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Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photoinsensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s).

Nanoparticles composed of a magnetic iron oxide core surrounded by a metal shell have utility in a broad range of biomedical applications. However, the presence of surface energy differences between the two components makes wetting of oxide with metal unfavorable, precluding a “core–shell” structure of an oxide core completely surrounded by a thin metal shell. Three-dimensional island growth followed by island coalescence into thick shells is favored over the two-dimensional layer-by-layer growth of a thin, continuous metal coating of a true core–shell. Aqueous synthesis of gold-coated magnetite nanoparticles with analysis by infrared, energy-dispersive X-ray, and electron energy loss spectroscopies; high-resolution transmission electron microscopy; selected area electron diffraction; and high-angle annular dark-field scanning transmission electron microscopy showed two distinct morphologies that are inconsistent with an idealized core–shell. The majority were isolated ~16–22-nm-diameter nanoparticles consisting of ~7-nm-diameter magnetite and a thick deposition of gold, most often discontinuous, with some potentially “sandwiched” morphologies. A minority were aggregates of agglomerated magnetite decorated with gold but displaying significant bare magnetite. Both populations were successfully conjugated to fibrinogen and targeted to surface-activated platelets, demonstrating that iron oxide–gold nanoparticles produced by aqueous synthesis do not require an ideal core–shell structure for biological activity in cell labeling and targeting applications.

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M3Rs (muscarinic M3 acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M3R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

Electrophilic tri-substituted ethylene monomers, alkyl ring-substituted 2-phenyl-1,1-dicyanoethylenes, RC6H4CH=C(CN)2 (where R is 2-OCH3, 3-OCH3, 4-OCH3, 2-OC2H5, 3-OC2H5, 4-OC2H5, 4-OC3H7, 4-OC4H9, 4-OC6H11), were synthesized by piperidine-catalyzed Knoevenagel condensation of ring-substituted benzaldehydes and malononitrile, and characterized by CHN elemental analysis, IR, 1H- and 13C-NMR. Novel co-polymers of the ethylenes and vinyl acetate were prepared at equimolar monomer feed composition by solution co-polymerization in the presence of a radical initiator (ABCN) at 70°C. The composition of the co-polymers was calculated from nitrogen analysis, and the structures were analyzed by IR, 1H- and 13C-NMR, GPC, DSC and TGA. High T g of the co-polymers, in comparison with that of polyvinyl acetate, indicates a substantial decrease in chain mobility of the co-polymer due to the high dipolar character of the tri-substituted ethylene monomer unit. The gravimetric analysis indicated that the co-polymers decomp...

Review of: Enterohemorrhagic Escherichia coli and Other Shiga Toxin-Producing E. coli; Vanessa Sperandio and Carolyn H. Hovde (ed.); (2015). ASM Press, Washington, DC. 553 pages.

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35−/−) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35−/− mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35−/− mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.

Anti-CD20 antibody immunotherapy effectively treats non-Hodgkin's lymphoma and autoimmune disease. However, the cellular and molecular pathways for B cell depletion remain undefined because human mechanistic studies are limited. Proposed mechanisms include antibody-, effector cell–, and complement-dependent cytotoxicity, the disruption of CD20 signaling pathways, and the induction of apoptosis. To identify the mechanisms for B cell depletion in vivo, a new mouse model for anti-CD20 immunotherapy was developed using a panel of twelve mouse anti–mouse CD20 monoclonal antibodies representing all four immunoglobulin G isotypes. Anti-CD20 antibodies rapidly depleted the vast majority of circulating and tissue B cells in an isotype-restricted manner that was completely dependent on effector cell Fc receptor expression. B cell depletion used both FcγRI- and FcγRIII-dependent pathways, whereas B cells were not eliminated in FcR common γ chain–deficient mice. Monocytes were the dominant effector cells for B cell depletion, with no demonstrable role for T or natural killer cells. Although most anti-CD20 antibodies activated complement in vitro, B cell depletion was completely effective in mice with genetic deficiencies in C3, C4, or C1q complement components. That the innate monocyte network depletes B cells through FcγR-dependent pathways during anti-CD20 immunotherapy has important clinical implications for anti-CD20 and other antibody-based therapies.

