Membrane Labeling Research Articles

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

Avian Papilloma and Squamous Cell Carcinoma: a Histopathological, Immunohistochemical and Virological study

In this retrospective study, we describe the histopathological findings in seven papillomas and 45 squamous cell carcinomas (SCCs) from psittacine birds, raptors and domestic fowl. The age of affected birds ranged from 3 to 40 years, with median age significantly higher in psittacines (P=0.014). The majority of tumours were located in the skin (24/52, 46.2%) or uropygial gland (10/52, 19.2%). Thirty of the SCCs (66.7%) were well differentiated and 15 (33.3%) were poorly-differentiated. SCCs exhibited a significantly higher degree of nuclear pleomorphism (P=0.005) and a greater proportion were ulcerated (P=0.001) compared with papillomas; however, there was no significant difference in mitotic count (MC) or inflammation score. The expression of cyclo-oxygenase (COX)-2 and E-cadherin was investigated by immunohistochemistry. The COX-2 total score (TS) was significantly higher in SCCs compared with papillomas (P=0.002), but the difference between COX-2 TS of well- and poorly-differentiated SCCs was not significant. COX-2 labelling was predominantly cytoplasmic, but some tumours had concurrent membranous and/or perinuclear labelling. SCCs with membranous labelling had a significantly higher MC (P=0.028). A significantly higher proportion of SCCs were negative for E-cadherin compared with papillomas (P=0.042), but there was no significant difference between well- and poorly-differentiated SCCs. Fourteen papillomas and SCCs from psittacines were also tested by polymerase chain reaction for the presence of Psittacus erithacus papillomavirus 1 and Psittacid herpesvirus 1, but all samples tested negative. We demonstrate for the first time the expression of COX-2 and E-cadherin in avian tissues, and suggest that these markers may be useful in differentiating papillomas from SCCs, particularly when sample size is small.

Enduring alterations in hippocampal astrocytesynaptic proximity following adolescent alcohol exposure: reversal by gabapentin.

Adolescent alcohol abuse is a substantive public health problem that has been the subject of intensive study in recent years. Despite reports of a wide range of effects of adolescent intermittent ethanol (AIE) exposure on brain and behavior, little is known about the mechanisms that may underlie those effects, and even less about treatments that might reverse them. Recent studies from our laboratory have indicated that AIE produced enduring changes in astrocyte function and synaptic activity in the hippocampal formation, suggesting the possibility of an alteration in astrocyte-neuronal connectivity and function. We utilized astrocyte-specific, membrane restricted viral labeling paired with immunohistochemistry to perform confocal single cell astrocyte imaging, three-dimensional reconstruction, and quantification of astrocyte morphology in hippocampal area CA1 from adult rats after AIE. Additionally, we assessed the colocalization of astrocyte plasma membrane labeling with immunoreactivity for AMPA-(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptor 1, an AMPA receptor subunit and established neuronal marker of excitatory synapses, as a metric of astrocyte-synapse proximity. AIE significantly reduced the colocalization of the astrocyte plasma membrane with synaptic marker puncta in adulthood. This is striking in that it suggests not only an alteration of the physical association of astrocytes with synapses by AIE, but one that lasts into adulthood - well after the termination of alcohol exposure. Perhaps even more notable, the AIE-induced reduction of astrocyte-synapse interaction was reversed by sub-chronic treatment with the clinically used agent, gabapentin (Neurontin), in adulthood. This suggests that a medication in common clinical use may have the potential to reverse some of the enduring effects of adolescent alcohol exposure on brain function. All animal experiments conducted were approved by the Duke University Institutional Animal Care and Use Committee (Protocol Registry Number A159-18-07) on July 27, 2018.

Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Verification and spatial mapping of TRPV1 and TRPV4 expression in the embryonic and adult mouse lens

