ABSTRACT
The human silencing hub (HUSH) complex is an epigenetic repressor complex whose role has emerged as an important guardian of genome integrity. It protects the genome from exogenous DNA invasion and regulates endogenous retroelements by recruiting histone methyltransferases catalyzing histone 3 lysine 9 trimethylation (H3K9me3) and additional proteins involved in chromatin compaction. In particular, its regulation of transcriptionally active LINE1 retroelements, by binding to and neutralizing LINE1 transcripts, has been well characterized. HUSH is required for mouse embryogenesis and is associated with disease, in particular cancer. Here we provide insights into the structural and biochemical features of the HUSH complex. Furthermore, we discuss the molecular mechanisms by which the HUSH complex is recruited to specific genomic regions and how it silences transcription. Finally, we discuss the role of HUSH complex members in mammalian development, antiretroviral immunity, and diseases such as cancer.
Subject(s)
Histones , Neoplasms , Humans , Animals , Mice , Histones/genetics , Histones/metabolism , Nuclear Proteins/metabolism , Gene Silencing , Retroelements , Neoplasms/genetics , Mammals/geneticsABSTRACT
BACKGROUND: Intracoronary thrombus formation is a main cause of acute myocardial infarction triggered by platelet activation. However, there are no data on the impact of different treatment strategies with antiplatelet agents before percutaneous coronary intervention (PCI) on histological characteristics of thrombus formation. OBJECTIVE: In this study, we investigate the impact of preinterventional administration of the P2Y12-inhibitors clopidogrel and prasugrel on thrombus composition, highlighting significant changes associated with the antiplatelet pre-treatment. METHODS: We prospectively enrolled 104 consecutive patients with ST-segment elevation myocardial infarction (STEMI) undergoing immediate PCI and thrombus aspiration by immunohistochemical staining along with RNA-sequencing employing Nanostring analysis. Fifty-two patients were treated with either prasugrel loading (60 mg) or clopidogrel loading (600 mg) prior to PCI, respectively. RESULTS: In Patients with STEMI, intracoronary thrombus architecture was significantly altered between patients pre-treated with prasugrel when compared to clopidogrel. Fibrin content of thrombi was significantly decreased (41.8 % versus 66.7 %, p = 0.009) after pre-treatment with prasugrel compared to clopidogrel. Furthermore, levels of MPO positive cells in intracoronary thrombi were significantly decreased in patients with prasugrel pre-treatment (90.5 versus 201.1, p = 0.014) indicating an association of antiplatelet pre-treatment and the inflammatory responses during thrombus formation. Most strikingly, we observed significant differences among both pre-treatment groups regarding altered RNA expression and signaling pathways of thrombo-inflammatory processes within the thrombotic material, which were independently associated with antiplatelet strategies. CONCLUSIONS: Our study elucidates the impact of antiplatelet pre-treatment on thrombus remodeling and architecture, thereby lowering the risk of recurrent adverse cardiovascular events in prasugrel-treated patients.
Subject(s)
Myocardial Infarction , Percutaneous Coronary Intervention , ST Elevation Myocardial Infarction , Thrombosis , Humans , Prasugrel Hydrochloride/pharmacology , Prasugrel Hydrochloride/therapeutic use , Clopidogrel/therapeutic use , ST Elevation Myocardial Infarction/drug therapy , ST Elevation Myocardial Infarction/etiology , Percutaneous Coronary Intervention/adverse effects , Treatment Outcome , Myocardial Infarction/drug therapy , Myocardial Infarction/etiology , Platelet Aggregation Inhibitors/adverse effects , Thrombosis/etiology , RNAABSTRACT
Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.
Subject(s)
Adult Stem Cells , CRISPR-Cas Systems , Animals , Mice , Cell Differentiation/genetics , Fetus , OrganoidsABSTRACT
A father consulted his general practitioner with his 3-years-old son who had swelling on his penis for several months. He experienced no miction problems. The swelling appeared to be a retention of smegma. This is harmless and will disappear spontaneously as the process of separation of the preputium continues.
