HEPES-buffered Saline Research Articles

HEPES-Buffered Saline (HBS) (Used for Dye Loading)

HEPES-Buffered Saline (HBS) (Used for Dye Loading)

Structural Basis for Universal Corrinoid Recognition by the Cobalamin Transport Protein Haptocorrin

Cobalamin (Cbl; vitamin B12) is an essential micronutrient synthesized only by bacteria. Mammals have developed a sophisticated uptake system to capture the vitamin from the diet. Cbl transport is mediated by three transport proteins: transcobalamin, intrinsic factor, and haptocorrin (HC). All three proteins have a similar overall structure but a different selectivity for corrinoids. Here, we present the crystal structures of human HC in complex with cyanocobalamin and cobinamide at 2.35 and 3.0 Å resolution, respectively. The structures reveal that many of the interactions with the corrin ring are conserved among the human Cbl transporters. However, the non-conserved residues Asn-120, Arg-357, and Asn-373 form distinct interactions allowing for stabilization of corrinoids other than Cbl. A central binding motif forms interactions with the e- and f-side chains of the corrin ring and is conserved in corrinoid-binding proteins of other species. In addition, the α- and β-domains of HC form several unique interdomain contacts and have a higher shape complementarity than those of intrinsic factor and transcobalamin. The stabilization of ligands by all of these interactions is reflected in higher melting temperatures of the protein-ligand complexes. Our structural analysis offers fundamental insights into the unique binding behavior of HC and completes the picture of Cbl interaction with its three transport proteins.

α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N-Methyl-d-aspartate (NMDA) Receptors Adopt Different Subunit Arrangements

Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.

Treatment with a Novel Dinitroazetidine, Abdnaz, Improves Nitrite Reductase Activity of Sickle Red Blood Cells

AbstractAbstract 3246 IntroductionVaso-occlusion is a hallmark of sickle cell disease (SCD). Congestion of blood vessels by sickle red blood cells (RBC) is caused by hemoglobin polymerization (sickling), increased adhesion, and vasoconstriction due to a disregulated nitric oxide (NO) metabolism. NO generated from L-arginine by NO synthases plays a key role in vasodilation. Under hypoxia hemoglobin has been shown to be a potent alternative source of NO by reducing nitrite (NO2−). In SCD, modification of hemoglobin may reduce sickling and increase its ability to generate NO, and as such improve blood flow. In this study, we examined hemoglobin modification and consequent change in nitrite reductase capacity by 1-bromoacetyl-3,3-dinitroazetidine (ABDNAZ). This is a compound that has recently been described to bind to hemoglobin (Scicinski et al. Drug Met Disp. 2012) and has promising anticancer activity by altering blood flow in hypoxic tumors (Ning et al. Cancer Res. 2012). MethodBlood samples from SCD patients and control blood from healthy volunteers was collected with IRB approval. Red blood cells were isolated and resuspended to physiological hemactocrit in HEPES buffered saline (HBS) containing 2% bovine serum albumin (BSA). Samples were incubated with ABDNAZ, hydroxyurea (HU) or iodoacetamide at a final concentration of 3 mM for 30 minutes at room temperature. Intact RBC, hemolysates or purified hemoglobin samples were exposed to a constant nitrogen (N2) flow (200 mL/minute) at 37°C in a tonometer. At time zero, sodium nitrite was added to a final concentration of 5 mM. The influx of NO2− into the RBC was measured using Griess reagent on supernatant samples. The gas outflow from the tonometer was collected in Mylar balloons at various time points, and NO released in the gas phase was measured using a Nitric Oxide Analyzer. ResultsUpon NO2− addition under deoxygenated conditions, influx in RBC was rapid. An equilibrium between supernatant and RBC was reached within 10 minutes and indicated active recruitment of RBC rather than a simple diffusion equilibrium. Release of NO from SCD patient RBC was increased compared to normal control (HbAA) RBC. Under these conditions, hemolysates with the identical hemoglobin concentration as compared to intact RBC showed a pronounced increase in NO release. Using purified hemoglobin NO release was reduced to 50% as compared to hemolysates. Blood samples from both normal control and sickle cell patients treated with ABDNAZ, showed a significant increase in NO release as compared to untreated samples. Iodoacetamide and HU treatment, described to increase NO (Gladwin et al. Br. J. Hematol. 2002), also showed increased NO release but at a markedly lower level as compared to ABDNAZ treatment. ConclusionsEpisodes of vaso-occlusion result in pain and organ damage in SCD. Local generation of NO will improve vasodilation, and is potentially beneficial. The novel compound ABDNAZ, currently in human clinical trials, greatly increases nitrite reductase capacity of hemoglobin, in both normal and SCD blood samples. Our results indicate that this agent improves NO release from RBC and suggests that modification of sickle patient blood by ABDNAZ in the presence of plasma nitrite should be considered as a potential therapeutic approach. Disclosures:Scicinski:RadioRx, Inc: Employment. Oronsky:RadioRx, Inc: Employment.

