Defining the subcellular distribution and metabolic channeling of phosphatidylinositol.

Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.

Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

Monitoring of nucleophosmin oligomerization in live cells.

Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.

PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

Negative charge and membrane-tethered viral 3B cooperate to recruit viral RNA dependent RNA polymerase 3D pol.

Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3Dpol enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3Dpol.

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein.

Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.

On mechanism of intermediate-sized circular DNA compaction mediated by spermine: contribution of fluorescence lifetime correlation spectroscopy.

The compaction of DNA plays a role in the nuclei of several types of cells and becomes important in the non-viral gene therapy. Thus, it is in the scope of research interest. It was shown, that spermine-induced compaction of large DNA molecules occurs in a discrete "all-or-non" regime, where the coexistence of free and folded DNA molecules was observed. In the case of intermediate-sized DNA molecules (approximately 10 kbp), so far, it was stated that the mechanism of folding is continuous. Here, we show, that neither a standard benchmark technique-dynamic light scattering, nor a single molecule technique such as fluorescence correlation spectroscopy, can decide what kind of mechanism is undertaken in the compaction process. Besides, we introduce an application of a new approach-fluorescence lifetime correlation spectroscopy. The method takes an advantage of a subtle lifetime change of an intercalating dye PicoGreen during the titration with spermine and based on that, it reveals the discrete mechanism of the process. Furthermore, we show that it allows for observation of the equilibrium state transition dynamics.

Fluorescence study of the solvation of fluorescent probes prodan and laurdan in poly(epsilon-caprolactone)-block-poly(ethylene oxide) vesicles in aqueous solutions with tetrahydrofurane.

Steady-state and time-resolved fluorescence measurements were used to study the relaxation of the microenvironment of hydrophobic probes 6-propionyl-2-(dimethylamino)naphthalene (prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (laurdan) in systems containing vesicles formed by the amphiphilic diblock copolymer poly(epsilon-caprolactone)-block-poly(ethylene oxide) (PCL-PEO) and water/tetrahydrofurane (THF) solvent mixtures. It was found that in case of prodan, both steady-state and time-resolved emission spectra were composed of two subspectra corresponding to the emission of prodan molecules located (i) in fairly rigid (effectively viscous) and hydrophobic domains of the vesicles close to the PCL/PEO interface and (ii) in a more polar and less viscous medium (in the bulk solution). The fraction of the emission from the more polar microenvironment increases with increasing content of THF in the system. Laurdan, in contrast to prodan, appeared to be solubilized preferentially in the hydrophobic domains up to 30 vol % of THF content, and its emission spectra changed only due to swelling of hydrophobic PCL domains by added THF. The study shows that the analysis of the time-resolved emission from a probe distributed in two media is, in principle, possible, but it is quite complex and appreciably less accurate, and the relaxation times are ill-defined averages of several processes. The bimodal or shoulder-containing time-resolved spectra have to be decomposed in pertinent time-resolved subspectra and treated separately. Another important result of the study is a piece of knowledge concerning the motion of the probe with respect to the vesicle. In the studied complex system, not only the relaxation of the solvent and reorganization of polymer segments around the fluorescent headgroup of the probe affect the emission but also a lateral motion of the probe with respect to the nanoparticle within the lifetime of the excited state contributes significantly to the relaxation and to the relatively slow time-resolved Stokes shift.

Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy.

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.

Reversible aggregation of polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) block copolymer micelles in acidic aqueous Solutions.

Micelles of polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) (PS-PVP-PEO) were studied in acidic aqueous solutions by static and dynamic light scattering, alkalimetric titration, fluorescence correlation spectroscopy, and after deposition on a mica surface by atomic force microscopy. The PS-PVP-PEO micelles prepared by dialysis in ternary 1,4-dioxane-methanol-acidic water mixtures have a very low association number and show a strong tendency to form aggregates. The aggregation, which is promoted at low pH, seems to be fully reversible. Possible mechanisms of the aggregation are discussed. Atomic force microscopy scans of PS-PVP-PEO micelles deposited on a mica surface reveal the formation of micellar aggregates and support the general concept of aggregation upon changes in conditions and deterioration of the stability of small micelles.

Diffusion of sphingomyelin and myelin oligodendrocyte glycoprotein in the membrane of OLN-93 oligodendroglial cells studied by fluorescence correlation spectroscopy.

Evidence has been accumulated that the plasma membrane of various mammalian cell types is heterogeneous in structure and may contain lipid microdomains (lipid rafts). This study focuses on the membrane organization of living oligodendrocytes, which are the myelin-producing cells of the central nervous system. Fluorescence correlation spectroscopy (FCS) was used to monitor the lateral diffusion of a lipid and of a protein in the oligodendroglial cell line OLN-93. The lipid was fluorescently labelled sphingomyelin (Bodipy FL-C5 SM). The protein was the myelin oligodendrocyte glycoprotein (MOG). In order to monitor the lateral diffusion of MOG, OLN-93 cells were transfected with a MOG-EGFP (enhanced green fluorescent protein) fusion plasmid. The measurements were performed at room temperature. FCS data were analyzed for two-dimensional (2D) diffusion according to three models which all included a triplet fraction: (a) 2D 1 component (2D1C), (b) 2D anomalous diffusion (2D1Calpha), and (c) 2D 2 components (2D2C). Preliminary results indicate that for the lipid case, the best fits are obtained with 2D2C. In the case of MOG-EGFP, 2D2C and 2D1Calpha give fits of similar quality. The parameter estimates obtained with 2D1Calpha, however, have a lower standard deviation. The anomaly parameter for MOG-EGFP is 0.59+/-0.01.