Live COS-7 Cells Research Articles

Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.

The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.

Roles of Cytoskeletal Components on the Membrane Movement of Individual Bk Ca Channels in Live COS-7 cell

Membrane dynamics of the large-conductance Ca2+-activated K+ channels (BKCa channels) was investigated in COS-7 cells by co-expressing the α subunit of the channel tagged with an acceptor peptide for biotin at its extracellular N-terminus and a genetically modified E. coli biotin ligase. We were able to visualize individual BKCa channels that had been biotinylated metabolically and then expressed on to the surface of the cells using quantum dots (QDs) coated with streptavidin. The time-lapse imaging on hundreds of homomeric BKCa channel revealed that the channels in live COS-7 cells exhibited a ‘confined diffusion’ in an area of 1.915 μm2 with an initial diffusion coefficient of 0.033 μm2/s. In order to understand the roles of cytoskeleton on the diffusional characteristics of the channel, we monitored the channel movement in the presence of drugs known to disrupt the filamentous actin and the microtubule, and quantified their effects on channel dynamics. It was found that both diffusion coefficient and the area of diffusion are differentially affected by such drug treatments. We are currently investigating the domain(s) of the channel responsible for cytoskeletal influence on the membrane dynamics of BKCa channel. Using this QD-based, single-molecule tracking technique, we wish to understand the cellular players influencing the diffusional movement of BKCa channel and their mechanism of action. ACKNOWLEDGEMENTS The authors are grateful to the members of the Laboratory of Molecular Neurobiology at GIST for their valuable comments and the Bio-Imaging Research Center at GIST for providing various imaging equipments. This research was supported by the grants from the Bio-Imaging Research Center to C-S Park.

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

Real Time Monitoring of Endogenous Messenger RNA Using Linear Antisense Probe

In eukaryotic cells mRNA plays a key role in gene regulation. However, the function of mRNA is not fully understood because direct analysis of endogenous mRNAs in living cells has been difficult. We developed a method for the observation of endogenous mRNA in living cells using two fluorescently labeled linear antisense 2′O-methyl RNA oligonucleotides. When those two antisense probes, each is labeled with different fluorescent dyes, are hybridized to an adjacent sequence of the target mRNA, the distance between two fluorophores becomes close and FRET occurs.Here we applied linear antisense probes to the real time monitoring of endogenous mRNA, which will be useful in understanding the function of mRNA as well as the intracellular localization. First, two kinds of linear antisense probes were microinjected into the cytoplasm of living COS7 cells and the FRET signal from cells was recorded over time to examine the kinetics of the hybridization reaction with c-fos mRNA. The hybridization reaction of linear antisense probes proceeded quickly and time constants of linear antisense probe was estimated to be less than one minute. When using Molecular Beacon, the conventional probe for endogenous mRNAs, it took more than one hour to complete the hybridization. Next, the induction of c-fos mRNA in the cytoplasm of COS7 cells was investigated in real time using linear antisense probes. As a result, the elevation of c-fos mRNA expressed in the cytoplasm was observed within one hour after the stimulation with PMA (phorbol 12-myristate 13-acetate). In conclusion, we showed the linear antisense probes are advantageous in monitoring of mRNAs due to their prominent kinetics in hybridizing with target mRNAs in living cells.

Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells

Phase imaging with a high-resolution wavefront sensor is considered. This is based on a quadriwave lateral shearing interferometer mounted on a non-modified transmission white-light microscope. The measurement technology is explained both in the scope of wave optics and geometrical optics in order to discuss its implementation on a conventional microscope. In particular we consider the effect of a non spatially coherent source on the phase-image signal-to-noise ratio. Precise measurements of the phase-shift introduced by microscopic beads or giant unilamellar vesicles validate the principle and show the accuracy of the methods. Diffraction limited images of living COS-7 cells are then presented, with a particular focus on the membrane and organelle dynamics.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones

BackgroundThe neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones.ResultsFollowing its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61β but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous.ConclusionThe lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue.