Endothelial connexin43 mediates acid-induced increases in pulmonary microvascular permeability

Acid aspiration, a common cause of acute lung injury, leads to alveolar edema. Increase in lung vascular permeability underlies this pathology. To define mechanisms, isolated rat lungs were perfused with autologous blood. Hydrochloric acid and rhodamine-dextran 70 kDa (RDx70) were coinstilled into an alveolus by micropuncture. RDx70 fluorescence was used to establish the spatial distribution of acid. Subsequently, FITC-dextran 20 kDa (FDx20) was infused into microvessels for 60 min followed by a 10-min HEPES-buffered saline wash. During the infusion, FITC fluorescence changes were recorded to quantify the ratio of peak to postwash fluorescence. The ratio, termed normalized fluorescence, was low for acid compared with buffer instillation both in microvessels abutting acid-treated alveoli and those located more than 700 μm away. In contrast, the normalized fluorescence was similar to buffer controls when a higher molecular weight tracer (FITC-dextran 70 kDa) was infused instead of FDx20, suggesting that normalized FDx20 fluorescence faithfully represented microvascular permeability. Inhibiting endothelial connexin43 (Cx43) gap junction communication with Gap27 blunted the acid-induced reduction in normalized fluorescence, although scrambled Gap27 did not have any effect. The blunting was evident not only in microvessels away from the site of injury, but also in those abutting directly injured alveoli. Thus the new fluorescence-based method reveals that acid increases microvascular permeability both at acid-instilled and away sites. Inhibiting endothelial Cx43 blocked the permeability increase even at the direct injury sites. These data indicate for the first time that Cx43-dependent mechanisms mediate acid-induced increases in microvascular permeability. Cx43 may be a therapeutic target in acid injury.

Structural and Functional Characterization of Monomeric EphrinA1 Binding Site to EphA2 Receptor

The EphA2 receptor is overexpressed in glioblastoma multiforme and has been to shown to contribute to cell transformation, tumor initiation, progression, and maintenance. EphrinA1 (eA1) is a preferred ligand for the receptor. Treatment with monomeric eA1, the form of eA1 found in the extracellular environment, causes receptor phosphorylation, internalization, and down-regulation with subsequent anti-tumor effects. Here, we investigated the structure-function relationship of a monomeric eA1 focusing on its G-H loop ((108)FQRFTPFTLGKEFKE(123)G), a highly conserved region among eAs that mediates binding to their receptors. Alanine substitution mutants of the G-H loop amino acids were transfected into U-251 MG glioblastoma multiforme cells, and functional activity of each mutant in conditioned media was assessed by EphA2 down-regulation, ERK and AKT activation and cellular response assays. Alanine substitutions at positions Pro-113 Thr-115, Gly-117, Glu-122, and also Gln-109 enhanced the EphA2 receptor down-regulation and decreased p-ERK and p-AKT. Substitution mutants of eA1 at positions Phe-108, Arg-110, Phe-111, Thr-112, Phe-114, Leu-116, Lys-118, Glu-119, and Phe-120 had a deleterious effect on EphA2 down-regulation when compared with eA1-WT. Mutants at positions Phe-108, Lys-18, Lys-121, Gly-123 retained similar properties to eA1-WT. Recombinant eA1-R110A, -T115A, -G117A, and -F120A have been found to exhibit the same characteristics as the ligands contained in the conditioned media mainly due to the differences in their binding to the receptor. Thus, we have identified variants of eA1 that possess either superagonistic or antagonistic properties. These new findings will be important in the understanding of the receptor/ligand interactions and in further design of anti-cancer therapies targeting the eA/EphA system.

Association of the Endosomal Sorting Complex ESCRT-II with the Vps20 Subunit of ESCRT-III Generates a Curvature-sensitive Complex Capable of Nucleating ESCRT-III Filaments

The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.

HEPES-buffered saline (HBS, 2×)

HEPES-buffered saline (HBS, 2×)

Structure and Dynamics of Highly PEG-ylated Sterically Stabilized Micelles in Aqueous Media

Molecular assemblies of highly PEG-ylated phospholipids are important in many biomedical applications. We have studied sterically stabilized micelles (SSMs) of self-assembled DSPE–PEG2000 in pure water and isotonic HEPES-buffered saline solution. The observed SSM sizes of 2–15 nm largely depend on the solvent and the lipid concentration used. The critical micelle concentration of DSPE–PEG2000 is 10 times higher in water than in buffer, and the viscosity of the dispersion dramatically increases with the lipid concentration. To explain the experimentally observed results, we performed atomistic molecular dynamics simulations of solvated SSMs. Our modeling revealed that the observed assemblies have very different aggregation numbers (N(agg) ≈ 90 in saline solution and N(agg) < 8 in water) because of very different screening of their charged PO4(–) groups. We also demonstrate that the micelle cores can inflate and their coronas can fluctuate strongly, thus allowing storage and delivery of molecules with different chemistries.

