Split Luciferase Complementation Research Articles

Biophysical validation of serotonin 5-HT2A and 5-HT2C receptor interaction.

The serotonin (5-HT) 5-HT2A receptor (5-HT2AR) and 5-HT2C receptor (5-HT2CR) in the central nervous system are implicated in a range of normal behaviors (e.g., appetite, sleep) and physiological functions (e.g., endocrine secretion) while dysfunctional 5-HT2AR and/or 5-HT2CR are implicated in neuropsychiatric disorders (e.g., addiction, obesity, schizophrenia). Preclinical studies suggest that the 5-HT2AR and 5-HT2CR may act in concert to regulate the neural bases for behavior. Here, we utilize three distinct biophysical and immunocytochemistry-based approaches to identify and study this receptor complex in cultured cells. Employing a split luciferase complementation assay (LCA), we demonstrated that formation of the 5-HT2AR:5-HT2CR complex exists within 50 nm, increases proportionally to the 5-HT2CR:5-HT2AR protein expression ratio, and is specific to the receptor interaction and not due to random complementation of the luciferase fragments. Using a proximity ligation assay (PLA), we found that cells stably expressing both the 5-HT2AR and 5-HT2CR exhibit 5-HT2AR:5-HT2CR heteroreceptor complexes within 40 nm of each other. Lastly, bioluminescence resonance energy transfer (BRET) analyses indicates the formation of a specific and saturable 5-HT2AR:5-HT2CR interaction, suggesting that the 5-HT2AR and 5-HT2CR form a close interaction within 10 nm of each other in intact live cells. The bioengineered receptors generated for the LCA and the BRET exhibit 5-HT-mediated intracellular calcium signaling as seen for the native receptors. Taken together, this study validates a very close 5-HT2AR:5-HT2CR interaction in cultured cells.

The mode of action of remorin1 in regulating fruit ripening at transcriptional and post-transcriptional levels.

Remorins are plant-specific and plasma membrane-associated proteins that display a variety of functions in plant growth, development, biotic and abiotic stresses, and signal transduction. However, little information is available for understanding their role in fruit ripening. Here, remorin 1 (SlREM1) is cloned from tomato and its localization is examined by co-localization analysis and immunoblotting. Functions of SlREM1 in fruit ripening are characterized based on gene expression, co-immunoprecipitation coupled with mass spectroscopy and split luciferase complementation imaging assays in SlREM1 overexpression and RNA interference (RNAi) lines. The results indicate that SlREM1 is localized at the plasma membrane. Overexpression of SlREM1 in tomato stimulates fruit ripening with an increase in ethylene production and lycopene accumulation as compared to the wild-type. Consistently, these genes involved in ethylene and lycopene biosynthesis and ripening regulators also are upregulated in SlREM1 overexpression lines. SlREM1 can interact with ethylene biosynthesis proteins SAM1, ACO1 and ACS2 and is degraded by ubiquitin-mediated proteolysis. Our findings reveal that SlREM1 serves as a positive regulator of fruit ripening and provide novel cues for understanding of the molecular regulation network of fruit ripening.

A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells.

Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53wt, p53Y220C, p53G245S and p53R282W. We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53.

Biophysical identification of the 5‐HT2A receptor:5‐HT2C receptor interaction in vitro

