Hydrodynamic flow in the cytoplasm of plant cells

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007) shows that active transport of organelles gives rise to a drag in the cytosol, setting up a hydrodynamic flow, which contributes to a fast distribution of proteins and nutrients in plant cells. Here, we show experimentally that actively transported organelles produce hydrodynamic flow that significantly contributes to the movement of the molecules in the cytosol. We have used fluorescence recovery after photobleaching and show that in tobacco Bright Yellow 2 (BY-2) suspension cells constitutively expressing cytoplasmic green fluorescent protein (GFP), free GFP molecules move faster in cells with active transport of organelles than in cells where this transport has been inhibited with the general myosin inhibitor BDM (2,3-butanedione monoxime). Furthermore, we show that the direction of the GFP movement in the cells with active transport is the same as that of the organelle movement and that the speed of the GFP in the cytosol is proportional to the speed of the organelle movement. In large BY-2 cells with fast cytoplasmic streaming, a GFP molecule reaches the other side of the cell approximately in the similar time frame (about 16 s) as in small BY-2 cells that have slow cytoplasmic streaming. With this, we suggest that hydrodynamic flow is important for efficient transport of cytosolic molecules in large cells. Hydrodynamic flow might also contribute to the movement of larger structures than molecules in the cytoplasm. We show that synthetic lipid (DOPG) vesicles and 'stealth' vesicles with PEG phospholipids moved in the cytoplasm.     

Quantitative imaging of green fluorescent protein in cultured cells: Comparison of microscopic techniques,use in fusion proteins and detection limits

To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background. In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence. Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488-nm excitation, a rapid cut-on dichroic mirror and a narrow-bandpass emission filter. Two-photon excitation of GFP fluorescence at the equivalent of ? 390 nm provided better absorption than did 488-nm excitation. This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching. Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST?GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone. In HeLa cells, the cytoplasmic GFP concentration must be greater than ? 1 μM to allow quantifiable discrimination over autofluorescence. However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.     

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.     

Quantitative microspectrometrical determination of the total protein and total protein-SH content of the nuclei of Ehrlich ascites tumor cells without prior isolation

The total protein and total protein-SH content of both the cytoplasmic fraction and the nuclei of Ehrlich ascites tumor cells (EATC) were determined macroscopically and microspectrometrically. The separation into cytoplasmic and nuclear fraction was performed by a modification of the method of Mamaril et al. [4]. The macroscopical determination of the total protein and the total protein-SH content were performed with the Folin method of Lowry et al. [2] and the DTNB method of Ellman [1], respectively. The quantitative microspectrometrical determinations of total protein content and of total protein-SH content were performed using the tetrazonium staining method of N?hammer [5] and N?hammer et al. [6] and the mercurochromcyanide (MCN) method of N?hammer et al. [7], respectively. Within the intact cells, fixed and stained histochemically, the total protein and the total protein-SH content of the nuclei were determined microspectrometrically. The so called "nuclear extinctions", measured as the sum of the extinctions of the nucleus and of the parts of the cytoplasm above and below the nucleus, were calculated into the true nuclear extinctions, which then show a good correspondence to the values measured both microspectrometrically and macroscopically on isolated nuclei. The calculation for the true nuclear extinctions is based on a special preparation of spherically shaped cells and nuclei.     

FRET analysis of transmembrane flipping of FM4-64 in plant cells: is FM4-64 a robust marker for endocytosis?

Although the styryl dye FM4–64 is now used routinely to monitor endocytosis in plants, the argument about its potential to cytoplasmically and non-endocytically relocate into a selective set of vesicular compartments persists. To address this question, we determined whether fluorescence resonance energy transfer (FRET) could occur between a cytoplasmically expressed, short-wavelength excitation green fluorescent protein (GFP) and FM4–64 in Nicotiana benthaminana . After exposure to FM4–64, the root hair plasma membrane and internal organelles became labelled. Under these conditions, no FRET with cytoplasmic GFP was seen. However, if the cells were treated with a low concentration of quillajasaponin, a membrane permeabilization agent, the cells continued to stream and FRET was detected. Thereby, we demonstrate that under conditions that do not severely compromise cell viability, the FM4–64 dye becomes a suitable FRET partner for the cytoplasmically localized GFP. Under normal conditions, FM4–64 does not significantly enter the cytosolic side of the membrane, but remains at the plasma membrane or trapped in the organelles of the endocytic pathway. Hence, when the structure or permeability of the plasma membrane is unaltered, FM4–64 dye is a robust marker for endocytosis.     