Subject(s)
Penis , Smegma , Male , Humans , Child, Preschool , Edema , PelvisABSTRACT
Remembering the location of food is essential for survival. Rodents and humans employ mainly hippocampus-dependent spatial strategies, but when being stressed they shift to striatum-mediated stimulus-based strategies. To investigate underlying brain circuits, we tested mice with a heightened stress susceptibility due to a lack of the GABA-synthetizing enzyme GAD65 (GAD65-/- mice) in a dual solution task. Here, GAD65-/- mice preferred to locate a food reward in an open field via a proximal cue, while their wildtype littermates preferred a spatial strategy. The analysis of cFos co-activation across brain regions and of stress-induced mRNA expression changes of GAD65 pointed towards the hippocampal dorsal dentate gyrus (dDG) as a central structure for mediating stress effects on strategy choices via GAD65. Reducing the GAD65 expression locally in the dDG by a shRNA mediated knock down was sufficient to replicate the phenotype of the global GAD65 knock out and to increase dDG excitability. Using DREADD vectors to specifically interfere with dDG circuit activity during dual solution retrieval but not learning confirmed that the dDG modulates strategy choices and that a balanced excitability of this structure is necessary to establish spatial strategy preference. These data highlight the dDG as a critical hub for choosing between spatial and non-spatial foraging strategies.
ABSTRACT
New approach methodologies (NAMs) that do not use experimental animals are, in certain settings, entirely appropriate for assuring the safety of chemical ingredients, although regulatory adoption has been slow. In this opinion article we discuss how scientific advances that utilize NAMs to certify systemic safety are available now and merit broader acceptance within the framework of next generation risk assessments (NGRA).
Subject(s)
Animal Testing Alternatives , Chemical Safety , Animals , Risk AssessmentABSTRACT
Both COVID-19 disease and cocaine consumption have prothrombotic and hypercoagulable effects and are associated with increased risk of cardiovascular events. We report the case of a patient with acute myocardial infarction in the setting of active COVID-19 disease and recent cocaine consumption. We hypothesize that COVID-19 and cocaine synergistically provoke cardiovascular events. Identifying COVID-19 disease and/or cocaine abuse as potential triggers of acute myocardial infarction can be crucial due to distinctive therapeutic consequences.
ABSTRACT
A father consulted his general practitioner with his 18-month-old son with several loose fingernails. We saw a toddler with nine fingernails that peeled off on the proximal side. The normal nails were visible under the loose nails. Diagnosis: onychomadesis.
Subject(s)
Nail Diseases , Nails, Malformed , Child, Preschool , Humans , Infant , Nail Diseases/diagnosis , Nail Diseases/surgery , Nails , Nails, Malformed/diagnosis , Nails, Malformed/etiologyABSTRACT
New Approach Methodologies (NAMs) promise to offer a unique opportunity to enable human-relevant safety decisions to be made without the need for animal testing in the context of exposure-driven Next Generation Risk Assessment (NGRA). Protecting human health against the potential effects a chemical may have on embryo-foetal development and/or aspects of reproductive biology using NGRA is particularly challenging. These are not single endpoint or health effects and risk assessments have traditionally relied on data from Developmental and Reproductive Toxicity (DART) tests in animals. There are numerous Adverse Outcome Pathways (AOPs) that can lead to DART, which means defining and developing strict testing strategies for every AOP, to predict apical outcomes, is neither a tenable goal nor a necessity to ensure NAM-based safety assessments are fit-for-purpose. Instead, a pragmatic approach is needed that uses the available knowledge and data to ensure NAM-based exposure-led safety assessments are sufficiently protective. To this end, the mechanistic and biological coverage of existing NAMs for DART were assessed and gaps to be addressed were identified, allowing the development of an approach that relies on generating data relevant to the overall mechanisms involved in human reproduction and embryo-foetal development. Using the knowledge of cellular processes and signalling pathways underlying the key stages in reproduction and development, we have developed a broad outline of endpoints informative of DART. When the existing NAMs were compared against this outline to determine whether they provide comprehensive coverage when integrated in a framework, we found them to generally cover the reproductive and developmental processes underlying the traditionally evaluated apical endpoint studies. The application of this safety assessment framework is illustrated using an exposure-led case study.