Disruption of serotonin 5‐HT2A receptor (5‐HT2AR) and 5‐HT2CR function is implicated in a range of neuropsychiatric disorders with these receptors being essential sites of action for anti‐psychotic and anti‐obesity medications. Based on evidence that the 5‐HT2AR and 5‐HT2CR co‐localize and co‐immunoprecipitate from neurons in the medial prefrontal cortex of the rat (Anastasio et al, ACS Chem. Neurosci. 15:1248, 2015), we hypothesize that these two receptors form a heteromeric complex in living cells. Here, we utilize three distinct biophysical and immunocytochemistry‐based approaches to identify and study this receptor complex in living cells. First, we generated a split luciferase complementation assay to test the hypothesis that the 5‐HT2AR and 5‐HT2CR are in close proximity (< 50 nm). We predicted that inactive luciferase fragments fused to each receptor would be brought into close proximity when receptors form a complex, thereby reconstituting the enzymatic activity of luciferase. A robust complementation (i.e., increased luminescence) was observed upon addition of the substrate D‐luciferin, an outcome that was dependent on protein levels assessed in radioligand binding studies (n=3, p<0.001). Co‐transfection with either the wildtype 5‐HT2AR or 5‐HT2CR inhibited the complementation (n=3, p<0.01). Using a proximity ligation assay, we demonstrate expression of the existence of 5‐HT2AR:5‐HT2CR heteroreceptor complexes in vitro, indicating the protomers are within 40 nm of each other (n=5, p<0.05). To further delineate the receptors are within very close proximity (< 10 nm) in living cells, we generated 5‐HT2AR and 5‐HT2CR constructs for use in a bioluminescence resonance energy transfer (BRET) assay. Preliminary results indicate that the 5‐HT2AR and 5‐HT2CR form a robust BRET interaction creating a maximal signal of 230 mBRET units and a BRET50 of 2.1, which was more than double the signal generated in control studies with the β2‐adrenergic receptor. Taken together, we have identified a very close 5‐HT2AR:5‐HT2CR interaction in living cells. Ongoing research will employ these technologies to further investigate the formation and function of the receptor complex.Support or Funding InformationNIDA T32 DA07287 “Neural and Pharmacological Mechanisms of Abused Drugs”This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Arabidopsis Mediator Complex Subunit MED19a is Involved in ABI5-mediated ABA Responses

Arabidopsis Mediator complex subunit 19a (MED19a), which mediates interactions between transcriptional regulators and RNA polymerase II, plays a critical role in plant response to infection by pathogens. However, the roles of MED19a in other signaling pathways are unknown. Here, we report that MED19a plays an important role in regulation of abscisic acid (ABA)-mediated transcriptional regulation in Arabidopsis. Plants deficient in MED19a showed reduced sensitivity to ABA inhibition of seed germination, cotyledon greening, root growth, and stomatal opening. MED19a-deficient mutants also had reduced resistance to drought stress, evidenced by high water-loss rates and low survival rates. Molecular genetic analysis revealed that MED19a mutants had down-regulated ABA-induced genes, including Em1, Em6, and RD29B, and MED19a could occupy the promoters of Em1 and Em6 in an ABA-dependent manner. Furthermore, MED19a interacted with the transcription factor ABA-insensitive 5 (ABI5) in split-luciferase complementation assays and co-immunoprecipitation assays. An analysis of double mutants (med19a-2 and abi5-7) suggested that the action of MED19a in ABA signaling was dependent upon ABI5. Furthermore, MED19a and ABI5 influenced each other in recruiting the promoters of the target genes Em1 and Em6, which are involved in embryonic development. Altogether, these results indicate that MED19a acts as a positive regulator in ABI5-mediated ABA responses.

Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP.

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (∼40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ( KD < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in E. coli. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems.

Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor.

The aggregation of alpha-synuclein (αS), natively unstructured presynaptic protein, is a crucial factor leading to the pathogenesis of Parkinson’s disease (PD) and other related disorders. Recent studies have shown prefibrillar and oligomeric intermediates of αS as toxic to the cells. Herein, split-luciferase complementation assay is used to design a “signal-on” biosensor to monitor oligomerization of A53T αS inside the cells. Then, the effect of carbon-based nanomaterials, such as graphene quantum dots (GQDs) and graphene oxide quantum dots (GOQDs), on A53T αS oligomerization in vitro and in living cells is investigated. In this work, for the first time, it was found that GQDs at a concentration of 0.5 μg/mL can promote A53T αS aggregation by shortening the nucleation process, which is the key rate-determining step of fibrillation, thereby making a signal-on biosensor. While these nanomaterials may cross the blood–brain barrier because of their small sizes, the interaction between αS and GQDs may contribute to PD etiology.