The transient receptor protein vanilloid channels, TRPV1 and TRPV4, have recently been shown to be mechanosensors in the ocular lens that act to transduce physical changes in lens volume and internal hydrostatic pressure into the activation of signalling pathways in lens epithelial cells. These pathways in turn regulate ion and water transport to ensure that the optical properties of the lens remain constant. Despite the functional evidence that implicate the roles of TRPV1 and TRPV4 in the lens, their respective cellular expression patterns in the different regions of the lens has to date not been fully characterised. Using Western blotting we have confirmed that TRPV1 and TRPV4 are expressed throughout all regions (epithelium, outer cortex, inner cortex/core) of the adult mouse lens. Subsequent immunolabeling of lens cryosections confirmed that TRPV1 and TRPV4 are expressed throughout all regions of the lens, but revealed differentiation-dependent differences in the subcellular expression of the two channels in the different regions. In the epithelium and outer cortex, intense TRPV1 and TRPV4 labeling was predominately associated with the cytoplasm. In a discrete zone in the inner cortex, labeling for both proteins was greatly diminished, but could be enhanced by incubating sections with the detergent Triton X-100 to reveal TRPV1 and TRPV4 labelling that was associated with the membrane. This suggests that in this region of the lens there is a potential interacting protein that masks the binding of the TRPV1 and TRPV4 antibodies to their respective epitopes in the lens inner cortex. In the core of the lens, which contains the embryonic nucleus, TRPV1 and TRPV4 labelling was associated exclusively with fibre cell membranes. This labelling in the lens core of the adult mouse lens appeared to originate in early development as a similar membrane labelling was observed at embryonic day 10 (E10) of the cells in the lens vesicle that subsequently forms the embryonic nucleus in the adult lens. During subsequent stages of embryonic development TRPV1 and TRPV4 remained membranous in the inner cortex and core, while showing labelling that was associated with the cytoplasm in the superficial outer cortical region. The extent of cytoplasmic labelling for TRPV4, but not TRPV1, in this cortical region could however be dynamically regulated by cutting the zonules that normally attach the lens to the ciliary body. We have shown an early onset and continuous expression of TRPV1 and TRPV4 across all lens regions, and that TRPV4 can be dynamically trafficked into the membranes of differentiating fibre cells, results that suggests that these mechanosensitive channels may also be functionally active in lens fibre cells.

Design and Applications of a Fluorescent Labeling Technique for Lipid and Surfactant Preformed Vesicles.

Amphiphilic molecules such as surfactants, lipids, and block copolymers can be assembled into bilayers and form vesicles. Fluorescent membrane labeling methods require the use of dye molecules that can be inserted into the bilayers at different stages of synthesis. To our knowledge, there is no generalized method for labeling preformed vesicles. Herein, we develop a versatile protocol that is suitable to both surfactant and lipid preformed vesicles and requires no separation or purification steps. On the basis of the lipophilic carbocyanine green dye PKH67, the methodology is assessed on zwitterionic phosphatidylcholine vesicles. To demonstrate its versatility, it is applied to dispersions of anionic or cationic vesicles, such as a drug administrated to premature infants with respiratory distress syndrome, or a vesicle formulation used as a fabric softener for home care applications. By means of fluorescence microscopy, we then visualize the interaction mechanisms of nanoparticles crossing live cell membranes and of surfactants adsorbed on a cotton fabric. These results highlight the advantages of a membrane labeling technique that is simple and applicable to a large number of soft matter systems.

Glutathione-capped quantum dots for plasma membrane labeling and membrane potential imaging

The plasma membrane of cells is a crucial biological membrane that involved in a variety of cellular processes including cell signaling transduction through membrane electrical activity. Recently, monitoring membrane electrical activity using fluorescence imaging has attracted numerous attentions for its potential applications in evaluating how the nervous system works. However, the development of ideal fluorescent voltage-sensitive probes with both high membrane labeling efficiency and voltage sensitivity is still retain a big challenge. Herein, glutathione-capped CdSe@ZnS quantum dots (CdSe@ZnS-GSH QDs) with a size of 2.5 nm and an emission peak at 520 nm are synthesized using a facile ligand exchange method for plasma membrane labeling and membrane potential imaging. The as-synthesized CdSe@ZnS-GSH QDs can effectively label cell membrane at neutral pH within 30 min and exhibit excellent optical stability in continuous imaging for up to 60 min. With the test concentration up to 200 nM, CdSe@ZnS-GSH QDs show high biocompatibility to cells and do not affect cell proliferation, disturb cell membrane integrity or cause apoptosis and necrosis of cells. Then, a two-component voltage sensor strategy based on fluorescence resonance energy transfer (FRET) between CdSe@ZnS-GSH QDs and the dipicrylamine (DPA) is successfully developed to monitor the membrane potential by the fluorescence of CdSe@ZnS-GSH QDs. This study offers a facile strategy for labeling plasma membrane and monitoring the membrane potential of cells and will hold great potential in the research of signaling within intact neuronal circuits.