Too Close for Molecular Comfort

Release of Ca 2+ from intracellular stores leads to the rapid translocation of the endoplasmic reticulum (ER)-resident protein STIM1 (stromal interacting molecule 1) to a region of the ER near the plasma membrane. This enables the interaction of STIM1 with the Orai1 channel and the subsequent entry of Ca 2+ across the plasma membrane. Várnai et al . investigated this process in living COS-7 cells in which they created molecular bridges between the plasma membrane and the adjacent ER. They targeted fluorescently labeled constructs containing the FRB fragment of mTOR (mammalian target of rapamycin) to the cytoplasmic face of the ER, and constructs containing the FKBP-12 protein to the inner face of the plasma membrane, and induced their heterodimerization with rapamycin. This allowed visualization of stable contact zones between the ER and the plasma membrane in conjunction with visualization of fluorescently labeled STIM1 and Orai1 (or both) before and after treatment with thapsigargin (to deplete Ca 2+ stores in the ER) or stimulation of P2Y receptors with ATP. These regions of juxtaposition coincided with the regions in which STIM1 and Orai1 interacted after store depletion; however, Orai1, and the STIM1-Orai1 complex, were excluded from cross-linked contact regions in which the gap between the ER and the plasma membrane was too narrow. The authors used linkers to create bridges of different length, which indicated that STIM1 could be accommodated when the gap between the two membranes was 4 to 6 nm, whereas Orai1 required a gap of 11 to 14 nm, suggesting that it exists as part of a large macromolecular complex. P. Várnai, B. Tóth, D. J. Tóth, L. Hunyady, T. Balla, Visualization and manipulation of plasma membrane-endoplasmic reticulum contact sites indicates the presence of additional molecular components within the STIM1-Orai1 complex. J. Biol. Chem. 282 , 29678-29690 (2007). [Abstract] [Full Text]

The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket

OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.

Characterization of cyclin L1 as an immobile component of the splicing factor compartment

Cyclin L1 and cyclin L2 are two closely related members of the cyclin family that contain C-terminal arginine- and serine-rich (RS) domains and are localized in the splicing factor compartment (nuclear speckles). Here we applied photobleaching techniques to show that a green fluorescent protein (GFP) fusion protein of cyclin L1, in contrast to cyclin L2, was not mobile within the nucleus of living COS7 cells. The objectives of this study were to 1) characterize the intranuclear localization and mobility properties of cyclin L1 in different cellular states, and 2) dissect the structural elements required for immobilization of cyclin L1. Transcriptional arrest by actinomycin D caused accumulation of GFP-cyclin L2 in rounded and enlarged nuclear speckles but did not affect the subnuclear pattern of distribution of GFP-cyclin L1. Although immobile in most phases of the cell cycle, GFP-cyclin L1 was diffusely distributed and highly mobile in the cytoplasm of metaphase cells. By analysis of a series of chimeras, deletion constructs, and a point mutant, a segment within the RS domain of cyclin L1 was identified to be necessary for the immobility of the protein in nuclear speckles. This study provides the first characterization of an immobile component of nuclear speckles.

Generation and functional characterization of fluorescent, N-terminally tagged CB 1 receptor chimeras for live-cell imaging

N-terminally tagged CB 1 receptor fusion proteins, incorporating enhanced green fluorescent protein (GFP) or super-ecliptic pHluorin (SEP), were generated to study CB 1 receptor trafficking and cell surface receptor expression in live COS7 and HEK293 cells and hippocampal neurons. An artificial signal sequence (SS) was required for efficient surface expression of CB 1 receptor chimeras, which behaved like wild-type CB 1 receptors in functional assays. Treatment with cannabinoid ligands led to a rapid down-regulation of SS-GFP-CB 1 from the plasma membrane in COS7 and HEK293 cells, associated with trafficking into cytosolic vesicles. Activation of CB 1 receptors was also linked with a time-dependent reduction in cell surface SEP-CB 1 fluorescence and incorporation of the construct into acidic endosomes, revealed following exposure to NH 4Cl. In live hippocampal neurons, SEP-CB 1 fluorescence was largely restricted to the axon, consistent with its polarised surface expression. Thus, these new molecular tools are well suited for studying CB 1 receptor trafficking and a new generation of GPCR chimeras incorporating SEP at the N-terminus will be especially useful for monitoring dynamic changes in cell surface receptor expression in living cells.

Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells

BackgroundPolyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.MethodsPEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.ResultsPEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependant, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.ConclusionPEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.

Single Nanoparticle Photothermal Tracking (SNaPT) of 5-nm Gold Beads in Live Cells

Tracking individual nano-objects in live cells during arbitrary long times is a ubiquitous need in modern biology. We present here a method for tracking individual 5-nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against single fluorescent molecule tracking in live COS7 cells on subsecond timescales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. Single Nanoparticle Photothermal Tracking has the unique potential to record arbitrary long trajectory of membrane proteins using nonfluorescent nanometer-sized labels.

Huntingtin inclusion bodies are iron‐dependent centers of oxidative events

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.