To systematically review medical and familial conditions in autistic subjects (AU) as compared to other developmentally disabled controls (DD). Data was gathered prospectively for 102 AUs and 106 DDs who were comparable for age, sex, and nonverbal intelligence. Chi-square and t test analyses were used to evaluate the data collected for AUs and DDs. Demographic data for AUs and DDs was similar for age, sex, and nonverbal IQ scores. The only statistically significant demographic differences were higher educational levels in mothers of AUs and increased number of firstborn AUs. Family history data revealed a greater number of reported maternal learning disabilities for DDs and increased paternal mental retardation for AUs but no differences in familial medical or psychiatric disorders. No prenatal or perinatal risk factors were identified in the AU group although increased alcohol use during pregnancy and decreased gestational age was found in the DD group. Review of developmental and behavioral issues revealed delayed toileting, tantrums, strong food preferences, and preoccupations to be significantly increased in AUs as compared to DDs. No significant differences were noted between the two groups in health problems or doctor visits. However, AUs had significantly larger mean head size than DDs. This study confirmed several previously established findings in autism including 4 to 1 male prevalence, increased head size, strong food preferences, and lack of prenatal/perinatal findings specifically associated with autism. However, the data suggested no increased prevalence of health problems among AUs or their family members compared to DDs. Several biologic interventions and etiologic theories of autism have been based on the assumption of medical differences in this population, an assumption called into question by current data.

The protein C/protein S system is known to regulate thrombin generation in vivo by cleaving factors Va and VIIIa. We have examined the activity of activated protein C in several tissue factor–initiated models of coagulation. We used 4 models: monocytes as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as both the tissue factor source and the thrombin-generating surface; and relipidated tissue factor with lipid vesicles providing the surface for thrombin generation. With the lipid surface, activated protein C dose-dependently reduced thrombin generation. Similarly, when endothelial cells provided the only surface for thrombin generation, activated protein C dose-dependently decreased thrombin generation significantly. By contrast, whenever platelets were present, activated protein C only minimally affected the amount of thrombin generated. When endothelial cells were the tissue factor source with platelets providing the surface for thrombin generation, activated protein C did increase the time until the burst of thrombin generation but had minimal effects on the total amount of thrombin generated. Activated protein C had essentially no effect on thrombin generation when monocytes were the tissue factor source with platelets providing the surface for thrombin generation. From the studies reported here, we conclude that in vivo, despite the important role of the protein C system in regulating thrombosis, activated protein C does not serve as a primary regulator of platelet-dependent thrombin generation.

SummaryTissue factor pathway inhibitor (TFPI) contains three Kunitz domains separated by two connecting regions. We have cloned another naturally occurring TFPI gene product from a mouse lung cDNA library which we have called TFPI β. TFPIβ is derived from alternative splicing of the TFPI gene. Analysis of the cDNA shows that mouse TFPIβ protein is identical to TFPI from the N’-terminus through the second connecting region. However, mouse TFPIβ possesses neither a third Kunitz domain nor an Arg, Lys-rich C’-terminus but instead has a completely different C’-terminal (β-domain) sequence which is not homologous to any known protein. Northern blot analyses show that the tissues for mouse TFPIβ synthesis are heart and lung; in contrast, TFPI appears in Northern blots of heart and spleen. Both TFPIβ and TFPI messages first appear in 7-day-old mouse embryos, but only the TFPI mRNA persists until 17 days. Purified recombinant TFPIβ shows an apparent molecular weight of 38 kDa. Kinetic studies indicate that mouse TFPIβ is a slow-binding enzyme inhibitor for human factor Xa. In addition, heparin does not enhance the inhibition of factor Xa by mouse TFPIβ although it does accelerate factor Xa inhibition by TFPI.

Abstract —Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor–initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.

SummaryTissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Rhodopsin is a prototypical G-protein coupled receptor that initiates photo-transduction in the retina of the eye. Like many other G protein-coupled receptors, rhodopsin appears to form oligomers in the membrane. The notion that rhodopsin forms oligomers in its native membrane, however, is still quite controversial. The clearest demonstration that rhodopsin forms oligomers comes from atomic force microscopy images displaying large arrays of rhodopsin monomers. It has been speculated, however, that oligomers observed in these images are due to phase separation occurring in membranes at temperatures below 37 C. In the work presented here, we explored in detail the oligomeric status of rhodopsin and its dependency on temperatures using spectrally resolved Resonance Energy Transfer (RET) in Chinese hamster ovary cells. As a control, we also investigated a G188R mutant rhodopsin. This mutation causes misfolding of rhodopsin and leads to the retinal degenerative disorder retinitis pigmentosa. Our results suggest that the quaternary structure of wild-type rhodopsin is vastly different compared to that of the misfolded mutant rhodopsin.

Tissue factor is the major initiator of coagulation. Both factor IX and factor X are activated by the complex of factor VIIa and tissue factor (VIIa/TF). The goal of this study was to determine the specific roles of factors IXa and Xa in initiating coagulation. We used a model system of in vitro coagulation initiated by VIIa/TF and that included unactivated platelets and plasma concentrations of factors II, V, VIII, IX, and X, tissue factor pathway inhibitor, and antithrombin III. In some cases, factor IX and/or factor X were activated by tissue factor- bearing monocytes, but in some experiments, picomolar concentrations of preactivated factor IX or factor X were used to initiate the reactions. Timed samples were assayed for both platelet activation and thrombin activity. Factor Xa was 10 times more potent than factor IXa in initiating platelet activation, but factor IXa was much more effective in promoting thrombin generation than was factor Xa. In the presence of VIIa/TF, factor X was required for both platelet activation and thrombin generation, while factor IX was only required for thrombin generation. We conclude that VIIa/TF-activated factors IXa and Xa have distinct physiologic roles. The main role of factor Xa that is initially activated by VIIa/TF is to activate platelets by generating an initial, small amount of thrombin in the vicinity of platelets. Factor IXa, on the other hand, enhances thrombin generation by providing factor Xa on the platelet surface, leading to prothrombinase formation. Only tiny amounts of factors IX and X need to be activated by VIIa/TF to perform these distinct functions. Our experiments show that initiation of coagulation is highly dependent on activation of small amounts of factors IXa and Xa in proximity to platelet surfaces and that these factors play distinct roles in subsequent events, leading to an explosion of thrombin generation. Furthermore, the specific roles of factors IXa and Xa generated by VIIa/TF are not necessarily reflected by the kinetics of factor IXa and Xa generation.

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that affects patients with cystic fibrosis and immunocompromised individuals. This bacterium coexpresses two unique forms of lipopolysaccharides (LPSs) on its surface, the A- and B-band LPS, which are among the main virulence factors that contribute to its pathogenicity. The polysaccharides in A-band LPSs are synthesized in the cytoplasm and translocated into the periplasm via an ATP-binding cassette (ABC) transporter consisting of a transmembrane protein, Wzm, and a cytoplasmic nucleotide-binding protein, Wzt. Most of the biochemical studies of A-band PSs in Pseudomonas aeruginosa are focused on the stages of the synthesis and ligation of PS, leaving the export stage involving the ABC transporter mostly unexplored. This difficulty is compounded by the fact that the subunit composition and structure of this bi-component ABC transporter are still unknown. Here we propose a simple but powerful method, based on Forster Resonance Energy Transfer (FRET) and optical micro-spectroscopy technology, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of the Wzm–Wzt complex in living cells. It is found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the structure and behavior of this ABC transporter will help develop antibiotics targeting the biosynthesis of the A-band LPS endotoxin.