ABSTRACT
Estimation of points of departure (PoDs) from high-throughput transcriptomic data (HTTr) represents a key step in the development of next-generation risk assessment (NGRA). Current approaches mainly rely on single key gene targets, which are constrained by the information currently available in the knowledge base and make interpretation challenging as scientists need to interpret PoDs for thousands of genes or hundreds of pathways. In this work, we aimed to address these issues by developing a computational workflow to investigate the pathway concentration-response relationships in a way that is not fully constrained by known biology and also facilitates interpretation. We employed the Pathway-Level Information ExtractoR (PLIER) to identify latent variables (LVs) describing biological activity and then investigated in vitro LVs' concentration-response relationships using the ToxCast pipeline. We applied this methodology to a published transcriptomic concentration-response data set for 44 chemicals in MCF-7 cells and showed that our workflow can capture known biological activity and discriminate between estrogenic and antiestrogenic compounds as well as activity not aligning with the existing knowledge base, which may be relevant in a risk assessment scenario. Moreover, we were able to identify the known estrogen activity in compounds that are not well-established ER agonists/antagonists supporting the use of the workflow in read-across. Next, we transferred its application to chemical compounds tested in HepG2, HepaRG, and MCF-7 cells and showed that PoD estimates are in strong agreement with those estimated using a recently developed Bayesian approach (cor = 0.89) and in weak agreement with those estimated using a well-established approach such as BMDExpress2 (cor = 0.57). These results demonstrate the effectiveness of using PLIER in a concentration-response scenario to investigate pathway activity in a way that is not fully constrained by the knowledge base and to ease the biological interpretation and support the development of an NGRA framework with the ability to improve current risk assessment strategies for chemicals using new approach methodologies.
Subject(s)
Toxicogenetics , Transcriptome , Bayes Theorem , Estrogens , Risk Assessment/methodsABSTRACT
Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous "nuclear exosome targeting" (NEXT) and "human silencing hub" (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.
Subject(s)
Exosome Multienzyme Ribonuclease Complex , Exosomes , Animals , Chromatin/genetics , Chromatin/metabolism , DNA Transposable Elements/genetics , Exosome Multienzyme Ribonuclease Complex/genetics , Exosomes/metabolism , Humans , Mice , Nuclear Proteins/metabolism , RNA/metabolism , RNA StabilityABSTRACT
Organ-on-chip (OoC) technology is full of engineering and biological challenges, but it has the potential to revolutionize the Next-Generation Risk Assessment of novel ingredients for consumer products and chemicals. A successful incorporation of OoC technology into the Next-Generation Risk Assessment toolbox depends on the robustness of the microfluidic devices and the organ tissue models used. Recent advances in standardized device manufacturing, organ tissue cultivation and growth protocols offer the ability to bridge the gaps towards the implementation of organ-on-chip technology. Next-Generation Risk Assessment is an exposure-led and hypothesis-driven tiered approach to risk assessment using detailed human exposure information and the application of appropriate new (non-animal) toxicological testing approaches. Organ-on-chip presents a promising in vitro approach by combining human cell culturing with dynamic microfluidics to improve physiological emulation. Here, we critically review commercial organ-on-chip devices, as well as recent tissue culture model studies of the skin, intestinal barrier and liver as the main metabolic organ to be used on-chip for Next-Generation Risk Assessment. Finally, microfluidically linked tissue combinations such as skin-liver and intestine-liver in organ-on-chip devices are reviewed as they form a relevant aspect for advancing toxicokinetic and toxicodynamic studies. We point to recent achievements and challenges to overcome, to advance non-animal, human-relevant safety studies.
Subject(s)
Lab-On-A-Chip Devices , Risk Assessment/methods , Toxicology/methods , Animal Testing Alternatives/methods , Animal Testing Alternatives/trends , Humans , Intestines/metabolism , Liver/metabolism , Risk Assessment/trends , Skin/metabolism , Tissue Culture Techniques , Toxicology/trendsABSTRACT
The tumor immune microenvironment is influenced by the epigenetic landscape of the tumor. Here, we have identified the SETDB1-TRIM28 complex as a critical suppressor of antitumor immunity. An epigenetic CRISPR-Cas9 screen of 1,218 chromatin regulators identified TRIM28 as a suppressor of PD-L1 expression. We then revealed that expression of the SETDB1-TRIM28 complex negatively correlated with infiltration of effector CD8+ T cells. Inhibition of SETDB1-TRIM28 simultaneously upregulated PD-L1 and activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway to increase infiltration of CD8+ T cells. Mechanistically, SETDB1-TRIM28 inhibition led to micronuclei formation in the cytoplasm, which is known to activate the cGAS-STING pathway. Thus, SETDB1-TRIM28 inhibition bridges innate and adaptive immunity. Indeed, SETDB1 knockout enhanced the antitumor effects of immune checkpoint blockade with anti-PD-L1 in a mouse model of ovarian cancer in a cGAS-dependent manner. Our findings establish the SETDB1-TRIM28 complex as a regulator of antitumor immunity and demonstrate that its loss activates cGAS-STING innate immunity to boost the antitumor effects of immune checkpoint blockade.