Cold-Adapted Protein Kinases and Thylakoid Remodeling Impact Energy Distribution in an Antarctic Psychrophile.

The Antarctic psychrophile Chlamydomonas sp. UWO241 evolved in a permanently ice-covered lake whose aquatic environment is characterized not only by constant low temperature and high salt but also by low light during the austral summer coupled with 6 months of complete darkness during the austral winter. Since the UWO241 genome indicated the presence of Stt7 and Stl1 protein kinases, we examined protein phosphorylation and the state transition phenomenon in this psychrophile. Light-dependent [γ-33P]ATP labeling of thylakoid membranes from Chlamydomonas sp. UWO241 exhibited a distinct low temperature-dependent phosphorylation pattern compared to Chlamydomonas reinhardtii despite comparable levels of the Stt7 protein kinase. The sequence and structure of the UWO241 Stt7 kinase domain exhibits substantial alterations, which we suggest predisposes it to be more active at low temperature. Comparative purification of PSII and PSI combined with digitonin fractionation of thylakoid membranes indicated that UWO241 altered its thylakoid membrane architecture and reorganized the distribution of PSI and PSII units between granal and stromal lamellae. Although UWO241 grown at low salt and low temperature exhibited comparable thylakoid membrane appression to that of C. reinhardtii at its optimal growth condition, UWO241 grown under its natural condition of high salt resulted in swelling of the thylakoid lumen. This was associated with an upregulation of PSI cyclic electron flow by 50% compared to growth at low salt. Due to the unique 77K fluorescence emission spectra of intact UWO241 cells, deconvolution was necessary to detect enhancement in energy distribution between PSII and PSI, which was sensitive to the redox state of the plastoquinone pool and to the NaCl concentrations of the growth medium. We conclude that a reorganization of PSII and PSI in UWO241 results in a unique state transition phenomenon that is associated with altered protein phosphorylation and enhanced PSI cyclic electron flow. These data are discussed with respect to a possible PSII-PSI energy spillover mechanism that regulates photosystem energy partitioning and quenching.

Modulation of biological responses to 2 ns electrical stimuli by field reversal

Nanosecond bipolar pulse cancellation, a recently discovered phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (>100 μs) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). Here we report 2 ns bipolar pulse cancellation, extending the scale of previously published results down to the time required to construct the permeabilizing lipid electropores observed in molecular simulations. We add new cancellation endpoints, and we describe new bipolar pulse effects that are distinct from cancellation. This new data, which includes transport of cationic and anionic permeability indicators, fluorescence of membrane labels, and patterns of entry into permeabilized cells, is not readily explained by the accelerated discharge mechanism. We suggest that multi-step processes that involve first charged species movement and then responses of cellular homeostasis and repair mechanisms are more likely to explain the broad range of reported results.

Membrane Radiolabelling of Exosomes for Comparative Biodistribution Analysis in Immunocompetent and Immunodeficient Mice - A Novel and Universal Approach.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin and inherent intercellular biomolecule delivery capability. An in-depth knowledge of their in vivo biodistribution is therefore essential. This work aimed to develop a novel, reliable and universal method to radiolabel exosomes to study their in vivo biodistribution.Methods: Melanoma (B16F10) cells were cultured in bioreactor flasks to increase exosome yield. B16F10-derived exosomes (ExoB16) were isolated using ultracentrifugation onto a single sucrose cushion, and were characterised for size, yield, purity, exosomal markers and morphology using nanoparticle tracking analysis (NTA), protein measurements, flow cytometry and electron microscopy. ExoB16 were radiolabelled using 2 different approaches - intraluminal labelling (entrapment of 111Indium via tropolone shuttling); and membrane labelling (chelation of 111Indium via covalently attached bifunctional chelator DTPA-anhydride). Labelling efficiency and stability was assessed using gel filtration and thin layer chromatography. Melanoma-bearing immunocompetent (C57BL/6) and immunodeficient (NSG) mice were injected intravenously with radiolabelled ExoB16 (1x1011 particles/mouse) followed by metabolic cages study, whole body SPECT-CT imaging and ex vivo gamma counting at 1, 4 and 24 h post-injection.Results: Membrane-labelled ExoB16 showed superior radiolabelling efficiency and radiochemical stability (19.2 ± 4.53 % and 80.4 ± 1.6 % respectively) compared to the intraluminal-labelled exosomes (4.73 ± 0.39 % and 14.21 ± 2.76 % respectively). Using the membrane-labelling approach, the in vivo biodistribution of ExoB16 in melanoma-bearing C57Bl/6 mice was carried out, and was found to accumulate primarily in the liver and spleen (~56% and ~38% ID/gT respectively), followed by the kidneys (~3% ID/gT). ExoB16 showed minimal tumour i.e. self-tissue accumulation (~0.7% ID/gT). The membrane-labelling approach was also used to study ExoB16 biodistribution in melanoma-bearing immunocompromised (NSG) mice, to compare with that in the immunocompetent C57Bl/6 mice. Similar biodistribution profile was observed in both C57BL/6 and NSG mice, where prominent accumulation was seen in liver and spleen, apart from the significantly lower tumour accumulation observed in the NSG mice (~0.3% ID/gT).Conclusion: Membrane radiolabelling of exosomes is a reliable approach that allows for accurate live imaging and quantitative biodistribution studies to be performed on potentially all exosome types without engineering parent cells.