The pathogenic Pseudomonas aeruginosa is a Gram-negative bacterium coexpressing two unique forms of lipopolysaccharides on its surface, the A and B bands. The A-band polysaccharides are thought to be translocated into the periplasm through an ATP-binding-cassette (ABC) transporter consisting of two subunits: a transmembranar protein, Wzm, and a nucleotide-binding protein, Wzt. Since Wzm and Wzt proteins possess no domains or motifs that are targeted for modifications by eukaryotic cellular machineries or exhibit any signal for organelles localization, we used Chinese hamster ovary (CHO) cells as a model system to study the protein complex structure and stoichiometry of Wzt protein in the absence or the presence of Wzm. We transfected the CHO cells with Wzt proteins fused to the green fluorescent protein (GFP2) or its variant yellow fluorescent protein (YFP) to study the protein complex structure of Wzt. A spectrally resolved two-photon microscope was used to obtain fluorescent images from transfected cells, and a novel spectral Fluorescence Resonance Energy Transfer (FRET) provided apparent FRET efficiency (Eapp) for each image pixel (Raicu et al., Nature Photonics, 2009). Distributions of FRET efficiencies were obtained from several cells, and the peak positions for the cells whose Eapp histograms showed single peak were binned to obtain a “meta-histogram”. The meta-histograms were fitted to simulated distributions obtained from various theoretical models (Raicu et. al., Nature Photonics, 2009), to determine the stoichiometry and geometry of Wzt oligomers. Wzt formed rhombus-shaped homo-tertamers, which became square shaped upon co-expression of untagged Wzm.

Background. The chemotherapeutic treatment of cancer suffers from poor specificity for targeting the tumor cells and often results in adverse effects such as systemic toxicity, damage to nontarget tissues, and development of drug-resistant tumors in patients. Increasingly, drug nanocarriers have been explored as a way of lessening or overcoming these problems. In this study, antibody-conjugated Au-coated magnetite nanoparticles, in conjunction with inductive heating produced by exposure to an oscillating magnetic field (OMF), were evaluated for their effects on the viability of tumor cells in a murine model of breast cancer. Treatment effects were evaluated by light microscopy and SEM.Results. 4T1 mammary epithelial carcinoma cells overexpressing the folate receptor were targeted with an anti-folate receptor primary antibody, followed by labeling with secondary antibody-conjugated Au-coated magnetite nanoparticles. In the absence of OMF exposure, nanoparticle labeling had no effect on 4T1 cell viability. However, following OMF treatment, many of the labeled 4T1 cells showed extensive membrane damage by SEM analysis, and dramatically reduced viability as assessed using a live/dead staining assay.Conclusions. These results demonstrate that Au-coated magnetite targeted to tumor cells through binding to an overexpressed surface receptor, in the presence of an OMF, can lead to tumor cell death.

Advances in two-photon microscopy with spectral resolution (TPM-SR) and the development of a simple theory of Forster Resonance Energy Tran sfer (FRET) for single molecular complexes recently lead to the development of a novel method for the determination of structure and localization in living cells of membrane protein complexes (Raicu et al., Nature Photon. , 3, 2009). An appealing feature of this method is its ability to provide such important information while being unaffected by spurious signals originating from stochastic FRET (Singh and Raicu, Biophys. J., 98 , 2010). We will present the results obtained from our recent studies of trimeric FRET calibration standards expressed in the cytoplasm of Chinese hamster ovary (CHO) cells, as well as a model G protein-coupled receptor expressed in the membrane of yeast. Emphasis will be placed on the measurement and analysis of single-molecular-complex FRET data for determination of the quaternary structure of some proteins (or the protein complex structure). Keywords: Forster (Fluorescence) Resonance Energy Transfer (FRET); two-photon microscopy; Chinese hamster ovary (CHO) cells; G protein-coupled receptor (GPCR); spectral resolution; sterile two alpha-factor receptor.

The ATP-binding-cassette (ABC) transporters are protein nanomachines associated with membranes and common to all living cells. ABC transporters utilize the energy of ATP hydrolysis to transport a variety of solutes across the membrane. Wzm (the transmembrane component) and Wzt (the nucleotide binding component) proteins constitute an ABC transporter that translocates the endotoxic polysaccharides through the inner membrane of the pathogenic bacterium Pseudomonas aeruginosa. To gain insights into the subunit stoichiometry and structural assembly of this ABC transporter, we reconstituted the ABC transporter in CHO cell lines. We expressed Wzm and Wzt proteins fused with green fluorescent protein (GFP2) or its variant, yellow fluorescent protein (YFP). The three-dimensional cellular localization of the expressed ABC components were constructed by stacking images of different sections of the cells utilizing a newly developed and spectrally-resolved two-photon microscope. We next used Fluorescence Resonance Energy Transfer (FRET) to obtain a quantitative understanding of the interaction between the ABC components by calculating the apparent FRET efficiencies for single pixels. When Wzm-GFP2 and Wzm-YFP were co-expressed, our FRET analysis indicated that Wzm self-associates within the cell membrane as an oligomer. This correlates with our biochemical characterization of membrane-extracted Wzm. When Wzt-GFP2 and Wzm-YFP were co-expressed, our FRET analysis indicated that Wzm and Wzt interact at the inner membrane surface. This correlates with our in vitro studies that show Wzm and Wzt interact with each other. The distribution of FRET efficiencies were compared when Wzt-GFP2 and Wzm-YFP or Wzt-YFP and Wzm-GFP2 were co-expressed to study the stoichiometry of Wzm and Wzt in the ABC transporter. The combination of FRET analysis and biochemical approaches will lead to a comprehensive investigation of the structural assembly and subunit composition of this ABC transporter and ultimately other transporters.