Spectral characterization of Dictyostelium autofluorescence

Dictyostelium discoideum is used extensively as a model organism for the study of chemotaxis. In recent years, an increasing number of studies of Dictyostelium chemotaxis have made use of fluorescence-based techniques. One of the major factors that can interfere with the application of these techniques in cells is the cellular autofluorescence. In this study, the spectral properties of Dictyostelium autofluorescence have been characterized using fluorescence microscopy. Whole cell autofluorescence spectra obtained using spectral imaging microscopy show that Dictyostelium autofluorescence covers a wavelength range from approximately 500 to 650 nm with a maximum at approximately 510 nm, and thus, potentially interferes with measurements of green fluorescent protein (GFP) fusion proteins with fluorescence microscopy techniques. Further characterization of the spatial distribution, intensity, and brightness of the autofluorescence was performed with fluorescence confocal microscopy and fluorescence fluctuation spectroscopy (FFS). The autofluorescence in both chemotaxing and nonchemotaxing cells is localized in discrete areas. The high intensity seen in cells incubated in the growth medium HG5 reduces by around 50% when incubated in buffer, and can be further reduced by around 85% by photobleaching cells for 5-7 s. The average intensity and spatial distribution of the autofluorescence do not change with long incubations in the buffer. The cellular autofluorescence has a seven times lower molecular brightness than eGFP. The influence of autofluorescence in FFS measurements can be minimized by incubating cells in buffer during the measurements, pre-bleaching, and making use of low excitation intensities. The results obtained in this study thus offer guidelines to the design of future fluorescence studies of Dictyostelium.     

New ways to look at axons in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, a well-established model organism for the analysis of nervous system development and function, nerve processes can be labelled in the intact animal with markers based on the "Green Fluorescent Protein" (GFP). The generation of GFP variants with improved brightness and modified emission spectra potentiated the use of this marker for in vivo labelling of subcellular structures. This made it possible to label different groups of neurons and their axons in the same animal with GFP variants of different spectral characteristics. Here I show with double labelling experiments that spatial relationships of axons in small axon bundles can now be resolved at the light microscopic level. In the future this will largely circumvent the need for time-consuming electron microscopic reconstructions to detect local defects in axon outgrowth. Furthermore, I demonstrate that neuronal processes can now be traced even in the head ganglia, an area of the nervous system that was previously almost inaccessible for analysis due to the compact arrangement of cell bodies and axons.     

不同理化条件对GFP发光特性影响的研究

为了优化可用于保持绿色荧光蛋白（GFP）发光特性的理化条件,本文采用不同离心转速、温度、pH分别在不同作用时间下对表达GFP的HepG2细胞进行处理,然后在荧光酶标仪测定细胞荧光值。结果表明：转速从1000r/min提升到10000r/min,作用时间从5min到15min,GFP的发光度变化也停留在4个百分点之内;在-70℃～50℃这个范围内不会明显的破坏GFP的发光特性,但当温度达到90℃时GFP的发光明显减弱,且随着作用时间的延长发光越弱;当pH由2～12的变化过程中,GFP的发光度也由弱变强再变弱,时间越长对GFP的破坏越大,发光度值也就越小。     

ReAsH: another viable option for in vivo protein labelling in Dictyostelium

Biarsenical-tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). However, there are no reports of the tetracysteine tagging system being used in Dictyostelium . In order to establish this tagging system in Dictyostelium , the filamin gene ( FLN ) was modified to express a C-terminal tetracysteine sequence and then transfected into cells. After addition of either FlAsH-EDT₂ or ReAsH-EDT₂, the fluorescence intensity of cells increased in a time-dependent manner and reached a plateau after 3 h of incubation. ReAsH had a much stronger and more specifically localized fluorescent signal compared with FlAsH. After removal of the ReAsH-EDT₂ reagent, the fluorescence signal remained detectable for at least 24 h. The localization of filamin labelled by ReAsH was similar to that of an FLN-mRFP fusion protein, but the fluorescence signal from the ReAsH-labelled protein was stronger. Our findings suggest that the ReAsH-tetracysteine tagging system can be a useful alternative for in vivo protein tagging in Dictyostelium .     

Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse*

Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP-tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP-tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.     

Mesoporous iron oxide nanoparticles prepared by polyacrylic acid etching and their application in gene delivery to mesenchymal stem cells

Novel monodisperse mesoporous iron oxide nanoparticles (m‐IONPs) were synthesized by a postsynthesis etching approach and characterized by electron microscopy. In this approach, solid iron oxide nanoparticles (s‐IONPs) were first prepared following a solvothermal method, and then etched anisotropically by polyacrylic acid to form the mesoporous nanostructures. MTT cytotoxicity assay demonstrated that the m‐IONPs have good biocompatibility with mesenchymal stem cells (MSCs). Owing to their mesoporous structure and good biocompatibility, these monodisperse m‐IONPs were used as a nonviral vector for the delivery of a gene of vascular endothelial growth factor (VEGF) tagged with a green fluorescence protein (GFP) into the hard‐to‐transfect stem cells. Successful gene delivery and transfection were verified by detecting the GFP fluorescence from MSCs using fluorescence microscopy. Our results illustrated that the m‐IONPs synthesized in this work can serve as a potential nonviral carrier in gene therapy where stem cells should be first transfected and then implanted into disease sites for disease treatment. Microsc. Res. Tech. 76:936–941, 2013. © 2013 Wiley Periodicals, Inc.     

Recognition,presence, and survival of locust central nervous glia in situ and in vitro

Insect glial cells serve functions for the formation, maintenance, and performance of the central nervous system in ways similar to their vertebrate counterparts. Characterization of physiological mechanisms that underlie the roles of glia in invertebrates is largely incomplete, partly due to the lack of markers that universally label all types of glia throughout all developmental stages in various species. Studies on primary cell cultures from brains of Locusta migratoria demonstrated that the absence of anti-HRP immunoreactivity, which has previously been used to identify glial cells in undissociated brains, can also serve as a reliable glial marker in vitro, but only in combination with a viability test. As cytoplasmic membranes of cultured cells are prone to degradation when they lose viability, only cells that are both anti-HRP immunonegative and viable should be regarded as glial cells, whereas the lack of anti-HRP immunoreactivity alone is not sufficient. Cell viability can be assessed by the pattern of nuclear staining with DAPI (4′,6-diamidino-2-phenylindole), a convenient, sensitive labeling method that can be used in combination with other immunocytochemical cellular markers. We determined the glia-to-neuron ratio in central brains of fourth nymphal stage of Locusta migratoria to be 1:2 both in situ and in dissociated primary cell cultures. Analysis of primary cell cultures revealed a progressive reduction of glial cells and indicated that dead cells detach from the substrate and vanish from the analysis. Such changes in the composition of cell cultures should be considered in future physiological studies on cell cultures from insect nervous systems. Microsc. Res. Tech. 2009. © 2008 Wiley-Liss, Inc.     

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.     

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high‐resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non‐polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR‐FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP‐vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR‐FM and wide‐field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR‐FSM affords major improvements in these parameters compared with wide‐field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low‐level GFP‐fusion protein expression is a useful tool for FSM imaging. When used in time‐lapse mode, TIR‐FSM of actin and GFP‐conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.     

The microspectrometrical determination of the nucleus-total-cell-protein-ratio: a possibility of objective cell type analysis

Cells from smears of the normal human squamous epithelium of the gingiva, fixed and stained for protein using the tetrazonium method optimized by N?hammer and calibrated by N?hammer et al., were investigated. The extinctions of both the total cells and of the rectangular areas circumscribing the nuclei, were measured microspectrometrically. Altogether 417 cells from 6 healthy persons of both sexes were investigated. 9 distinct subgroups of cells were found showing an exact linear correlation between nuclear and total cell extinctions. In the graph of both the nuclear and the total cell extinctions the 9 subgroups can be seen as 9 distinct linear groups of points, defined exactly by their regression lines. Thus, every squamous epithelial cell within the smear can be typed definitely and objectively in respect to its membership of one of these 9 linear groups of points. The obviously definite, legitimate connection between the extinctions of the total cells and of their nuclei affords a glimpse into the processes of cellular differentiation and allows the definition of the so-called stem cell in terms of protein content of the total cell and of the nucleus.     