203 Newly designed liposome nanoparticles for drug delivery into boar spermatozoa

Liposomes are spherical vesicles with a membrane composed of at least one phospholipid bilayer. They have been successfully applied for intracellular drug or genetic material delivery in many biomedical areas. In agriculture, however, their use to produce transgenic farm animals through exogenous DNA transfection of spermatozoa remains unsatisfactory. Here, we tested a newly designed liposome preparation for effective and harmless interactions with boar spermatozoa. Extended fresh boar semen samples were gently centrifuged (N=3 independent replicates), and sperm pellets were resuspended in PBS. Sperm concentrations were then adjusted to 2×108 mL−1 and aliquoted in 0.5mL for labelling with various doses of fluorescent liposomes (0, 15, 30, or 60µg), for plasma membrane labelling (Experiment 1) or with nonfluorescent liposomes loaded with doxorubicin (0 or 60µg) having natural fluorescence property, for nucleus labelling (Experiment 2). After co-incubation for 45 to 60min at 37°C, sperm aliquots of each treatment were immediately evaluated for motility and morphology characteristics using a computer-assisted sperm analyzer. Remaining sperm-liposome mixtures were centrifuged to remove the excess of liposomes, and pelleted spermatozoa were imaged with the In vivo Imaging System (IVIS: PerkinElmer, Waltham, MA, USA) and fluorescence confocal laser microscope (LSCM; Zeiss, Oberkochen, Germany). Data were statistically analysed using one-way ANOVA or Student’s t-test, with P<0.05 indicating a significant difference. In Experiment 1, the proportions of motile and forward progressive spermatozoa were significantly (P<0.05) increased by the presence of 30µg (65±4% and 46±3%, respectively) and 60µg (66±1% and 48±1%, respectively) of liposomes compared with 0µg, the control (51±3% and 36±3%, respectively). In Experiment 2, doxorubicin-loaded liposomes significantly (P<0.05) improved the proportions of motile and progressive spermatozoa (87±2% and 69±8% v. 71±8% and 55±8% for the control, respectively). The percentages of static and abnormal spermatozoa (bent tail or distal cytoplasmic droplet) were significantly decreased by the presence of liposomes in both experiments (P<0.05). In contrast, the velocity parameters of spermatozoa were not significantly affected by the presence of liposomes (P>0.05). The microscope imaging revealed sperm-liposome interactions with displayed sperm membrane and nucleus fluorescence in Experiments 1 and 2, respectively. The proportion of stained cells in each experiment is in evaluation by flow cytometry. In conclusion, the newly designed liposomes were effective for simple interactions with the plasma membrane and molecule delivery to boar spermatozoa, while improving sperm motility. Further studies are ongoing with the replacement of doxorubicin with a DNA fragment. This work was supported by USDA-ARS Biophotonics Initiative #58-6402-3-018.