Some components of biological systems can be readily identified solely by their unique structure or other intrinsic physical properties which are evident when visualized in brightfield or various types of interference-based light microscopy (LM). However, for the unambiguous identification and localization of most biological molecular or macromolecular elements within a structural framework, some type of staining/labeling must be employed. This is important for a variety of applications including identification of particular tissues, identification of cells and subcellular components/structures, tracking of cells or subcellular components, and co-localization of cells and cellular components on or within tissues or cells. Labeling is also used to provide quantitative comparisons of epitope density, cell numbers, organelle numbers or volume, and a variety of other types of quantitative data. However, considerable caution must be taken when attempting quantitative or even semi-quantitative analyses. Efficiencies of labeling for different epitopes and antibodies or antibody mixtures vary. The exact relationship of color density, particle numbers, or fluorescence intensity (which can fade during observation), to the actual numbers of labeled sites is critical and often not known. These factors often make quantitative estimations or comparisons very risky.

The immunogold technique is a valuable method for labeling cellular macromolecules. However, multiple labeling using colloidal gold (cAu) nanoparticles of different sizes presents certain drawbacks; namely, as particle size increases, there is a decreased labeling efficiency and diminished spatial resolution with respect to the locations of labeled epitopes. Both concerns also limit the utility of heavy metal particles for comparative analysis of labeling densities. To minimize the variables due to differential labeling efficiencies, the best solution would be to conduct multiple labeling with particles of similar size. Consequently, some parameter other than size is necessary to distinguish each label type. In this study, we report the synthesis of colloidal palladium (cPd) nanoparticles of similar size but having two distinct shapes, umbonate and faceted, which are readily distinguishable from spherical colloidal gold particles. Their utility and fidelity as labels using a human platelet whole-mount model is also demonstrated.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

CD20 plays a role in human B cell proliferation and is an effective target for immunotherapy. In this study, mouse CD20 expression and biochemistry were assessed for the first time using a new panel of CD20-specific mAb, with CD20 function assessed using CD20-deficient (CD20(-/-)) mice. CD20 expression was B cell restricted and was initiated during late pre-B cell development. The frequency and density of CD20 expression increased during B cell maturation in the bone marrow, with a subpopulation of transitional IgM(hi) B cells expressing higher CD20 levels than the majority of mature recirculating B cells. Transitional T1 B cells in the spleen also expressed high CD20 levels, providing a useful new marker for this B cell subset. In CD20(-/-) mice, immature and mature B cell IgM expression was approximately 20-30% lower relative to B cells from wild-type littermates. In addition, CD19-induced intracellular calcium responses were significantly reduced in CD20(-/-) B cells, with a less dramatic effect on IgM-induced responses. These results reveal a role for CD20 in transmembrane Ca(2+) movement in mouse primary B cells that complements previous results obtained using human CD20 cDNA-transfected cell lines. Otherwise, B cell development, tissue localization, signal transduction, proliferation, T cell-dependent antibody responses and affinity maturation were normal in CD20(-/-) mice. Thus, mouse and human CD20 share similar patterns of expression and function. These studies thereby provide an animal model for studying CD20 function in vivo and the molecular mechanisms that influence anti-CD20 immunotherapy.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins in nature. GPCRs cascade signals from outer environment into cells and hence, are the target of more than 60% of modern clinical drugs. Determination of oligomerization of GPCRs is subject to significant controversy in the literature. We have investigated the quaternary structure of human muscarinic acetylcholine receptor type 3 (hM3) in living cells using Forster Resonance Energy Transfer (FRET) and two-photon excitation in an optical micro-spectroscopic set-up [Raicu et al, Nature Photonics, 2009]. The wild-type form of the hM3 receptor was fused to Cerulean while its mutated form, activated solely by a synthetic ligand (RASSL), was fused to Citrine and expressed constitutively in Flp-InTM T-RExTM 293 cells [Alvarez-Curto et al, Journal of Biological Chemistry, 2010]. When WT-hM3-Citrine and RASSL-hM3-Cerulean were co-expressed in same cells, excited donor (Cerulean), transferred its energy to nearby acceptors (Citrine) through FRET. Apparent FRET efficiency (Eapp) distribution maps were obtained for the imaged section of the cells by unmixing acquired spectrally resolved images using elementary donor and acceptor spectra. By selecting pixels in the Eapp image corresponding to the plasma membrane, a FRET efficiency histogram was obtained displaying the number of pixels in a particular range of Eapp values versus the corresponding Eapp value. The Eapp histograms were analyzed using a FRET theory [V. Raicu, Journal of Biological Physics, 2007], predicting various numbers and positions of peaks in the histograms for various sizes and geometry of oligomers. Eapp histograms of all cells were best fitted by a rhombus tetramer model. By also simulating the amplitudes of the peaks, we determined that hM3 receptors form both dimers and rhombus tetramers at the plasma membrane, and that their proportion remained largely unaffected by CNO binding.

