allowing us to visualize tissue-specific, dynamic SnRK1 activation upon energy deprivation in stable transgenic Arabidopsis plants. The applications of the SYMPL cloning toolbox lay the foundation for the exploration of PPIs, phosphorylation and other post-translational modifications with unprecedented ease and sensitivity.

Biosensors based on Förster resonance energy transfer (FRET) have revolutionized cellular biology by allowing the direct measurement of biochemical processes in situ. Many genetically encoded sensors make use of fluorescent proteins that are limited in spectral versatility and that allow few ways to change the spectral properties once the construct has been created. In this work, we developed genetically encoded FRET biosensors based on the chemigenetic SNAP and HaloTag domains combined with matching organic fluorophores. We found that the resulting constructs can display comparable responses, kinetics, and reversibility compared to their fluorescent protein-based ancestors, but with the added advantage of spectral versatility, including the availability of red-shifted dye pairs. However, we also find that the introduction of these tags can alter the sensor readout, showing that careful validation is required before applying such constructs in practice. Overall, our approach delivers an innovative methodology that can readily expand the spectral variety and versatility of FRET-based biosensors.

ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Radiotherapy is commonly used as a cytotoxic treatment of a wide variety of tumors. Interestingly, few case reports underlined its potential to induce immune-mediated abscopal effects, resulting in regression of metastases, distant from the irradiated site. These observations are rare, and apparently depend on the dose used, suggesting that dose-related cellular responses may be involved in the distant immunogenic responses. Ionizing radiation (IR) has been reported to elicit immunogenic apoptosis, necroptosis, mitotic catastrophe, and senescence. In order to link a cellular outcome with a particular dose of irradiation, we performed a systematic study in a panel of cell lines on the cellular responses at different doses of X-rays. Remarkably, we observed that all cell lines tested responded in a similar fashion to IR with characteristics of mitotic catastrophe, senescence, lipid peroxidation, and caspase activity. Iron chelators (but not Ferrostatin-1 or vitamin E) could prevent the formation of lipid peroxides and cell death induced by IR, suggesting a crucial role of iron-dependent cell death during high-dose irradiation. We also show that in K-Ras-mutated cells, IR can induce morphological features reminiscent of methuosis, a cell death modality that has been recently described following H-Ras or K-Ras mutation overexpression.