Subject(s)
Epigenomics/methods , Histone-Lysine N-Methyltransferase/metabolism , Immunity, Innate/immunology , Immunotherapy/methods , Ovarian Neoplasms/genetics , Tripartite Motif-Containing Protein 28/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Female , Humans , Mice , Transfection , Tumor MicroenvironmentABSTRACT
In this case report we present a previously healthy 21-year-old male with extensive thromboembolism in the setting of asymptomatic COVID-19 infection and heterozygous factor V Leiden mutation with no additional thrombophilic risk factors.
Subject(s)
COVID-19/complications , Factor V/genetics , SARS-CoV-2 , Thromboembolism/complications , Thromboembolism/genetics , Asymptomatic Infections , COVID-19/diagnosis , COVID-19/diagnostic imaging , Computed Tomography Angiography , Heterozygote , Humans , Male , Thromboembolism/therapy , Young AdultABSTRACT
Duplications and deletions of short chromosomal fragments are increasingly recognized as the cause for rare neurodevelopmental conditions and disorders. The NDR2 gene encodes a protein kinase important for neuronal development and is part of a microduplication region on chromosome 12 that is associated with intellectual disabilities, autism, and epilepsy. We developed a conditional transgenic mouse with increased Ndr2 expression in postmigratory forebrain neurons to study the consequences of an increased gene dosage of this Hippo pathway kinase on brain circuitry and cognitive functions. Our analysis reveals reduced terminal fields and synaptic transmission of hippocampal mossy fibers, altered hippocampal network activity, and deficits in mossy fiber-dependent behaviors. Reduced doublecortin expression and protein interactome analysis indicate that transgenic Ndr2 disturbs the maturation of granule cells in the dentate gyrus. Together, our data suggest that increased expression of Ndr2 may critically contribute to the development of intellectual disabilities upon gene amplification.
ABSTRACT
Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together, our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.
Subject(s)
Epigenesis, Genetic , Phosphoproteins/genetics , Phosphoproteins/metabolism , Pluripotent Stem Cells/metabolism , Animals , CRISPR-Cas Systems , Cell Proliferation , DNA Methylation , Gene Knock-In Techniques , HEK293 Cells , Histone-Lysine N-Methyltransferase , Humans , Long Interspersed Nucleotide Elements/genetics , Mice , Mouse Embryonic Stem Cells , Tumor Suppressor Protein p53/geneticsABSTRACT
BACKGROUND: Gremlin-1 is a cystine knot protein and is expressed in organs developing fibrosis. Transient ischaemia leads to myocardial fibrosis, a major determinant of impaired myocardial function. MATERIALS AND METHODS: Expression of Gremlin-1 was investigated in infarcted myocardium by real-time PCR, Western blot analysis, histological and immunohistochemistry staining. We further elaborated the colocalization of Gremlin-1 and TGF-ß proteins by confocal microscopy and co-immunoprecipitation experiments. The interaction between Gremlin-1 and TGF-ß was analysed by photon correlation spectroscopy. Gremlin-1 modulation of the TGF-ß-dependent collagen I synthesis in fibroblasts was investigated using ELISA and immunohistochemistry experiments. The effect of prolonged administration of recombinant Gremlin-1 on myocardial function following ischaemia/reperfusion was accessed by echocardiography and immunohistochemistry. RESULTS: Gremlin-1 is expressed in myocardial tissue and infiltrating cells after transient myocardial ischaemia (P < .05). Gremlin-1 colocalizes with the pro-fibrotic cytokine transforming growth factor-ß (TGF-ß) expressed in fibroblasts and inflammatory cell infiltrates (P < .05). Gremlin-1 reduces TGF-ß-induced collagen production of myocardial fibroblasts by approximately 20% (P < .05). We found that Gremlin-1 binds with high affinity to TGF-ß (KD = 54 nmol/L) as evidenced by photon correlation spectroscopy and co-immunoprecipitation. intravenous administration of m Gremlin-1-Fc, but not of equivalent amount of Fc control, significantly reduced infarct size by approximately 20%. In the m Gremlin-1-Fc group, infarct area was reduced by up to 30% in comparison with mice treated with Fc control (I/LV: 4.8 ± 1.2% vs 6.0 ± 1.2% P < .05; I/AaR: 15.2 ± 1.5% vs 21.1 ± 5%, P < .05). CONCLUSIONS: The present data disclose Gremlin-1 as an antagonist of TGF-ß and presume a role for Gremlin-1/TGF-ß interaction in myocardial remodelling following myocardial ischaemia.