Secretory Leukocyte Protease Inhibitor (SLPI) Expression and Tumor Invasion in Oral Squamous Cell Carcinoma

Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies.

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.

Polymyxin B self-associated with phospholipid nanomicelles

Context: Although Polymyxin B (PXB) is an effective antibiotic for Gram-negative bacterial infections, clinical use is hampered by toxicity and protein binding, which may be overcome by delivering PXB using a safe nanocarrier.Objective: To determine whether PXB self-associates with long-circulating biocompatible/biodegradable PEGylated phospholipid nanomicelles (SSM) and change the PXB in vitro bioactivity.Materials and methods: PXB and SSM (15 nm) composed of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were prepared in 10 mM HEPES-buffered saline. Interactions between PXB and SSM were determined by dynamic light scattering and fluorescence spectroscopy. Anti-infective effects of PXB-SSM were tested against Pseudomonas aeruginosa strain PA01 in vitro.Results: Approximately four PXB molecules self-associated with each SSM. However, significant decrease in P. aeruginosa killing was observed with PXB-SSM relative to PXB alone (P < 0.05). Empty SSM had no significant effect on bacterial growth.Discussion: PXB’s self-association with SSM resulted in mitigation of the in vitro antibacterial activity. This phenomenon could be attributed, in part, to PEG2000 hindering electrostatic interactions between cationic PXB and anionic bacterial cell wall.Conclusion: PXB association with SSM formed a stable nanomedicine, resulting in decreased bioactivity of the drug in vitro. Effectiveness of this nanomedicine in vivo is yet to be studied.

STIM1 and STIM2 Are Located in the Acidic Ca2+ Stores and Associates with Orai1 upon Depletion of the Acidic Stores in Human Platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.

Disease-associated N-terminal Complement Factor H Mutations Perturb Cofactor and Decay-accelerating Activities

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19–20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1–4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1–4V62 and FH1–4I62 bind immobilized C3b with similar affinities (KD = 10–14 μm), whereas FH1–4I62 is slightly more effective than FH1–4V62 as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1–4V62 binds to C3b with comparable affinity (KD ∼12 μm) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1–4V62, binds poorly to immobilized C3b (KD >35 μm) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Homophilic Adhesion Mechanism of Neurofascin, a Member of the L1 Family of Neural Cell Adhesion Molecules

The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.

Anionic and cationic dextran hydrogels for post-loading and release of proteins

In this study, post-loading of proteins in and release from chemically crosslinked dextran hydrogels exploiting reversible electrostatic interactions was investigated. Methacrylated dextran (Dex-MA) was co-polymerized with methacrylic acid (MA) or dimethylaminoethyl methacrylate (DMAEMA) to form negatively and positively charged hydrogels, respectively. Incubation of negatively charged hydrogels in a low ionic strength (10 mM HEPES, pH 7.4) solution of cytochrome C (isoelectric point ( pI) 10.2) led to quantitative absorption of the protein in the hydrogel. BSA ( pI 4.8) and myoglobin ( pI 7.2) were post-loaded into positively charged gels at neutral pH and negatively charged gels at pH 5, respectively. Loading efficiency and protein distribution in the gels were dependent on network charge (maximum loading efficiency at 100–150 μmol charged monomer/g gel) and crosslink density (higher and more homogenous loading at lower crosslink density) and on the ionic strength during loading (lower but more homogenous loading at higher ionic strength). Diffusion controlled release of the loaded protein was triggered by incubation of the hydrogel in HEPES buffered saline (HBS) pH 7.4. The amount of released cytochrome C in HBS varied from 94% to 70% from gels containing 60 and 150 MA/g, respectively. Importantly, quantitative release was obtained in 1 M NaCl, indicating that post-loading led to neither the formation of insoluble protein aggregates nor irreversible immobilization of the protein in the matrix. ESI-MS analysis of the released cytochrome C revealed that post-loading did not result in oxidation of the protein, as opposed to loading during preparation of the gels. In conclusion, this paper shows that post-loading of proteins in dextran hydrogels and release exploiting reversible charge interactions can be applied for efficient loading of proteins that are negative, positive or neutral at physiological pH. Importantly, our data demonstrate that using this loading method no chemical modification to the protein occurred.

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Zmynd15 Encodes a Histone Deacetylase-dependent Transcriptional Repressor Essential for Spermiogenesis and Male Fertility

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In(TM) T-REx(TM) 293 cells expressing a wild type human M(3) muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M(3) receptor expressed stably in Flp-In(TM) TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC(50) values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M(3) muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.