G-protein-coupled receptors (GPCRs) such as human muscarinic acetylcholine receptor 3 (hM3) assume dimeric/oligomeric forms in living cells while maintaining their ability to bind and activate G-proteins. The precise stoichiometry, quaternary organization, and stability of these receptor complexes in living cells remain a subject of significant controversy. We have investigated the organization of hM3 receptors in living cells using spectrally resolved two photon microscopy (SR-TPM). Wild type hM3 and a synthetic-ligand-regulated form of this receptor (RASSL), were expressed either constitutively or in an antibiotic dependent manner in Flp-InTM T-RExTM 293 cells [Alvarez-Curto et al, Journal of Biological Chemistry, 2010]. A so-called donor of energy (Cerulean Fluorescent Protein) fused to RASSL can get excited by laser light and transfers its energy to nearby (

Exotic Rubus taxa in Australia have been revised following consultation with European and North American experts in Rubus, allied with studies of variation in patterns of DNA restriction fragments and morphology. Many of these taxa have names that are applied for the first time in Australia (prefaced with a †). The major focus of the work was the Rubus fruticosus L. aggregate and taxa of this aggregate covered here are R. anglocandicans A. Newton, R. cissburiensis W.C. Barton & Ridd., †R. echinatus Lindl., †R. erythrops Edees & A. Newton, R. laciniatus Willd., R. leightonii Lees ex Leight. †R. leucostachys Schleich. ex Sm., †R. phaeocarpus W.C.R. Watson, R. polyanthemus Lindeb., †R. riddelsdellii Rilstone, †R. rubritinctus W.C.R. Watson, R. ulmifolius Schott (including R. ulmifolius var. ulmifolius and †R. ulmifolius var. anoplothyrsus Sudre), and R. vestitus Weihe, along with two undescribed taxa, Rubus sp. Scott Creek (D.E. Symon 16504) and Rubus sp. Tasmania (J.R. Hosking 1551). Other naturalised taxa are R. alceifolius Poir., R. ellipticus Sm., R. idaeus L., †R. laudatus A. Berger, †R. loganobaccus L.H. Bailey, †R. philadelphicus Blanch., R. roribaccus (L.H. Bailey) Rydb. and R. rugosus Sm. Patterns of morphological and molecular variation among individuals of the R. fruticosus agg. in Australia were examined. In phenetic analyses based on examination of 137 herbarium specimens and 27 morphological characters, taxa showed varying degrees of separation. Some taxa, for example R. anglocandicans and the two varieties of R. ulmifolius, formed distinct groups in these analyses whereas there was considerable overlap among individuals of other species. Fifty M13/HaeIII DNA-banding patterns (phenotypes) were identified among 198 collections from the R. fruticosus agg. across Australia. Thirty-five DNA phenotypes were correlated with 15 taxa of the R. fruticosus agg.; the remaining 15 DNA types correlated poorly or were determined with only a moderate level of confidence. R. anglocandicans, R. echinatus, R. leightonii, R. leucostachys, R. sp. Tasmania, R. ulmifolius and R. vestitus had two or more DNA phenotypes whereas only one DNA phenotype was observed for the remaining eight taxa. Taxa that were more distinct with respect to their DNA phenotypes also tended to be more distinct with respect to morphology based on a Mantel matrix correlation test. Within taxa that were difficult to tell apart morphologically, those sharing the same DNA phenotype were considered members of the same Rubus taxon. These results are discussed in the context of the evolution and ecology of the R. fruticosus agg. in Australia and in relation to the incomplete taxonomy of Rubus in Europe and North America.