RIPK3 kinase-mediated phosphorylation of MLKL pseudokinase is the execution event of necroptosis. Two independent reports—in Immunity (Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) and Cell (Gong et al., 2017Gong Y.N. Guy C. Olauson H. Becker J.U. Yang M. Fitzgerald P. Linkermann A. Green D.R. Cell. 2017; 169: 286-300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar)—reveal that MLKL affects homeostatic membrane trafficking and necroptosis-enhanced bubble formation involving interaction with the ESCRT machinery. RIPK3 kinase-mediated phosphorylation of MLKL pseudokinase is the execution event of necroptosis. Two independent reports—in Immunity (Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) and Cell (Gong et al., 2017Gong Y.N. Guy C. Olauson H. Becker J.U. Yang M. Fitzgerald P. Linkermann A. Green D.R. Cell. 2017; 169: 286-300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar)—reveal that MLKL affects homeostatic membrane trafficking and necroptosis-enhanced bubble formation involving interaction with the ESCRT machinery. For several decades, necrotic cell death was thought to be accidental and uncontrolled. Necroptosis, a form of programmed cell death, can be stimulated by members of the TNF family (TNF, Fas, TRAIL), by Toll-like receptor (TLR) ligands (LPS, poly(I:C)), and during viral infection by the sensor Z-DNA binding protein-1 (ZBP-1), also called DNA-dependent activator of IRFs (interferon responsive factors) (DAI) (Vanden Berghe et al., 2014Vanden Berghe T. Linkermann A. Jouan-Lanhouet S. Walczak H. Vandenabeele P. Nat. Rev. Mol. Cell Biol. 2014; 15: 135-147Crossref PubMed Scopus (1149) Google Scholar). The induction of necroptosis is mediated by Rip homotypic interaction motif (RHIM) domain-containing proteins such as RIPK1 kinase, RIPK3 kinase, Toll-like receptor interacting factor (TRIF), and DAI. During TNF-induced signaling, RIPK1 has a double role. As a scaffold it becomes K63 and linearly ubiquitylated by inhibitor of apoptosis proteins (IAPs) or linear ubiquitin chain assembly complex (LUBAC) serving as a platform for the binding of the inhibitor of κB kinase (IKK) complex, leading to NF-κB activation, or binding of the TGFβ-activated kinase-1 (TAK1) complex leading to MAPK cascades (Pasparakis and Vandenabeele, 2015Pasparakis M. Vandenabeele P. Nature. 2015; 517: 311-320Crossref PubMed Scopus (1222) Google Scholar). This is called complex I. However, under conditions of IAP, IKK, or TAK1 inhibition, the pro-death function of RIPK1 is unleashed, requiring its kinase activity. Nevertheless, there exists a brake operated by caspase-8-mediated prevention of necroptosis induction. These events happen in so-called complex II or the ripoptosome complex. From the above it is obvious that there exist several mechanisms that keep RIPK1 in a survival mode devoid of kinase activity. Once activated, RIPK3 will phosphorylate MLKL leading to exposure of the N-terminal 4 helical bundle domain and recruitment to the plasma membrane by phosphatidylinositol phosphate interactions. There are three non-exclusive models of how MLKL would exert the execution phase of necroptosis: (1) as a cation channel itself, (2) by engaging Ca2+ and Na+ channels, or (3) by multiple interactions with phospholipids, destabilizing locally plasma membrane integrity (Petrie et al., 2017Petrie E.J. Hildebrand J.M. Murphy J.M. Immunol. Cell Biol. 2017; 95: 152-159Crossref PubMed Scopus (52) Google Scholar). Interestingly, two recent papers now show an additional brake at this final stage of necroptosis execution: the activation of the endosomal sorting complexes required for transport (ESCRT)-III machinery resulting in exosome formation (Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) and ectosome budding (Gong et al., 2017Gong Y.N. Guy C. Olauson H. Becker J.U. Yang M. Fitzgerald P. Linkermann A. Green D.R. Cell. 2017; 169: 286-300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) (Figure 1). The different types of extracellular vesicles (EVs) between these groups may be related to the fact that a bulk of the experimental setup by Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar include studies at homeostasis conditions and after TNF stimulation, while in the Gong et al., 2017Gong Y.N. Guy C. Olauson H. Becker J.U. Yang M. Fitzgerald P. Linkermann A. Green D.R. Cell. 2017; 169: 286-300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar paper the core of the data is based on the use of inducible and dimerizable active MLKL, which biases largely the endogenous machinery and directly engages the end point of necroptosis. However, despite this, the conclusion from both studies is that MLKL directly engages the ESCRT-III system leading to the removal and reduced cytoxicity of MLKL allowing the cells to produce more cytokines and chemokines, especially in the case of bone marrow-derived dendritic cells (BMDCs). Interestingly, Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar report that in LPS-stimulated BMDCs from caspase-8- and MLKL-deficient mice, the products of inflammasome activation (processed IL-1β and activated caspase-1) are not present in the EVs, and moreover not even pro-IL-1β was present, while procaspase-1 was. This would imply that RIPK3-mediated activation of MLKL is required for the insertion of pro-IL-1β in EVs. This all illustrates how phospho-MLKL-associated endosomal trafficking and its association with the ESCRT-III system plays a role not only in postponing the necroptosis execution but also in inflammasome-mediated cytokine activation. The ESCRT machinery exerts a highly conserved multipurpose process involved in receptor sorting, membrane remodeling, and membrane scission, with ESCRT-III as the major membrane-remodeling component. Cellular membrane scission processes mediated by ESCRT-III include biogenesis of multivesicular endosomes, budding of enveloped viruses, cytokinetic abscission, neuron pruning, plasma membrane wound repair, nuclear pore quality control, nuclear envelope reformation, and nuclear envelope repair (Christ et al., 2017Christ L. Raiborg C. Wenzel E.M. Campsteijn C. Stenmark H. Trends Biochem. Sci. 2017; 42: 42-56Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The essence of necrotic cell death is plasma membrane permeabilization, a condition that is subject to ESCRT-III repair machinery. Recently, it was reported that necrosis-causing bacterial toxins such as cholesterol-dependent cytolysines (e.g., streptolysine O from S. pyrogenes, intermedilysin from S. intermedius, or perfringolysin O from C. perfringens) also stimulate the ESCRT-III-mediated repair by lipid-dependent extracellular vesicle shedding of the toxins. The ESCRT machinery determines the sensitivity of the cell to the toxins (Romero et al., 2017Romero M. Keyel M. Shi G. Bhattacharjee P. Roth R. Heuser J.E. Keyel P.A. Cell Death Differ. 