Subject(s)
Fibroblasts/metabolism , Heart/physiopathology , Intercellular Signaling Peptides and Proteins/genetics , Myocardial Infarction/genetics , Myocardial Reperfusion Injury/genetics , Myocardium/pathology , Transforming Growth Factor beta/metabolism , Animals , Collagen Type I/metabolism , Echocardiography , Endothelial Cells/metabolism , Fibroblasts/drug effects , Fibrosis , Heart/diagnostic imaging , Heart/drug effects , Humans , Immunoprecipitation , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Mice , Microscopy, Confocal , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/physiopathology , Recombinant Proteins , Transforming Growth Factor beta/drug effects , Ventricular Remodeling/geneticsABSTRACT
Adversities during juvenility increase the risk for stress-related disorders, such as post-traumatic stress disorder (PTSD) and alcohol use disorder. However, stress can also induce coping mechanisms beneficial for later stressful experiences. We reported previously that mice selectively bred for high alcohol preference (HAP) exposed to stress during adolescence (but not during adulthood) showed enhanced fear-conditioned responses in adulthood, as measured by fear-potentiated startle (FPS). However, HAP mice also showed enhanced responding to safety cues predicting the absence of foot shocks in adulthood. Here, we pursue these findings in HAP mice by investigating in further detail how juvenile stress impacts the acquisition of safety and fear learning. HAP mice were subjected to three days of juvenile stress (postnatal days 25, 27, 28) and discriminative safety/fear conditioning in adulthood. FPS was used to assess safety versus fear cue discrimination, fear learning, and fear inhibition by the safety cue. Both stressed and unstressed HAP mice were able to discriminate between both cues as well as learn the fear cue-shock association. Interestingly, it was only the previously stressed mice that were able to inhibit their fear response when the fear cue was co-presented with the safety cue, thus demonstrating safety learning. We also report an incidental finding of alopecia in the juvenile stress groups, a phenotype seen in stress-related disorders. These results in HAP mice may be relevant to understanding the influence of juvenile trauma for individual risk and resilience toward developing PTSD and how individuals might benefit from safety cues in behavioral psychotherapy.
Subject(s)
Alcoholism/physiopathology , Conditioning, Classical/physiology , Fear/physiology , Stress, Psychological/physiopathology , Age Factors , Animals , Cues , Disease Models, Animal , Female , Male , Mice , Reflex, Startle/physiologyABSTRACT
In agriculture, diversifying production implies picking up, in the wild biodiversity, species or populations that can be domesticated and fruitfully produced. Two alternative approaches are available to highlight wild candidate(s) with high suitability for aquaculture: the single-trait (i.e. considering a single phenotypic trait and, thus, a single biological function) and multi-trait (i.e. considering multiple phenotypic traits involved in several biological functions) approaches. Although the former is the traditional and the simplest method, the latter could be theoretically more efficient. However, an explicit comparison of advantages and pitfalls between these approaches is lacking to date in aquaculture. Here, we compared the two approaches to identify best candidate(s) between four wild allopatric populations of Perca fluviatilis in standardised aquaculture conditions. Our results showed that the single-trait approach can (1) miss key divergences between populations and (2) highlight different best candidate(s) depending on the trait considered. In contrast, the multi-trait approach allowed identifying the population with the highest domestication potential thanks to several congruent lines of evidence. Nevertheless, such an integrative assessment is achieved with a far more time-consuming and expensive study. Therefore, improvements and rationalisations will be needed to make the multi-trait approach a promising way in the aquaculture development.
Subject(s)
Aquaculture , Domestication , Perches/genetics , Quantitative Trait Loci/genetics , Animals , Breeding , Humans , Perches/growth & development , Phenotype , Polymorphism, Single Nucleotide/geneticsABSTRACT
CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and invoking the host-encoded homology-directed repair program through the concomitant introduction of large repair templates. Here, we present a system for the rapid study of any protein-of-interest in two neuronal cell models following its inducible expression from the human AAVS1 safe harbor locus. With lox-flanked foundation cassettes in the AAVS1 site and a tailor-made plasmid for accepting coding sequences-of-interest in place, the system allows investigators to produce their own neuronal cell models for the inducible expression of any coding sequence in less than a month. Due to the availability of preinserted enhanced green fluorescent protein (EGFP) coding sequences that can be fused to the protein-of-interest, the system facilitates functional investigations that track a protein-of-interest by live-cell microscopy as well as interactome analyses that capitalize on the availability of exquisitely efficient EGFP capture matrices.