All-Trans Retinoic Acid (ATRA) up-Regulates Cell Surface CD38 Expression Which Promotes Pro-Tumoral Mitochondrial Trafficking from Stromal Cells to Multiple Myeloma

BackgroundMultiple myeloma (MM) is malignancy highly reliant on its microenvironment. In this study, we investigated whether mitochondrial transfer occurred between bone marrow stromal cells (BMSC) and malignant plasma cells. We then used our observations as a platform to investigate the mechanisms controlling pro-tumoral mitochondrial transfer with a view to identifying druggable targets.MethodsPrimary MM cells were obtained from patients' bone marrow after informed consent and under approval from the United Kingdom Health Research Authority. Animal experiments were conducted under approvals from the UK Home Office and the University of East Anglia Animal Welfare and Ethics Review Board. Primary BMSC were also obtained from patient bone marrow, using adherence and characterised using flow cytometry. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining of the BMSC (in-vitro), rLV.EF1.AcGFP-Mem9 labelling of the MM plasma membrane with MitoTracker CMXRos staining of the BMSC (in-vitro) and an in vivo MM NSG xenograft model. CD38 expression on MM cells was tested after ATRA treatment, using RT-qPCR and flow cytometry. Mitochondrial transfer levels were assessed when CD38 was over expressed using ATRA or inhibited using lentivirus targeted shRNA.ResultsWe report that mitochondria are transferred from BMSC to MM cells. First, we cultured MM cells on MitoTracker Green labelled BMSC and found increased MitoTracker Green fluorescence in the MM cells. We then transduced MM with rLV.EF1.AcGFP-Mem9 lentivirus and stained BMSC with MitoTracker CMXRos and used wide field microscopy to show MM derived tunnelling nanotubles (TNT) formed between MM cells and BMSC, with red mitochondria located within the GFP-tagged TNT. Next, we engrafted the MM cell lines MM1S and U266 into NSG mouse, after isolation we detected the presence of mouse mitochondrial DNA in the purified MM population. Together, these data show that mitochondria are transferred from BMSC to MM cells.We next analysed OXPHOS levels in MM cells grown on BMSC, using the seahorse extracellular flux assay. We found that the MM cells had increased levels of OXPHOS after culture with BMSC, which was also the case for MM cell lines analysed after isolation from NSG mice, showing the micro-environment of MM can alter the metabolism of the malignant cell. To examine whether the mitochondrial transfer process was controlled by CD38, we knocked down CD38 in MM cells using lentiviral targeted shRNA. We found reduced levels of mitochondrial transfer in CD38KD MM cells, with a consequent reduction of OXPHOS in the malignant cells.Finally, as ATRA has previously been shown to increase CD38 expression in AML, we next quantified CD38 mRNA and surface glycoprotein level on malignant plasma cells with and without ATRA treatment. We found ATRA increased CD38 expression at the mRNA and protein levels and this resulted in an increase in mitochondrial transfer from BMSC to MM cells.ConclusionHere we show that CD38 mediated mitochondrial transfer in the MM micro-environment forms part of the malignant phenotype of multiple myeloma. This finding develops our understanding of the mechanisms which underpin the efficacy of CD38 directed therapy in MM. DisclosuresNo relevant conflicts of interest to declare.

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.

Abstract 2455: The long non-coding RNA (lncRNA) DANCR in acute myeloid leukemia (AML) stem cells (LSC)