Synthesis of a New Water-Soluble Rhodamine Derivative and Application to Protein Labeling and Intracellular Imaging

Synthesis of a new fluorescent rhodamine derivative, dye 1, is reported. This probe is different from other rhodamines insofar as it has several (four) carboxylic acid functionalities to promote water solubility and facilitate conjugation to proteins. It also has an aryl bromide functionality that could, in principle, be used to further functionalize the system for specialized applications. Dye 1 was conjugated to a model protein called ACBP (acyl-CoA binding protein). The properties of this conjugate were tested to establish that the label does not significantly perturb the binding function of the protein to its natural ligand in vitro and to confirm that its secondary structure was not significantly perturbed (circular dichroism). Experiments were performed to test if the labeled protein could be imported into living COS-7 cells (using the Chariot-peptide delivery system) and, if so, to observe, via fluorescence microscopy, which of the labeled protein was able to migrate to the nucleus, as expected for ACBP in cells. In the event, all these postulates were confirmed.

Opsin Oligomerization in a Heterologous Cell System

Using bioluminescence resonance energy transfer (BRET) we studied opsin oligomerization in heterologous expression systems and quantitatively assessed its oligomerization state. BRET2 saturation and competition experiments were performed with live COS-7 cells expressing Rluc-and GFP2-tagged receptor constructs. BRET2 saturation curves obtained were hyperbolic, and the calculated oligomerization state (N = 1 for dimers) suggested that opsin (N = 1.34 ± 0.25) forms higher oligomers. Very high BRET2 values obtained for the opsin homo-dimer pair indicated a large energy transfer efficiency (E) and for cases where E ≫ 0.1 a modified saturation curve was proposed. The existence of homo-dimer complexes was additionally supported by competition assay results and was also observed in HEK-293 cells. Furthermore, evidence was provided for homo-and hetero-dimerization of family A (β2-adrenergic) and B (gastric inhibitory polypeptide, GIP) receptors. In summary, these experiments demonstrate homo-and hetero-dimerization for opsin, β 2-adrenergic, and GIP receptors.

Intercellular trafficking of the major tegument protein VP22 of bovine herpesvirus-1 and its application to improve a DNA vaccine

Intercellular spread of bovine herpesvirus-1 (BHV-1) VP22 was demonstrated in living COS-7 cells transfected with a plasmid expressing VP22-YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) bicistronically. The intercellular trafficking property of VP22 was localized to the C-terminal portion of the molecule (amino acids 121-258; VP22-C). Plasmids encoding a truncated form of BHV-1 glycoprotein D (tgD) fused to VP22, VP22-C, or the N-terminal portion of VP22 (amino acids 1-120; VP22-N) were constructed. Mice immunized with plasmid encoding tgD-VP22 or tgD-VP22-C developed stronger immune responses when compared to animals immunized with plasmid encoding tgD or tgD fused to tgD-VP22-N.

Sialylation of Human Thyrotropin Receptor Improves and Prolongs Its Cell-Surface Expression

Glycosylation of the thyrotropin receptor (TSHR) has been shown to be essential for correct protein folding and for cell-surface targeting. In a recent study, we detected increased expression of beta-galactoside alpha(2,6)-sialyltransferase (SIAT1) in toxic thyroid adenomas where gain-of-function mutations of the TSHR have been invoked as one of the major causes. To investigate the physiological meaning of these findings, we designed experiments to evaluate the consequences of sialylation for the expression of the TSHR. Hence, we investigated the effect of coexpressing the TSHR and different sialyltransferases (SIAT1, SIAT4a, and SIAT8a) for cell-surface expression of the receptor. Coexpression of each of the three SIAT isoforms and the TSHR in COS-7 cells increased TSHR expression on the cell surface in the range of 50 to 100%. Moreover, Western blot analysis with lectins specific for alpha(2,3) and alpha(2,6)-linked sialic acids and lectin-binding enzyme-linked immunosorbent assay support a direct effect on TSHR cell-surface expression mediated by sialic acid transfer to the TSHR. Finally, we treated living COS-7 cells after cotransfection of TSHR and SIAT8a with neuraminidase for 30 min to remove covalently linked sialic acid. Subsequent loss of TSHR cell-surface expression suggests that sialylation prolongs the resting time of the TSHR on the cell surface. Our data demonstrate for the first time that the transfer of sialic acid can improve and prolong cell-surface expression of a transmembrane receptor.

Fluorescence Imaging Reveals the Nuclear Behavior of Peroxisome Proliferator-activated Receptor/Retinoid X Receptor Heterodimers in the Absence and Presence of Ligand*♦

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.

We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.