Here we present simple staining protocols suited to label intact plant tissues and protoplasts using fluorescent probes of FM4-64, LRB-PE, DiIC12, DiIC18, DiD, BD-SM and Laurdan. After initial incubations of Arabidopsis epidermal strips with either FM4-64 or BD-SM strong autofluorescence signals were detected that did not allow discriminations between dye-specific signals and unspecific ones coming from deeper leaf tissues (Figure1). Some parts of the autofluorescence signal resulted from accumulations of the dyes within the cell wall microfibril texture. Therefore the plasma membrane participation of each dye in protoplasts was documented employing one- and two-photon microscopy. FM4-64 turned out to be a stable plasma membrane marker (Figure2, C), just as LRB-PE (Figure2, E) and BD-SM (Figure3, A). DiIC12 (Figure2, G) and DiIC18 (Figure2, I) were strongly taken up into the cytosol which led to decreasing fluorescence signals at the plasma membrane over time. In contrast, a weak plasma membrane fluorescence was achieved using DiD (Figure2, K). The dye hardly incorporated into the bilayer. To detect possible differences in the lipid composition of Arabidopsis, protoplast dual staining experiments were performed (Figure5). There, we found evidence for lipid polarization in the plasma membrane. Plasma membrane lipids underwent redistribution events within 15 to 20 h after cell wall digestion. Using BD-SM as marker for sphingolipid enriched membrane compartments and other fluorescent markers like FM4-64 and LRB-PE for phospholipid enriched areas, lipid redistributions could be visualized (Figure5, A-L). Lipid polarization was statistically allocated employing Pearson and Spearman correlation coefficients (Figure5, M-N;). In freshly isolated protoplasts BD-SM and FM4-64 showed colocalization in the plasma membrane, displayed by high correlation coefficients (Figure5, M). Time dependent lipid polarization became evident by a strong decrease of the Pearson and Spearman correlation coefficients after 15 h (Figure5, N), which indicated a more random distribution of dye molecules within the plane of the membrane. To ensure that the dyes labeled inhomogeneous lipid phases the partitioning of BD-SM in the Arabidopsis DRM fraction was confirmed by Triton X-100 treatment of purified Arabidopsis plasma membranes (Figure3, C). Detergent treatment revealed that BD-SM mixed up with natural phytosterols and sphingolipids, which are thought to self-aggregate to form lipid clusters. In DRMs isolated from Arabidopsis callus cultures sterols and sphingolipids showed a 4- to 5-fold increase relative to the total plasma membrane[14, 48]. In tobacco a similar increase of sterols and sphingolipids in DRMs was determined. This indicated that BD-SM is likely labeling PM areas of elevated sterol/sphingolipid content in vivo. It was shown for sphingomyelins in model bilayer membranes that attached saturated acyl chains promoted the formation of raft-like lipid ordered phases that merged with each other to form larger domains.
Except for FM4-64 all dyes have been dissolved in dimethylsulfoxide (DMSO). DMSO/water or DMSO/buffer mixtures were used to stain Arabidopsis tissues and protoplasts. DMSO has been reported to have a strong influence on the structure of lipid membranes when used even at small mole concentrations, probably by displacing water and thereby modifying the structure of lipid bilayers. Concerning cell biology, the amphiphilic DMSO molecule is known for its function to enhance membrane penetration, to induce cell fusion and for its role as cryoprotectant. Depending on DMSO concentration, membrane thickness can be affected, allowing for the formation of water pores to be induced and for the destruction of the bilayer structure of membranes. In atomic-scale molecular dynamics simulations it was revealed for liquid disordered dipalmitoylphosphatidylcholine (DPPC/DMSO/water) systems that concentrations of 10-20 mol% of DMSO led to pore formations within timescales of nanoseconds. Here we used DMSO concentrations in the range of 0.25-3% (v/v) which should not significantly alter the PM structure. Nevertheless there are reports using alfalfa protoplasts that a DMSO concentration of about 1% was already sufficient to induce changes in the transcript levels of certain genes, as reported for instance for cold acclimatization-specific (cas) genes. By increasing membrane rigidity, DMSO could possibly have induced calcium influxes, leading to a pronounced cold acclimation at room temperature. Even so it cannot be ruled out that DMSO induced the expression of certain genes that could have influence on intracellular calcium concentrations as well as on the plasma membrane protein composition. In accompanied control experiments, however, there were no DMSO-induced effects on membrane polarization detectable when incubating Arabidopsis protoplasts for 24 h in buffer media containing up to 3% DMSO.