We have determined the oligomeric size and configuration of fluorophore-tagged M1, M2, β1, and β2 receptors in the plasma membrane of Chinese hamster ovary (CHO) cells by examining the distribution of FRET efficiencies measured at the level of single pixels. The receptors were fused at the N-terminus to enhanced green or yellow fluorescent protein, and complementary pairs were co-expressed at different ratios of donor to acceptor (i.e., eGFP2-M1 and eYFP-M1, eGFP2-M2 and eYFP-M2, eGFP2-β1 and eYFP-β1, or eGFP2-β2 and eYFP-β2). Pixel-level emission spectra were recorded from images captured in a single plane; the relative contribution of each fluorophore then was determined by spectral deconvolution and used to calculate the corresponding apparent FRET efficiency. The distributions of efficiencies from 20 cells were analyzed in concert as a sum of Gaussians, with the number of components (n) and the relationships among the means taken as predicted for a dimer (n = 1), a triangular trimer (n = 2), and a tetramer configured as a square (n = 3) and a rhombus (n = 5). Distributions from each of the four receptors required 5 Gaussians, the means of which were related in the manner predicted for a rhombus. The inverse agonists atropine and timolol were without effect on the number of components or the relationship among the means detected for the M2 muscarinic receptor and the β2 adrenergic receptor, respectively. Homo-oligomers of the M1, M2, β1, and β2 receptors therefore appear to be rhombic tetramers in CHO cells. The configuration is unaffected by inverse agonists, at least in the case of the M2 and β2 receptors.

Forster Resonance Energy Transfer (FRET) is a process in which a donor (D) in the excited state transfers its energy nonradiatively to an acceptor (A) in the ground state. The underlying theory has been confirmed countless times, particularly with regard to the dependence of the FRET efficiency on the sixth power of the distance between D and A. In contrast, a complete FRET theory for multiple donors and acceptors in oligomeric complexes has been developed only recently (Raicu, 2007, J. Biol. Phys. 33:109-127), in parallel with technology of sufficient accuracy for tests in living cells (Raicu et al., 2009, Nature Photon. 3:107-113). This novel approach now has been applied to linked fluorescent proteins located in the cytoplasm and at the plasma membrane. The cytoplasmic probes were fused combinations of a donor (Cerulean, C), an acceptor (Venus, V), and a chromophore-deficient, Venus-like molecule that cannot absorb or transfer energy (Amber, A) (Koushik et al., 2009, PLoS ONE 4(11):e8031): namely, ACVA, ACAV, VCAA, and VCVV. The membrane-bound probes were fused dimers and trimers of eGFP2 (G2) and eYFP (Y): namely, G2Y, YG2, G2YG2, and YG2Y. According to the theory (Raicu, 2007), the FRET efficiency of a tetramer such as VCVV can be predicted from that of analogues that contain a single acceptor (e.g., ACVA, ACAV, VCAA); also, the apparent FRET efficiency of a trimer such as G2YG2 or YG2Y can be predicted from the pair-wise efficiency that corresponds to that of dimers such as G2Y and YG2. These predictions have been confirmed for FRET efficiencies measured by means of two-photon microspectroscopy (Raicu et al., 2009), in accord with the theory and underlying assumptions for FRET within multimers.