Noninvasive Imaging of Ras Activity by Monomolecular Biosensor Based on Split-Luciferase Complementary Assay

Deregulated activity of Ras GTPases has been observed in many types of human cancers, and contributes to the diverse aspects of carcinogenesis. Although the significance in tumorigenesis has been widely accepted and many therapeutic drugs are under development, little attention has been dedicated to the development of sensors for the Ras activity in vivo. Therefore, based on the split firefly luciferase complementation strategy, we developed a monomolecular bioluminescent biosensor to image endogenous Ras activity in living subject. In this biosensor, two inactive luciferase fragments are sandwiched by Raf-1, whose conformation changes upon GTP-Ras binding. Thus, the Ras activity can be surrogated by the intensity of the complementary luciferase. The bioluminescence analyses demonstrated that this novel biosensor behaved the robust and sensitive reporting efficiency in response to the dynamical changes of Ras activity, both in living colorectal cancer cells and in vivo. Compared to the traditional method, such as the pull-down assay, the bioluminescent sensor is simply, noninvasive, faster and more sensitive for the analysis of the endogenous Ras activity. This innovative work opens up the way for monitoring the preclinical curative effect and high-throughput screening of therapeutic drugs targeting Ras pathways.

Subdomain 2, Not the Transmembrane Domain, Determines the Dimerization Partner of Growth Hormone Receptor and Prolactin Receptor.

Growth hormone receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane class I cytokine receptors. In humans, GH interacts with GHR homodimers or PRLR homodimers and PRL interacts with only PRLR homodimers to promote signaling. In human breast cancer cells endogenously expressing both receptors, GHR and PRLR specifically coimmunoprecipitate. We previously devised a split luciferase complementation assay to study GHR and PRLR assemblages. In this technique, firefly luciferase is split into two fragments (N- and C-terminal fragments of the luciferase), each without enzyme activity and tethered to the tails of two receptors. The fragments restore luciferase activity when brought close to each other by the receptors. Real-time ligand-induced complementation changes reflect the arrangement of receptors and indicate that GHR/PRLR is arranged as a heteromultimer comprised of GHR-GHR homodimers and PRLR-PRLR homodimers. We now dissect determinants for GHR and PRLR homodimerization versus heteroassociation. GHR and PRLR have extracellular domains comprised of the ligand-binding N-terminal subdomain 1 and a membrane-proximal subdomain 2 (S2), which fosters receptor-receptor contact. Based on previous studies of S2 versus the transmembrane domain (TMD) in GHR dimerization, we constructed GHR(PRLRS2), GHR(PRLRS2-TMD), and GHR(PRLRTMD), replacing GHR's S2 alone, S2 plus TMD, and TMD alone with PRLR's counterpart. We tested by complementation the ability of these chimeras and GHR or PRLR to homodimerize or heteroassociate. Comparing various combinations, we found GHR(PRLRS2) and GHR(PRLRS2-TMD) behaved as PRLR, whereas GHR(PRLRTMD) behaved as GHR regarding their dimerization partners. We conclude that S2 of GHR and PRLR, rather than their TMDs, determines their dimerization partner.

Abstract 2027: Preclinical evaluation of AMG 176, a novel, potent and selective Mcl-1 inhibitor with robust anti-tumor activity in Mcl-1 dependent cancer models