The FRAP-experiments left striking hints that there are at least two lipid populations resident in polarized poles of the plasma membrane. Lipid fractions that predominantly harbored fluorescent BD-SM molecules showed a significant decrease in recovery time compared to fractions in which FM4-64 was participating (Figure6). The BD-SM labeled lipid fraction showed a diffusion coefficient [D] of 8.01 × 10-4 μm2/s with a mobile fraction of only 28% (a 72% immobile fraction), whereas for lipids in the FM4-64 labeled pool a [D] of 0.084 μm2/s with an 84% mobile fraction (a 16% immobile fraction) was determined. These numbers indicated that lipids of the FM4-64 labeled fraction are able to move more than 10 times faster than lipids of the mobile BD-SM labeled fraction, and the FM4-64 was predominantly mobile, whereas the BD-SM labeled fraction was predominantly immobile. In ternary systems consisting of cholesterol, sphingomyelin and dioleoylphosphatidylcholine (DOPC) diffusion coefficients were characterized with respct to lipid lateral diffusion rates. It was revealed that lipids of the Lo-phase appeared to recover 2–3 times slower than lipids of the Ld-phase. In single gold particle tracking experiments on artificial giant unilamellar vesicles (GUV’s) an increase in the sterol lipid content up to 50% of all lipids in planar lipid bilayers led to a nearly two fold reduction of [D]. At given temperature [D] strongly depends on the movement of surrounding lipids as well as from the observation period. In the fluid, lipid disordered phase of ternary model membranes large diffusion coefficients of about 10-6 cm2/s have been reported at short time scales of 1 ns, whereas at longer time scales diffusion coefficients decreased to 10-8 to 10-7 cm2/s[55, 56]. With an assumed [D] of 10-7 cm2/s, an individual lipid would cover a lateral distance of about 6 nm within a time period of 1 μs. The applied FRAP-technique for the measurement of lateral diffusion rates of different lipid phases in viable protoplasts however did not allow time and length resolutions of this magnitude since this method is based on optical systems, whose spatial resolving power is limited by the chosen scan method and by the diffraction of light. Acquired FRAP datasets are, therefore, not directly comparable to diffusion coefficients calculated from atomistic simulations mimicking lipid diffusions in model membranes. Nevertheless, there have been efforts made in defining lipid diffusion coefficients in model membranes employing FRAP; in pure liquid disordered, dimyristoylphosphatidylcholine (DMPC) systems a [D] of 7.5x10-8 cm2/s was measured at 35°C, decreasing to 6.0× 10-8 cm2/s when temperature dropped to 26°C. As cholesterol was added to the same system the liquid ordered phase formed; at 35°C [D] was measured to be 3.0×10-8 cm2/s in the Lo-phase, being further reduced to 1.8×10-8 cm2/s at 26°C. FRAP measurements in protoplasts were carried out at room temperatures of about 20-22°C. Taking the pure numbers, lipid diffusion coefficients in protoplasts appear to be two to three orders of magnitude slower compared to those found in artificial membranes.
To our best knowledge no similar FRAP-datasets are available describing lateral lipid diffusion coefficients in viable Arabidopsis protoplasts to this date. In analogy to findings in model membranes it is assumable for polarized protoplasts that the slower fluorescence recovery rates of BD-SM phases are caused by an accumulation of sterols at distinct sites of the plasma membrane (Figure5, G, K; Figure6).
Different dye loadings and FRAP-experiments revealed plasma membrane lipid heterogeneity in Arabidopsis protoplasts. One further possibility to prove this finding was the employment of a lipid environment sensitive fluorescent probe like Laurdan. Since in Lo-phases sterols and sphingolipids are tightly packed these regions have a less water content compared to Ld-phases, in which lipids are more densely packed. Laurdan can be applied to visualize such differences in the water content. In Experiments on A. thaliana protoplasts, a final Laurdan concentration of 5 mM was enough to successfully stain viable plasma membranes. For mammalian cells in contrast concentrations in the micromolar range are used. This might be explained by the more complex sterol profile of plant plasma membranes.