Abstract Introduction: Prognosis for AML patients (pts) is inadequate. Therapeutic strategies are needed to target not only bulk blasts but also the distinct LSCs, which are more resistant to chemotherapy & cause relapse. lncRNAs are non-coding RNA transcripts longer than 200 nucleotides that regulate cellular processes. A functional role in LSCs has yet to be elucidated. Methods: We derived a LSC-specific lncRNA profile using whole transcriptome sequencing (RNA-seq) data of 377 pts (<60 years) with cytogenetically normal (CN) AML. These data were correlated with a 'core enriched' (CE) gene expression signature representing 44 genes deregulated in LSCs (Eppert, Nat.Med. 2011). DANCR expression in the LSC-enriched compartment was validated using qRT-PCR. Transferrin/antibody conjugated nanoparticle (NP) knock down (KD) was used as an efficient & less toxic avenue to deliver siRNA against DANCR or scramble (SCR) in vitro & in vivo. LSC features (self-renewal, quiescence, engraftment) were analyzed & frequency determined in 3 primary AML samples and a CN-AML (Flt3ITD/WT/MllPTD/WT) mouse model. Results: 161 lncRNAs were consistently deregulated in CEhigh pts with a p<.001 & a correlation coefficient >.5 (Spearmen correlation test). We chose DANCR to further analyze because it was among the top lncRNAs upregulated in CEhigh pts, is highly conserved between human & mouse, & was previously shown to have a role in promoting stemness in hepatocellular carcinoma (Yuan, Hepatology 2016). We confirmed that DANCR expression was significantly higher in functionally validated LSC enriched populations (p=.05). To assess the role in self-renewal, we KD DANCR in pts cells & found a decrease in colony numbers after replating (average decrease vs SCR: 38.9%, p=.03). Using membrane labeling assays, we found a decrease of quiescent cells after DANCR KD (average decrease: 36.1%, p=.04). Long term colony-initiating cell cultures were performed to determine the LSC frequency & we found significant decreases in the number of LSCs in primary pts samples after DANCR KD (average decrease of LSC frequency: 79.2%, p=.001). Mice transplanted with murine AML cells (CD45.2+) were treated with NP (n=3 per group) for 5 days (d). 2d later, bone marrow was harvested & retransplantated (Tx) using limiting dilutions (2x106, 1x106, 5x105) into irradiated BoyJ (CD45.1+) recipients (n=30). 14d post-Tx, anti-Dancr recipient mice showed a lower engraftment (CD45.2/CD45.1: 37% vs 47%, p=.03) and also a concomitant improvement in survival (p<.001). Conclusion: We show for the first time that LSCs have a distinct lncRNA profile. Among the highly expressed lncRNAs associated with LSCs we identified & validated DANCR. Our data show that DANCR has an impact on LSC function (self-renewal, quiescence, engraftment) & regulates LSC frequency. DANCR is the first lncRNA shown to have a functional role in LSCs & represents a novel target for AML treatment. Citation Format: Marius Bill, Malith Karunasiri, Jessica Kohlschmidt, Allison E. Walker, Zachary Brannan, Stefano Volinia, Clara D. Bloomfield, Ramiro Garzon, Adrienne M. Dorrance. The long non-coding RNA (lncRNA) DANCR in acute myeloid leukemia (AML) stem cells (LSC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2455.

A Large Endoplasmic Reticulum-Resident Pool of TRPM1 in Retinal ON-Bipolar Cells.

The chemical signal of light onset, a decrease in glutamate release from rod and cone photoreceptors, is processed by a postsynaptic G protein signaling cascade in ON-bipolar cells (BPCs). The metabotropic glutamate receptor mGluR6, along with other cascade elements, is localized synaptically at the BPC dendritic tips. The effector ion channel protein transient receptor potential melastatin-1 (TRPM1), in contrast, is located not only at the dendritic tips but also in BPC bodies and axons. Little is known about the intracellular localization of TRPM1, or its trafficking route to the dendritic tip plasma membrane. Recombinant TRPM1 expressed in mammalian cells colocalized with endoplasmic reticulum (ER) markers, with little or none detected at the plasma membrane. In mouse retina, somatic TRPM1 was similarly intracellular, and not at the plasma membrane. Labeling of ER membranes by expression of a fluorescent marker showed that in BPCs the ER extends into axons and dendrites, but not dendritic tips. In cell bodies, TRPM1 colocalized with the ER, and not with the Golgi apparatus. Fluorescence protease protection (FPP) assays with TRPM1-GFP fusions in heterologous cells revealed that the N and C termini are both accessible to the cytoplasm, consistent with the transmembrane domain topology of related TRP channels. These results indicate that the majority of TRPM1 is present in the ER, from which it can potentially be transported to the dendritic tips as needed for ON light responses. The excess of ER-resident TRPM1 relative to the amount needed at the dendritic tips suggests a potential new function for TRPM1 in the ER.

Analysis of membranous Ki-67 staining in breast cancer and surrounding breast epithelium