Ultrastructural and cytochemical characteristics of megakaryoblastic leukemic cells

Transmission electron microscopy, scanning electron microscopy, and ultrastructural cytochemistry were utilized to study megakaryoblastic cells from four patients suffering from megakaryoblastic leukemia. The results show that megakaryoblastic leukemic cells have a unique ultrastructural appearance, surface architecture, and cytochemical activity. The cells are positive for platelet peroxidase cytochemical reaction, which is localized in the perinuclear space and endoplastic reticulum, but not in the Golgi apparatus and cytoplasmic granules. They have a rather smooth surface and display blebs or tuberculi which are different from those in other types of leukemic cells as seen under the scanning electron microscope. The megakaryoblastic leukemic cells also show a special appearance under the transmission electron microscope, such as a cytoplasm which contains numerous small mitochondria, mostly concentrated in one pole of the cell. These ultrastructural and cytochemical characteristics of the megakaryoblastic leukemic cells revealed by the combined techniques are very useful in the diagnosis of megakaryoblastic leukemia.     

Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells

Fluorescent protein-based FRET is a powerful method for visualizing protein-protein interactions and biochemical reactions in living cells. It can be difficult, however, to avoid photobleaching when observing fluorescent cells under the microscope, especially those expressing CFP. We compared the sensitivity of two protein-based FRET pairs to light-induced fluorescence changes in the donor, on FRET determination by fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low excitation light levels of the time- and space-correlated single photon counting (TSCSPC) method, FLIM acquisitions were achieved without donor photobleaching. Here, we show that photobleaching of CFP by a mercury lamp under the microscope induced a decrease in the mean fluorescence lifetime, which interfered with FRET determination between CFP and YFP. Importantly, the range of light-induced variation of the mean fluorescence lifetime of CFP was not proportional to the decrease in the steady state fluorescence intensity and varied from cell to cell. The choice of the CFP/YFP pair therefore requires that the cells be observed and analyzed at very low light levels during the whole FRET experiment. In contrast, the GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if some GFP photobleaching took place. We thus demonstrate that CFP can be an unreliable donor for FRET determination in living cells, due to its photosensitivity properties. We demonstrate that the GFP/mCherry pair is better suited for FRET measurement by FLIM in living cells than the CFP/YFP pair.     

Improving autofluorescent proteins: comparative studies of the effective brightness of Green Fluorescent Protein (GFP) mutants

We study the photophysical behavior of 8 mutants of Green Fluorescent Protein (GFP) using fluorescence correlation spectroscopy (FCS) on the single molecule level and double resonance excitation of bulk samples. Experimental data reported here and the previously published data on the RH/R(-) equilibrium and fluorescence quantum yields Phi(Fl) (Jung et al., 2005; Biophys J 88:1932-1947) are analyzed with respect to single molecule as well as conventional fluorescence microscopy. The fraction of GFP molecules in a dark state, [D], reduces the effective absorption cross section under photostationary conditions. The determination of the excitable fraction [B] and its fluorescence quantum yield Phi(Fl) gives the effective brightness Phi(eff). Our results show that in its wavelength range, eGFP is, among the GFPs, the best fluorophore for most microscopic applications. However, in the red shifted YFP-proteins, there is still potential for improvement, since a pronounced dark state population is detectable in all mutants investigated so far. We propose to use the mutant T203Y/E222Q in imaging studies, whenever the expression yield is not a limiting factor. In FCS experiments, where the useful concentration range of the expressed molecules is restricted to concentrations below micromolarity, our data suggest the use of wt-GFP or mutant T203Y, as these represent photochemical buffers. Both mutants might surpass the limitations given by out-of-focus bleaching in live cell microscopy.     

Stromules and the dynamic nature of plastid morphology

Investigation of plastids via green fluorescent protein (GFP) has led to the rediscovery of tubular extensions of the plastid membrane, termed stromules, for stroma‐filled tubules. These unique structures are challenging our understanding of plastid structure and function. Stromules are highly dynamic, branching and elongating across the plant cell. Recent experiments indicate that cytoplasmic microtubules and microfilaments control the shape and motility of stromules. Whether stromule formation involves plastid‐specific structural systems, such as the plastid division machinery, remains open to debate. Fluorescence photobleaching experiments have revealed that GFP can traffic between plastids joined by stromules. As a result, interest has grown in whether other macromolecules can also travel through these connections. Although the function of stromules is unknown, several aspects of their biology suggest they play a role in molecular exchange between plastids and other organelles.