Abstract Evasion of apoptosis represents an essential hallmark in the progression of many cancers. The Bcl-2 family of proteins plays a central role in regulating the apoptotic process. Targeting pro-survival Bcl-2 family members like Mcl-1 with small molecule inhibitors represents a viable therapeutic approach for the treatment of cancer. This study evaluated the in vitro and in vivo activity of AMG 176, a potent and selective Mcl-1 inhibitor currently in Phase I clinical development. AMG 176 binds with high affinity and selectivity to the BH3-binding groove of Mcl-1. In a cell based split-luciferase complementation assay, AMG 176 disrupted the interaction between Mcl-1 and Bak, leading to downstream activation of the intrinsic apoptosis pathway as measured by increased caspase activity and subsequent effects on viability. Oral administration of AMG 176 to mice bearing OPM-2 multiple myeloma xenografts resulted in a dose-dependent increase in activated Bak with a clear PK/PD relationship. Dosing regimens (20-60 mg/kg PO, QD) evaluating discontinuous and continuous administration of AMG 176 in established OPM2 xenografts demonstrated robust tumor growth inhibition with complete tumor regression at an elevated dose. Efficacy in this model was achieved at doses in agreement with those eliciting induction of apoptotic markers. Treatment of tumor cell lines with Compound A, a close structural analog of AMG 176, revealed a dose- and time-dependent increase in Mcl-1 protein levels that was reversible upon compound washout. Subsequent experiments performed with cycloheximide suggested that elevations in Mcl-1 protein levels were due to an increase in Mcl-1 protein half-life, likely driven by the compounds ability to disrupt proteasome-mediated degradation. Compound A was also used to characterize the kinetics of activating apoptosis. These studies revealed a rapid induction of apoptosis and loss of viability in Mcl-1 dependent multiple myeloma and AML cell lines. In cell lines highly dependent on Mcl-1, treatment with Compound A for as little as two hours was sufficient to achieve complete cell killing. Cell line profiling studies (&amp;gt;200 lines) revealed robust effects on cell viability in a subset of solid tumor cell lines and cell lines of hematological origin, including multiple myeloma, acute myeloid leukemia and non-Hodgkin lymphoma. Subsequent association analysis with molecular profiling endpoints identified an inverse correlation between BCLxL expression and sensitivity to Mcl-1 inhibition. Combination screens with Compound A revealed multiple highly synergistic combinations including compounds targeting the MAPK pathway, standard of care chemotherapeutics and agents targeting additional pro-survival members of the BCL-2 family. In conclusion, AMG 176 is a potent and selective Mcl-1 inhibitor, with significant in vitro and in vivo activity in Mcl-1 dependent cancer models. Citation Format: Sean R. Caenepeel, Brian Belmontes, Jan Sun, Angela Coxon, Gordon Moody, Paul E. Hughes. Preclinical evaluation of AMG 176, a novel, potent and selective Mcl-1 inhibitor with robust anti-tumor activity in Mcl-1 dependent cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2017-2027

A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer's disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of Gi/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated Gi/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα13·R7-RGS complexes. Because Gα13/R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ5 with or without R7BP. We found that neurite retraction evoked by Gα12/13-dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα12/13 but not Gαi/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function.

Split luciferase complementation assay for the analysis of G protein-coupled receptor ligand response in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and β-arrestin, which are independent of the endogenous S. cerevisiae GPCR signaling pathways. We applied the split luciferase complementation assay method to S. cerevisiae and found that it can be used to analyze the ligand response of the human somatostatin receptor in S. cerevisiae. On the contrary, the response of the pheromone receptor Ste2 was not observed by the assay. Thus, the split luciferase complementation should be free from the effect of the endogenous GPCR signaling. Biotechnol. Bioeng. 2017;114: 1354-1361. © 2017 Wiley Periodicals, Inc.

A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions

A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

The release and trans-synaptic transmission of Tau via exosomes

BackgroundTau pathology in AD spreads in a hierarchical pattern, whereby it first appears in the entorhinal cortex, then spreads to the hippocampus and later to the surrounding areas. Based on this sequential appearance, AD can be classified into six stages (“Braak stages”). The mechanisms and agents underlying the progression of Tau pathology are a matter of debate. Emerging evidence indicates that the propagation of Tau pathology may be due to the transmission of Tau protein, but the underlying pathways and Tau species are not well understood. In this study we investigated the question of Tau spreading via small extracellular vesicles called exosomes.MethodsExosomes from different sources were analyzed by biochemical methods and electron microscopy (EM) and cryo-EM. Microfluidic devices that allow the culture of cell populations in different compartments were used to investigate the spreading of Tau.ResultsWe show that Tau protein is released by cultured primary neurons or by N2a cells overexpressing different Tau constructs via exosomes. Neuron-derived exosomal Tau is hypo-phosphorylated, compared with cytosolic Tau. Depolarization of neurons promotes release of Tau-containing exosomes, highlighting the importance of neuronal activity. Using microfluidic devices we show that exosomes mediate trans-neuronal transfer of Tau depending on synaptic connectivity. Tau spreading is achieved by direct transmission of exosomes between neurons. In organotypic hippocampal slices, Tau-containing exosomes in conditioned medium are taken up by neurons and microglia, not astrocytes. In N2a cells, Tau assemblies are released via exosomes. They can induce inclusions of other Tau molecules in N2a cells expressing mutant human Tau. We also studied exosomes from cerebrospinal fluid in AD and control subjects containing monomeric and oligomeric Tau. Split-luciferase complementation reveals that exosomes from CSF can promote Tau aggregation in cultured cells.ConclusionOur study demonstrates that exosomes contribute to trans-synaptic Tau transmission, and thus offer new approches to control the spreading of pathology in AD and other tauopathies.

