Evaluation of pulsed-FRAP and conventional-FRAP for determination of protein mobility in prokaryotic cells

Background

Macromolecule mobility is often quantified with Fluorescence Recovery After Photobleaching (FRAP). Throughout literature a wide range of diffusion coefficients for GFP in the cytoplasm of Escherichia coli (3 to 14 µm²/s) is reported using FRAP-based approaches. In this study, we have evaluated two of these methods: pulsed-FRAP and “conventional”-FRAP.

Principal Findings

To address the question whether the apparent discrepancy in the diffusion data stems from methodological differences or biological variation, we have implemented and compared the two techniques on bacteria grown and handled in the same way. The GFP diffusion coefficients obtained under normal osmotic conditions and upon osmotic upshift were very similar for the different techniques.

Conclusions

Our analyses indicate that the wide range of values reported for the diffusion coefficient of GFP in live cells are due to experimental conditions and/or biological variation rather than methodological differences.     

Reduced protein diffusion rate by cytoskeleton in vegetative and polarized dictyostelium cells

Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells, the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied by altered intracellular protein mobility.     

Impact of osmotic stress on protein diffusion in Lactococcus lactis

We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram‐positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12‐transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ～ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram‐positive and Gram‐negative bacteria.     

Biophysical properties of Saccharomyces cerevisiae and their relationship with HOG pathway activation

Parameterized models of biophysical and mechanical cell properties are important for predictive mathematical modeling of cellular processes. The concepts of turgor, cell wall elasticity, osmotically active volume, and intracellular osmolarity have been investigated for decades, but a consistent rigorous parameterization of these concepts is lacking. Here, we subjected several data sets of minimum volume measurements in yeast obtained after hyper-osmotic shock to a thermodynamic modeling framework. We estimated parameters for several relevant biophysical cell properties and tested alternative hypotheses about these concepts using a model discrimination approach. In accordance with previous reports, we estimated an average initial turgor of 0.6 ± 0.2 MPa and found that turgor becomes negligible at a relative volume of 93.3 ± 6.3% corresponding to an osmotic shock of 0.4 ± 0.2 Osm/l. At high stress levels (4 Osm/l), plasmolysis may occur. We found that the volumetric elastic modulus, a measure of cell wall elasticity, is 14.3 ± 10.4 MPa. Our model discrimination analysis suggests that other thermodynamic quantities affecting the intracellular water potential, for example the matrix potential, can be neglected under physiological conditions. The parameterized turgor models showed that activation of the osmosensing high osmolarity glycerol (HOG) signaling pathway correlates with turgor loss in a 1:1 relationship. This finding suggests that mechanical properties of the membrane trigger HOG pathway activation, which can be represented and quantitatively modeled by turgor.     

Importance of intracellular water apparent diffusion to the measurement of membrane permeability

The exchange of water across biological membranes is of fundamental significance to both animal and plant physiology. Diffusional membrane permeability (P(d)) for the Xenopus oocyte, an important model system for water channel investigation, is typically calculated from intracellular water pre-exchange lifetime, cell volume, and cell surface area. There is debate, however, whether intracellular water motion affects water lifetime, and thereby P(d). Mathematical modeling of water transport is problematic because the intracellular water diffusion rate constant (D) for cells is usually unknown. The measured permeability may be referred to as the apparent diffusional permeability, P, to acknowledge this potential error. Herein, we show that magnetic resonance (MR) spectroscopy can be used to measure oocyte water exchange with greater temporal resolution and higher signal-to-noise ratio than other methods. MR imaging can be used to assess both oocyte geometry and intracellular water diffusion for the same single cells. MR imaging is used to confirm the dependence of intracellular water lifetime on intracellular diffusion. A model is presented to relate intracellular lifetime to true membrane diffusional permeability. True water diffusional permeability (2.7 +/- 0.4 microm/s) is shown to be 39 +/- 6% greater than apparent diffusional permeability for 8 oocytes. This discrepancy increases with cell size and permeability (such as after water channel expression) and decreases with increasing intracellular water D.     

Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility

A significant role of zinc-binding motifs on metal mobility in Escherichia coli was explored using a chimeric metal-binding green fluorescent protein (GFP) as an intracellular zinc indicator. Investigation was initiated by co-transformation and co-expression of two chimeric genes encoding the chimeric GFP carrying hexahistidine (His6GFP) and the zinc-binding motif fused to outer membrane protein A (OmpA) in E. coli strain TG1. The presence of these two genes was confirmed by restriction endonucleases analysis. Co-expression of the two recombinant proteins exhibited cellular fluorescence activity and enhanced metal-binding capability of the engineered cells. Incorporation of the zinc-binding motif onto the membrane resulted in 60-fold more binding capability to zinc ions than those of the control cells. The high affinity to metal ions of the bacterial surface influenced influx of metal ions to the cells. This may affect the essential ions for triggering important cell metabolism. A declining of fluorescent intensity of GFP has been detected on the cell expressed of zinc binding motif. Meanwhile, balancing of metal homeostasis due to the presence of cytoplasmic chimeric His6GFP enhanced the fluorescent emission. These findings provide the first evidence of real-time monitoring of intracellular mobility of zinc by autofluorescent proteins.     

Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein

All biological reactions depend on the diffusion and re-localization of biomolecules. Our understanding of biological processes requires accurate measurement of biomolecule mobility in living cells. Currently, approaches for investigating the mobility of biomolecules are generally restricted to measuring either fast or slow diffusion kinetics. We describe the development and application of a photoconvertible fluorescent protein, Phamret, that can be highlighted by UV light stimulation inducing a change in fluorescence emission from cyan fluorescent protein (CFP) to photoactivated GFP (PA-GFP). Phamret can be monitored by single excitation-dual emission mode for visualization of molecular dynamics for a broad range of kinetics. We also devised a microscopy-based method to measure the diffusion coefficient from the fluorescence decay after photostimulation of Phamret, enabling analysis of diffusion kinetics ranging from less than 0.1 microm2/s up to approximately 100 microm2/s, and found significant changes in free protein movement during cell-cycle progression.     

Cytoplasmic protein mobility in osmotically stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.     

Anisotropy and temperature dependence of myoglobin translational diffusion in myocardium: implication for oxygen transport and cellular architecture

Pulsed field gradient NMR methods have determined the temperature-dependent diffusion of myoglobin (Mb) in perfused rat myocardium. Mb diffuses with an averaged translational diffusion coefficient (DMb) of 4.24-8.37x10(-7)cm2/s from 22 degrees C to 40 degrees C and shows no orientation preference over a root mean-square displacement of 2.5-3.5 microm. The DMb agrees with the value predicted by rotational diffusion measurements. Based on the DMb, the equipoise diffusion PO2, the PO2 in which Mb-facilitated and free O2 diffusion contribute equally to the O2 flux, varies from 2.72 to 0.15 in myocardium and from 7.27 to 4.24 mmHg in skeletal muscle. Given the basal PO2 of approximately 10 mmHg, the Mb contribution to O2 transport appears insignificant in myocardium. In skeletal muscle, Mb-facilitated diffusion begins to contribute significantly only when the PO2 approaches the P50. In marine mammals, the high Mb concentration confers a predominant role for Mb in intracellular O2 transport under all physiological conditions. The Q10 of the DMb ranges from 1.3 to 1.6. The Mb diffusion data indicate that the postulated gel network in the cell must have a minimum percolation cutoff size exceeding 17.5 A and does not impose tortuosity within the diffusion root mean-square displacement. Moreover, the similar Q10 for the DMb of solution versus cell Mb suggests that any temperature-dependent alteration of the postulated cell matrix does not significantly affect protein mobility.     

Compartmentalization of mammalian proteins produced in Escherichia coli

We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.     

