Luciferase, when fused to an N-terminal signal peptide, is secreted from transfected Plasmodium falciparum and transported to the cytosol of infected erythrocytes

Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a rate-limiting step.     

Diffusion of green fluorescent protein in three cell environments in Escherichia coli

Surprisingly little is known about the physical environment inside a prokaryotic cell. Knowledge of the rates at which proteins and other cell components can diffuse is crucial for the understanding of a cell as a physical system. There have been numerous measurements of diffusion coefficients in eukaryotic cells by using fluorescence recovery after photobleaching (FRAP) and related techniques. Much less information is available about diffusion coefficients in prokaryotic cells, which differ from eukaryotic cells in a number of significant respects. We have used FRAP to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin. GFP was expressed in the cytoplasm, exported into the periplasm using the twin-arginine translocation (Tat) system, or fused to an integral plasma membrane protein (TatA). We show that TatA-GFP diffuses in the plasma membrane with a diffusion coefficient comparable to that of a typical eukaryotic membrane protein. A previous report showed a very low rate of protein diffusion in the E. coli periplasm. However, we measured a GFP diffusion coefficient only slightly smaller in the periplasm than that in the cytoplasm, showing that both cell compartments are relatively fluid environments.     

A nonfitting method using a spatial sine window transform for inhomogeneous effective-diffusion measurements by FRAP

Determining averaged effective diffusion constants from experimental measurements of fluorescent proteins in an inhomogeneous medium in the presence of ligand-receptor interactions poses problems of analytical tractability. Here, we introduced a nonfitting method to evaluate the averaged effective diffusion coefficient of a region of interest (which may include a whole nucleus) by mathematical processing of the entire cellular two-dimensional spatial pattern of recovered fluorescence. Spatially and temporally resolved measurements of protein transport inside cells were obtained using the fluorescence recovery after photobleaching technique. Two-dimensional images of fluorescence patterns were collected by laser-scanning confocal microscopy. The method was demonstrated by applying it to an estimation of the mobility of green fluorescent protein-tagged heterochromatin protein 1 in the nuclei of living mouse embryonic fibroblasts. This approach does not require the mathematical solution of a corresponding system of diffusion-reaction equations that is typical of conventional fluorescence recovery after photobleaching data processing, and is most useful for investigating highly inhomogeneous areas, such as cell nuclei, which contain many protein foci and chromatin domains.     

Protein diffusion in mammalian cell cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.     

In Situ Quantification of Protein Binding to the Plasma Membrane

This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus.     

Expression and function of P-glycoprotein and absence of multidrug resistance-related protein in rat and beige mouse peritoneal mast cells

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT–PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.     

A Highlights from MBoC Selection: Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface.     

Dynamics of Natural Killer Cell Receptor Revealed by Quantitative Analysis of Photoswitchable Protein

Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 μm² s⁻¹) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein.     

ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tζζ– GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.     

Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS) in Nitella Internodal Cells

Cytoplasmic streaming (CPS) is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor–Aris dispersion more than by Brownian diffusion.     

In-Cell Protease Assay Systems Based on Trans-Localizing Molecular Beacon Proteins Using HCV Protease as a Model System

This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s). The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.     

Unusual Centrosome Cycle in Dictyostelium: Correlation of Dynamic Behavior and Structural Changes

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a γ-tubulin–green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyostelium centrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G₁/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of γ-tubulin–green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.     

Rosella: A fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast

We have developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Its mode of action relies upon differences in pH between different cellular compartments and the vacuole. Here we demonstrate its utility in yeast (Saccharomyces cerevisiae) by expression in the cytosol, and targeting to mitochondria or to the nucleus. When cells were cultured in nitrogen depleted medium, uptake of the compartment labelled with the biosensor (i.e. cytosol, mitochondria, or nucleus) into the vacuole was observed. We showed that this vacuolar uptake was, for cytosol and mitochondria, an ATG8-dependent process while the uptake of the nucleus was significantly reduced in the absence of Atg8p and can be said to be partially ATG8-dependent. We further demonstrated the value of the biosensor as a reporter of autophagy by employing fluorescence-activated cell sorting of discrete populations of cells undergoing autophagy.     

Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast

We have developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Its mode of action relies upon differences in pH between different cellular compartments and the vacuole. Here we demonstrate its utility in yeast (Saccharomyces cerevisiae) by expression in the cytosol, and targeting to mitochondria or to the nucleus. When cells were cultured in nitrogen depleted medium, uptake of the compartment labelled with the biosensor (i.e., cytosol, mitochondria, or nucleus) into the vacuole was observed. We showed that this vacuolar uptake was, for cytosol and mitochondria, an ATG8-dependent process while the uptake of the nucleus was significantly reduced in the absence of Atg8p and can be said to be partially ATG8-dependent. We further demonstrated the value of the biosensor as a reporter of autophagy by employing fluorescence-activated cell sorting of discrete populations of cells undergoing autophagy.     

Nuclear localization of the major vault protein in U373 cells

The major vault protein (MVP) is the predominant member of a large ribonucleoprotein particle, named vault. Vaults are abundant in the cytosol of mammalian cells. Mammalian MVP has previously been reported to be associated with the nucleus, particularly its cytosolic surface on which vaults are thought to dock at or near the nuclear pore complex. To date the presence of vault particles inside the nucleus has been convincingly reported only for sea urchin cells. We have addressed the potential nuclear localization of MVP in mammalian cells by employing confocal laser microscopy and cryo-immunoelectron microscopy. As revealed by immunostaining and by analysis of cells transfected with a construct encoding MVP and green fluorescent protein, MVP is present in both the cytosol and in the nucleus. Cryo-electron microscopy of human astroglioma U373 cells reveals clusters of immunogold particles at nuclear pores and in the nucleoplasm suggesting that nuclear MVP is associated with particulate structures. Quantification of the fluorescence observed in the cytosol and in the nuclei reveals that about 5% of the MVP in U373 cells is localized inside the nucleus. Our results further support the notion that part of the cellular MVP can enter the nucleus.     

Human Gyrovirus Apoptin shows a similar subcellular distribution pattern and apoptosis induction as the chicken anaemia virus derived VP3/Apoptin

The chicken anaemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumour-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from a human skin swab. It shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin or CAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. These findings indicate a potential anti-cancer role for HGyV-Apoptin.     

Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.     

Dimerization of N-methyltransferases involved in caffeine biosynthesis

Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed.     

Fluorescence correlation spectroscopy close to a fluctuating membrane

Compartmentalization of the cytoplasm by membranes should have a strong influence on the diffusion of macromolecules inside a cell, and we have studied how this could be reflected in fluorescence correlation spectroscopy (FCS) experiments. We derived the autocorrelation function measured by FCS for fluorescent particles diffusing close to a soft membrane, and show it to be the sum of two contributions: short timescale correlations come from the diffusion of the particles (differing from free diffusion because of the presence of an obstacle), whereas long timescale correlations arise from fluctuations of the membrane itself (which create intensity fluctuations by modulating the number of detected particles). In the case of thermal fluctuations this second type of correlation depends on the elasticity of the membrane. To illustrate this calculation, we report the results of FCS experiments carried out close to a vesicle membrane. The measured autocorrelation functions display very distinctly the two expected contributions, and allow both to recover the diffusion coefficient of the fluorophore and to characterize the membrane fluctuations in term of a bending rigidity. Our results show that FCS measurements inside cells can lead to erroneous values of the diffusion coefficient if the influence of membranes is not recognized.