GHR/PRLR Heteromultimer Is Composed of GHR Homodimers and PRLR Homodimers.

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane cytokine receptors. Each prehomodimerizes and ligand binding activates Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by GH, whereas PRLR is activated by both GH and PRL. We previously devised a split luciferase complementation assay, in which 1 receptor is fused to an N-terminal luciferase (Nluc) fragment, and the other receptor is fused to a C-terminal luciferase (Cluc) fragment. When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cells by coimmunoprecipitation, suggesting that, in addition to forming homodimers, these receptors form hetero-assemblages with functional consequences. We now extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL augments PRLR-PRLR complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL caused decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers.

Defining the Protein:Protein Interaction Interface of FGF14:Nav1.6 Complex

The voltage-gated Na+ (Nav) channel is composed of transmembrane spanning domains and of a cytosolic C-terminal tail which regulates channel function through protein:protein interactions (PPI) with auxiliary proteins, including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF (iFGF) family. In addition to binding to the Nav C-tail, FGF14 forms homodimers and previous structural studies have proposed a conserved surface common for the Nav channel and the iFGF homodimer formation. Seeking for potential differences between the FGF14:Nav1.6-C-tail complex and the FGF14:FGF14 dimer interface, we have engineered model-based amino acid residue mutations at predicted FGF14 hot-spots and begun to screen for their impact on the protein complex stability. Using the in-cell split-luciferase complementation assay to reconstitute the FGF14:Nav1.6-C-tail and the FGF14:FGF14 complex, we identified a point of divergence at the FGF14V160 residue whose mutation led to opposite effects on the relative binding affinity to the FGF14 monomer versus the Nav1.6-C-tail. Functional studies using whole-cell patch-clamp electrophysiology indicated that V160 is a critical residue for FGF14 modulation of Nav1.6-mediated currents that can be abolished by Ala mutation. Initial studies using intrinsic fluorescence showed efficient interaction between purified FGF14 and the Nav1.6 C-tail. Surface plasmon resonance and isothermal titration calorimetry measurements to evaluate the role of V160 in regulating binding affinity of FGF14 to the Nav1.6 C-tail are underway. With its unique role in the regulating the FGF14 binding and function, the V160 residue is well-positioned as target site for PPI-based drug development against Nav channels.Supported by: R01MH095995 (FL) and the Gulf Coast Consortia NIGMS Grant No.1 T32 GM089657-04 (SRA).

Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice.

Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them.

ICE1 of Pyrus ussuriensis functions in cold tolerance by enhancing PuDREBa transcriptional levels through interacting with PuHHP1.

ICE1 transcription factor plays an important role in plant cold stress via regulating the expression of stress-responsive genes. In this study, a PuICE1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. The expression levels of the PuICE1 were induced by cold, dehydration and salt, with the greatest induction under cold conditions. PuICE1 was localized in the nucleus and could bind specifically to the MYC element in the PuDREBa promoter. The PuICE1 fused to the GAL4 DNA-binding domain to have transcriptional activation activity. Ectopic expression of the PuICE1 in tomato conferred enhanced tolerance to cold stress at cold temperatures, less electrolyte leakage, less MDA content, higher chlorophyll content, higher survival rate, higher proline content, higher activities of enzymes. In additon, steady-state mRNA levels of six stress-responsive genes coding for either functional or regulatory genes were induced to higher levels in the transgenic lines by cold stress. Yeast two-hybrid, transient assay, split luciferase complementation and BiFC assays all revealed that PuHHP1 protein can physically interact with PuICE1. Taken together, these results demonstrated that PuICE1 plays a positive role in cold tolerance, which may be due to enhancement of PuDREBa transcriptional levels through interacting with the PuHHP1.