Dynamics of the Ins(1,4,5)P3 receptor during polarization of MDCK cells

BACKGROUND INFORMATION: The uneven distribution of the Ins(1,4,5)P3R [Ins(1,4,5)P3 receptor] within the ER (endoplasmic reticulum) membrane generates spatially complex Ca2+ signals. The ER is a dynamic network, which allows the rapid diffusion of membrane proteins from one part of the cell to another. However, little is known about the localization and the dynamics of the Ins(1,4,5)P3R in the ER of living cells. We have used a MDCK (Madin-Darby canine kidney) clone stably expressing the Ins(1,4,5)P3R1-GFP (where GFP stands for green fluorescent protein) to investigate the effect of cell polarity on the lateral mobility of the Ins(1,4,5)P3R. RESULTS: In non-confluent MDCK cells, the chimaera is homogeneously distributed throughout the ER and the nuclear envelope. FRAP (fluorescence recovery after photobleaching) experiments showed that the receptor can move freely in the ER with a diffusion constant (D=0.01 microm2/s) approx. ten times lower than other ER membrane proteins. In confluent polarized cells, two populations of receptor can be defined: one population is distributed in the cytoplasm and is mobile but with a slower diffusion constant (D=0.004 microm2/s) compared with non-confluent cells, whereas the other population is concentrated at the periphery of the cells and is apparently immobile. CONCLUSIONS: The observed differences in the mobility of the Ins(1,4,5)P3R are most probably due to its interactions with stable protein complexes that form at the periphery of the polarized cells.     

Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium cells has been studied using fluorescence correlation spectroscopy to gain a better understanding of the functioning of the domains and to assess the effect of initiation of chemotaxis on these domains in the cell. PH2-GFP was homogeneously distributed in vegetative as well as chemotaxing cells, whereas PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were very similar; however, PH10-GFP exhibited slower diffusion. Photon counting histogram statistics show that this slow diffusion was not due to aggregation. Diffusion of the three PH domains was affected to similar extents by intracellular heterogeneities in vegetative as well as chemotaxing cells. From the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP and PH-CRAC-GFP remained unchanged.     

Protein Mobility in the Cytoplasm of Escherichia coli

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient, D_a, of GFP in E. coli DH5α was found to be 7.7 ± 2.5 μm²/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with D_a of 2.5 ± 0.6 μm²/s. In addition, GFP mobility can depend strongly on at least two factors: first, D_a is reduced to 3.6 ± 0.7 μm²/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces D_a to 4.0 ± 2.0 μm²/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of D_a reported here. These measurements have implications for the understanding of intracellular biochemical networks.     

Mobility of creatine phosphokinase and beta-enolase in cultured muscle cells.

The diffusion of beta-enolase and creatine phosphokinase in muscle cells has been studied by modulated fringe pattern photobleaching. Beta-enolase is mobile in the sarcoplasm. At 20 degrees C, the diffusion coefficient is 13.5 +/- 2.5 microm2 s(-1) in the cytosol and 56 microm2 s(-1) in aqueous media. As in the case of dextrans of the same hydrodynamic radius, its mobility is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments. A fraction of creatine phosphokinase is mobile in the sarcoplasm. Its diffusion coefficient in the cytosol, 4.5 +/- 1 microm2 s(-1), is lower than that of the dextran of equivalent size. The other fraction (20 to 50%) is very slightly mobile, with an apparent diffusion coefficient varying from 0.0035 to 0.043 microm2 s(-1). This low mobility might be attributed to exchange between free and bound creatine phosphokinase. The bound fraction of the endogenous enzyme was localized by immunocytofluorescence on the cultured muscle cells. Our results favor a localization of bound cytosolic creatine phosphokinase on the M-line and a diffuse distribution in all myotubes.     

Changes in osmotic pressure and structure at the initial stages of zygote formation inClosterium ehrenbergii

Summary The behavior of gamete cells ofClosterium ehrenbergii in hypertonic solutions was observed and the significance of changes in osmotic pressure of the protoplasts is discussed in relation to zygote formation. The osmotic pressure of fusing gamete protoplasts was calculated to be 0.063 Osm at the original cell volume. The osmotic pressure of immature gamete protoplasts was 0.24 Osm at incipient plasmolysis. This lowering of cell osmotic pressure may serve to protect the rupture of the plasma membrane during migration of protoplasts in the conjugation tube after dissolution of cell walls. During maturation of gamete cells, chloroplasts and dictyosomes differed greatly in their ultrastructure from those of vegetative cells. These structural changes may be induced by changes of the physiological condition including osmotic pressure in the cells.     

Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress

To obtain turgor pressure, intracellular osmolalities, and cytoplasmic water activity of Escherichia coli as a function of osmolality of growth, we have quantified and analyzed amounts of cell, cytoplasmic, and periplasmic water as functions of osmolality of growth and osmolality of plasmolysis of nongrowing cells with NaCl. The effects are large; NaCl (plasmolysis) titrations of cells grown in minimal medium at 0.03 Osm reduce cytoplasmic and cell water to approximately 20% and approximately 50% of their original values, and increase periplasmic water by approximately 300%. Independent analysis of amounts of cytoplasmic and cell water demonstrate that turgor pressure decreases with increasing osmolality of growth, from approximately 3.1 atm at 0.03 Osm to approximately 1.5 at 0.1 Osm and to less than 0.5 atm above 0.5 Osm. Analysis of periplasmic membrane-derived oligosaccharide (MDO) concentrations as a function of osmolality, calculated from literature analytical data and measured periplasmic volumes, provides independent evidence that turgor pressure decreases with increasing osmolality, and verifies that cytoplasmic and periplasmic osmolalities are equal. We propose that MDO play a key role in periplasmic volume regulation at low-to-moderate osmolality. At high growth osmolalities, where only a small amount of cytoplasmic water is observed, the small turgor pressure of E. coli demonstrates that cytoplasmic water activity is only slightly less than extracellular water activity. From these findings, we deduce that the activity of cytoplasmic water exceeds its mole fraction at high osmolality, and, therefore, conclude that the activity coefficient of cytoplasmic water increases with increasing growth osmolality and exceeds unity at high osmolality, presumably as a consequence of macromolecular crowding. These novel findings are significant for thermodynamic analyses of effects of changes in growth osmolality on biopolymer processes in general and osmoregulatory processes in particular in the E. coli cytoplasm.     

Productive phage infection in Escherichia coli with reduced internal levels of the major cations.

Bacteriophage-induced changes in the intracellular levels of the major cations of Escherichia coli were studied to investigate the role of ion concentrations for bacteriophage assembly in vivo. Infection of E. coli by phage T4, P1, or lambda caused a transient reduction of intracellular levels of potassium, magnesium, and polyamines. Phages T3 and T7, however, had no detectable effect on the cation concentrations within the cell. In all cases, any reduction in the ion concentrations was restored later in infection. When the intracellular potassium concentration was lowered from 325 to 150 mM with a different osmotic growth medium, the number of phage progeny was only slightly reduced (by a factor of two). On additional reduction of the intracellular magnesium concentration from 100 to 50 mM by adding the antibiotic polymyxin B to the infected cells, T4 infections, but not T3 or T7, were markedly affected. These studies show that T3, T4, and T7 phage assembly can efficiently occur in vivo over a broad spectrum of ion concentrations.     

The DNA binding activity of p53 displays reaction-diffusion kinetics

The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k1 and k2, respectively. The diffusion constant of p53-GFP was estimated to be Dp53-GFP=15.4 microm2 s-1, significantly slower than that of GFP alone, DGFP=41.6 microm2 s-1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k1=0.3 s-1 and k2=0.4 s-1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding.     

Characterization of the ER-located zinc transporter ZnT1 and identification of a vesicular zinc storage compartment in Hebeloma cylindrosporum

Metal tolerance of filamentous fungi is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the ectomycorrhizal fungal model Hebeloma cylindrosporum, we carried out a functional screening of an H. cylindrosporum cDNA library in the zrc1Δ mutant strain of Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. This strategy allowed the isolation of HcZnT1, a gene belonging to the cation diffusion facilitator family, which induced tolerance to Zn, but not to other metals. HcZnT1 was constitutively expressed in Hebeloma cells, whatever the Zn status of the medium and the fungal cell type (mycelia, sporocarps, mycorrhizas). A HcZnT1:GFP fusion protein was expressed in yeast and the corresponding fluorescence was recorded on endoplasmic reticulum membranes. Taken together, these different findings suggest a dual role of HcZnT1 in Zn homeostasis of fungal cells, by supplying requested Zn ions for the functioning of the endoplasmic reticulum as well as by detoxifying the cytosol under Zn stress. Zn pools were also investigated by using the Zn-specific fluorophore zinquin in H. cylindrosporum cells. Zinquin labeling revealed compartmentalization in intracellular vesicles interspersed throughout the cytoplasm that do not correspond to vacuolar compartments. Altogether the present data represent the first steps into the understanding of Zn homeostasis and tolerance in Hebeloma.