In Lo-phases fluorescence is shifted into the more blue spectrum of light (false colored green in Figure4), in polar Ld-phases accordingly more into the red. Immediately after cell wall removal there were no lipid phase polarizations detectable (Figure4, A-C), confirmed by a calculation of the general polarization (GP-) value in a pixel to pixel analysis of Laurdan images (Figure4, E). The GP-value can be used as indicator for the water content of lipid phases and accordingly as a degree for the predominant state of lipid order. A GP-value of −1 indicates an aqueous phase, whereas a GP-value of +1 indicates a fully ordered phase. GP-values in Figure4 E ranged from −0.3 to 0.2. Experimentally these values depend on the lipid composition and on temperature. In the liquid disordered phase of model membranes GP-values ranged from −0.3 to 0.3 while in liquid ordered phases these values are typically in the range of 0.5 to 0.6. In liposomes with equal molecular ratios of DOPC, cholesterol and sphingomyelin the GP-values of the liquid disordered phase ranged from −0.05 to 0.25 whereas a GP of 0.25 to 0.55 indicated the coexistence of a liquid ordered phase. It has been reported that “GP-values of living cells may not always directly correspond to those obtained in artificial membrane systems”, but that GP-values allow comparisons in order to rule out fluidity differences within plasma membranes. Recently Laurdan has been used to examine the degree of lipid order in different tissues of vertebrates. In vital zebrafish embryos the quantification of membrane order ranged between GP-values of 0 to 0.2 in more ordered apical membranes compared to basolateral membranes exhibiting a decreased lipid order with GP-values of 0 to 0.13. In mammalian MDCKII and RAW264.7 cell lines GP-values ranged from −0.1 to 0.3. Using the sterol-depleting agent methyl-ß-cyclodextrin GP-values decreased with decreasing sterol content of the plasma membrane, examined by a fluorescent live imaging approach. Using Laurdan on native BY2 plasma membranes GP-values dropped from 0.65 (at 4°C) to 0.55 (at 22°C) down to 0.30 (at 40°C). In analogy to findings in model membranes the BY2 plasma membrane constitution would accordingly be consisting of gel-like, liquid ordered domains below 20°C (GP > 0.55); above this temperature the relative order of the membrane progressively decreased.
Laurdan treatments of Arabidopsis protoplasts revealed that 15 h after cell wall removal redistributions of lipid phases occur (Figure4, F-H). With respect to the Laurdan fluorescence characteristics there were lipid phases with different water content emerging (Figure4, F, G). GP-values of up to 0.6 indicated high levels of lipid order for regions of the plasma membrane (Figure4 J, arrows). After 20 h polarization was even more pronounced, resulting in one distinct lipid ordered pole covering the lateral side of the protoplast (Figure4, K-O, arrows). Likely this pole arose from the coalescence of smaller regions of lipid order, as seen in Figure4, J. For model membranes it has been reported that Lo-phases, predominantly harboring lipids with saturated acyl chains like sterols and sphingomyelins, merged with each other to form larger domains.
Lipid polarization could be confirmed by several independent non-invasive tests like staining experiments with different lipid analogues (Figure5 A-L), FRAP-measurements (Figure6) and Laurdan labeling of plasma membranes including a calculation of GP-values (Figure4). At this point it is unclear what exactly caused plasma membrane resident lipids to relocate into distinct poles. Polarizations of lipid domains are reported for several biological systems across species, especially during cytokinesis. Experiments in fission yeast (Schizosaccharomyces pombe) revealed that there are sterols enriched at the growing cell tips and at sites of cytokinesis. In pollen tubes lipid microdomain polarization was found to be essential for polarized tube growth involving reactive oxygen species (ROS) signaling. ROS-producing NADPH oxidases were reported to be associated with DRMs and to depend on the presence of sterols. ROS signaling plays important roles in plants since reactive oxygen species help controlling processes of growth, development, biotic and abiotic stress response and programmed cell death. In the experiments performed here there were in contrast neither cell divisions nor any kinds of developmental processes detectable during observation periods of up to 24 h.
It has also previously been reported that plant protoplasts start regenerating their cell walls in suited cultivation media. In Nicotiana tabacum protoplast cell wall regeneration started within 30 minutes after removing cell wall components, and cell wall regeneration processes were indicated by formations of cellulose microfibril depositions at distinct sites at the protoplasts surface. The spatial organization of cellulose microfibrils defines the cell wall texture and is achieved by an oriented microfibril deposition. Transmembrane cellulose-synthase-complexes are directly linked to components of the cytoskeleton and enable an organized deposition of cellulose microfibrils. Some of the cell wall building components like chitin- and ß-D-glycan-synthases have been shown to be resident in detergent resistant membrane domains of Oomycetes. Callose and cellulose synthases have also found to be strongly enriched in DRM fractions isolated from plasma membranes of the tree species Populus, indicating that active proteins involved in cell wall biosynthesis associate with sphingolipid/sterol enriched, lipid ordered phases in vivo. It is likely that lipid redistributions in plasma membranes of Arabidopsis protoplasts reflect cellular attempts to regenerate absent cell walls. In tobacco protoplast cell walls fully regenerated within a time period of up to six hours. Observations were made, that the period in which the regeneration processes finished, strongly depended on the method that was used before when isolating the protoplasts; depending on the isolation procedure the regeneration processes could take up to 16 hours. Nevertheless there were no indicators for cell wall regenerations found in Arabidopsis protoplasts during observation periods of up to 24 h. This could be due to the medium used for cultivating the protoplasts; the medium still contained intact proteolytic enzymes like cellulases and pectolyases, which could have hindered cell wall regenerations. In Convolvus arvensis protoplasts it was shown that the ability of single cells to regenerate their cell walls was strongly diminished in the presence of active proteolytic enzymes.