2017; 24: 798-808Crossref PubMed Scopus (62) Google Scholar), and BMDMs particularly can deal very well with toxins in this way. An examination of the types of vesicles identified in the studies of Gong et al., 2017Gong Y.N. Guy C. Olauson H. Becker J.U. Yang M. Fitzgerald P. Linkermann A. Green D.R. Cell. 2017; 169: 286-300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar and Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar are instructive. In Gong et al., 2017Gong Y.N. Guy C. Olauson H. Becker J.U. Yang M. Fitzgerald P. Linkermann A. Green D.R. Cell. 2017; 169: 286-300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar they are large (500 nm), permeable ("broken") structures, which are phosphatidylserine (PS) positive and contain phospho-MLKL but have no other obvious content. As mentioned above they probably represent ectosomes directly shed from the membrane by the ESCRT system as a direct detoxification mechanism (Figure 1). In Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar EVs are small (∼150 nm) and correspond to exosomes derived from intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) which contain phospho-MLKL but also intracellular content (Figure 1), representing an endosomal trafficking process. Yet no information has been provided regarding the PS exposure status of these exosomes. As already mentioned above, siRNA silencing of the ESCRT-III complex components allowed cells to produce more cytokines, thus providing a functional context for a brake on the execution of necroptosis. The production of PS and MLKL-positive extracellular necroptotic vesicles will impact the immune system in view of the anti-inflammatory role of PS and its role in phagocytosis recognition, as recently reported (Zargarian et al., 2017Zargarian S. Shlomovitz I. Erlich Z. Hourizadeh A. Ofir-Birin Y. Croker B.A. Regev-Rudzki N. Edry-Botzer L. Gerlic M. PLoS Biol. 2017; 15: e2002711Crossref PubMed Scopus (114) Google Scholar). In this respect, the interaction of MLKL with the ESCRT-III system allows endosomal trafficking and release of EVs to form a continuum with the recognition and uptake by monocytes or dendritic cells. Such a continuum of endosomal trafficking, EVs, and phagocytosis could play a role in antiviral and antitumor responses. Interestingly, Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar report also on the day function of MLKL, besides its night job in necroptosis. The housekeeping role of MLKL involves endosomal transport and lysosomal degradation of TNF, EGF, and EGFR, which is decreased in MLKL knockout cells. This results in dramatically enhanced gene expression by both receptor systems. Altogether these data suggest that MLKL's role in endosomal trafficking and its interaction with the ESCRT-III system plays a role in setting not only the cellular lifespan for cytokine production but also the intensity of cytokine production by controlling the receptor turnover. This day job of MLKL also depends on the phosphorylation of Thr357 and Ser358 as demonstrated by the use of phosphomimetics, but RIPK3 seems not to be required for that, suggesting a role for another kinase. Yoon et al., 2017Yoon S. Kovalenko A. Bogdanov K. Wallach D. Immunity. 2017; 47 (this issue): 51-65Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar also showed that the structural requirements for constitutive and induced endosomal trafficking by MLKL are almost identical with those for necroptosis induction (involving the crucial Thr357 and Ser358 phosphorylation sites and the positively charged patch needed for the recruitment to the phosphatidylinositol phosphates). However, the N-terminal 4 helical bundle domain (4HBD), which is sufficient and more potent to induce necroptosis, apparently is not sufficient for efficient endosomal trafficking. The latter may also explain why, in the case of Gong et al., 2017Gong Y.N. Guy C. Olauson H. Becker J.U. Yang M. Fitzgerald P. Linkermann A. Green D.R. Cell. 2017; 169: 286-300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, enforced dimerization of the 4HBD of MLKL caused mainly ectosomes to be formed, resulting from direct budding of the damaged membranes (Figure 1). The housekeeping function of MLKL in endosomal trafficking and the functional interaction with ESCRT system suggests that the essence of necroptosis induction in viral infected cells, stressed cells, or cancer cells is eventually the inefficacy of the plasma membrane repair system combined or not with a metabolic crisis to generate the required phospholipids, eventually resulting in ballooning and explosion of the cell. The ESCRT-III system in such a constellation puts a timer on the cellular system and the bubbles that are released as a cell death subroutine program may already pre-alert the immune system and other cells in the organism of such explosive events to happen and trigger the induction of an adaptive response. Therefore it would be of interest to know whether the presence of MLKL in the EVs really modulates such an adaptive response and whether by its phospholipid interacting properties affect the physico-chemical features of the vesicles and the functional outcome on target cells (inflammatory responses, cell death, other). The two stories illustrate again that deadly factors, as is also the case for the executioner caspases, are integrated in a network of pleiotropic functions. The question is how to separate both functions of MLKL for therapeutic targeting but also how cells that do not express MLKL (e.g., neuronal cells) or how complete organism such as our dogs and cats, and maybe Carnivora in general, survive their challenging life without having MLKL genes (Dondelinger et al., 2016Dondelinger Y. Hulpiau P. Saeys Y. Bertrand M.J.M. Vandenabeele P. Trends Cell Biol. 2016; 26: 721-732Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). MLKL, the Protein that Mediates Necroptosis, Also Regulates Endosomal Trafficking and Extracellular Vesicle GenerationYoon et al.ImmunityJune 27, 2017In BriefWhen phosphorylated by RIPK3, MLKL triggers necroptotic death. Yoon et al. show that MLKL also contributes to endosomal trafficking and generation of extracellular vesicles. This function is independent of RIPK3 but can be enhanced by it, yielding phospho-MLKL release within the vesicles, thereby apparently withholding death mediation by MLKL. Full-Text PDF Open Archive

RIPK3 kinase-mediated phosphorylation of MLKL pseudokinase is the execution event of necroptosis. Two independent reports—in Immunity (Yoon et al., 2017) and Cell (Gong et al., 2017)—reveal that MLKL affects homeostatic membrane trafficking and necroptosis-enhanced bubble formation involving interaction with the ESCRT machinery.

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.