Membranous Ki-67 staining with the MIB-1 antibody has been described in hyalinising trabecular adenomas of the thyroid and sclerosing haemangiomas of the lung. Its relatively rare occurrence in breast tumours has also been documented. The aim of the present study was to assess the rate of any membranous MIB-1 staining in breast specimens. The staining was performed at room temperature with 1:100 dilution of the antibody. One hundred four core needle biopsies and 41 operative specimens were analysed. Membranous staining was noted in 36/144 invasive carcinomas, 20/42 in situ carcinomas and 46/99 cases of peritumoural benign/normal breast epithelium. Most often, it presented as focal and partial polarised luminal membranous staining although complete circumferential staining also occurred, and membranous labelling was sometimes accompanied by cytoplasmic staining, too. In a few cases tested, greater dilution of the primary antibody did not abolish the membranous staining, which was absent with the SP6 monoclonal Ki-67 antibody. The membranous staining of invasive tumours showed no association with histological grade, lumen formation, oestrogen or progesterone receptor status or the Ki-67 nuclear labelling. In contrast, it was associated with a HER2-positive status, although it occurred in all molecular subtypes approached by immunohistochemistry. The background of this membranous staining remains elusive. It is unlikely to represent an artefact. At least partial sharing of an epitope of the nuclear Ki-67 protein with an unidentified membranous protein and some functional differences between membranous staining producing tumours and tumours lacking this pattern of staining may both contribute to some extent.

Synthesis and Evaluation of the First Fluorescent Antagonists of the Human P2Y2 Receptor Based on AR-C118925.

The human P2Y2 receptor (hP2Y2R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y2R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y2R antagonists, based on the known, non-nucleotide hP2Y2R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y2R (pKd = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y2R. These properties make 98 one of the first tools for studying hP2Y2R distribution and organization.

24S-hydroxycholesterol suppresses neuromuscular transmission in SOD1(G93A) mice: A possible role of NO and lipid rafts

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the initial denervation of skeletal muscle and subsequent death of motor neurons. A dying-back pattern of ALS suggests a crucial role for neuromuscular junction dysfunction. In the present study, microelectrode recording of postsynaptic currents and optical detection of synaptic vesicle traffic (FM1-43 dye) and intracellular NO levels (DAF-FM DA) were used to examine the effect of the major brain-derived cholesterol metabolite 24S-hydroxycholesterol (24S-HC, 0.4 μM) on neuromuscular transmission in the diaphragm of transgenic mice carrying a mutant superoxide dismutase 1 (SODG93A). We found that 24S-HC suppressed spontaneous neurotransmitter release and neurotransmitter exocytosis during high-frequency stimulation. The latter was accompanied by a decrease in both the rate of synaptic vesicle recycling and activity-dependent enhancement of NO production. Inhibition of NO synthase with L-NAME also attenuated synaptic vesicle exocytosis during high-frequency stimulation and completely abolished the effect of 24S-HC itself. Of note, 24S-HC enhanced the labeling of synaptic membranes with B-subunit of cholera toxin, suggesting an increase in lipid ordering. Lipid raft-disrupting agents (methyl-β-cyclodextrin, sphingomyelinase) prevented the action of 24S-HC on both lipid raft marker labeling and NO synthesis. Together, these experiments indicate that 24S-HC is able to suppress the exocytotic release of neurotransmitter in response to intense activity via a NO/lipid raft-dependent pathway in the neuromuscular junctions of SODG93A mice.

Sodium functions as a negative allosteric modulator of the oxytocin receptor

The oxytocin receptor, a class A G protein coupled receptor (GPCR), is essentially involved in the physiology of reproduction. Two parameters are crucially important to support high-affinity agonist binding of the receptor: Mg2+ and cholesterol, both acting as positive modulators. Using displacement assays with a high-affinity fluorescent antagonist (OTAN-A647), we now show that sodium functions as a negative allosteric modulator of the oxytocin receptor. In membranes from HEK293 cells stably expressing the oxytocin receptor, oxytocin binding occurred with about 15-fold lower affinity when sodium chloride was increased from 0 to 300 mM, whereas antagonist binding remained largely unchanged. The effect was concentration-dependent, sodium-specific, and it was also observed for oxytocin receptors endogenously expressed in Hs578T breast cancer cells. A conserved Asp (Asp 85) is known to stabilize the sodium binding site in other GCPRs. Mutations of this residue into Ala or Asn are known to yield non-functional oxytocin receptors. When Asp 85 was exchanged for Glu, most of the oxytocin receptors were localized in intracellular structures, but a faint plasma membrane labeling with OTAN-A647 and the appearance of oxytocin-induced calcium responses indicated that these receptors were functional. However, a sodium effect was not detectable for the mutant D85E oxytocin receptors. Thus, the oxytocin receptor is allosterically controlled by sodium similar to other GPCRs, but it behaves differently concerning the involvement of the conserved Asp 85. In case of the oxytocin receptor, Asp 85 is obviously essential for proper localization in the plasma membrane.