3.1. Kinetic studies of FM-dye internalization
In fungal cells, FM-dyes rapidly labelled the plasma membrane and have been used to measure the kinetics of membrane internalization (Atkinson et al., 2002). In order to stain fully the majority of membrane-bounded organelles in a cell, it is usually necessary to immerse cells for 1 h or more in dye. In fungal hyphae, FM1-43 and FM4-64 dyes are taken up by both apical and subapical hyphal compartments. Interestingly, differences were observed in the patterns of organelle staining obtained with each dye (Fischer-Parton et al., 2000), suggesting that they do not necessarily follow the same trafficking pathways (see section 5.4).
In higher plant cells, kinetic studies of FM1-43 (Carroll et al., 1998; Emans et al., 2002) and FM4-64 (Ueda et al., 2001; Kutsuna & Hasezawa, 2002; Bolte et al., 2004; Figs 3–7) broadly present similar staining patterns in roots (Geldner et al., 2003), protoplasts (Ueda et al., 2001; Fig. 7) or whole cells (Kutsuna & Hasezawa, 2002; Figs 3–6). In each case, staining of the plasma membrane is immediate (Fig. 3a). Moreover, the cell wall does not act as a barrier and does not slow the staining process, as kinetic studies gave similar results between whole cells and freshly made protoplasts from the same cell line (data not shown). Osmotic shock provoking the retraction of the plasma membrane in plasmolysed cells clearly showed that, for FM4-64, the dye was not retained in cell walls. However, for FM1-43, a light staining of the cell wall was observed (data not shown). After 10 min in dye-containing medium, localized fluorescent thickenings occurred on the internal side of the plasma membrane (Fig. 3b), and mobile fluorescent organelles began to be seen at the periphery of the cytoplasm. With increasing time (20–60 min), fluorescent structures were observed throughout the whole cytoplasm (Fig. 3c). In Arabidopsis protoplasts, ring-like structures sometimes emerged following the appearance of punctuate organelles (Ueda et al., 2001). A light diffuse staining of the cytoplasm was often detected as well, which may be due to hydrophobic protein adsorption. After 1 h, numerous organelle membranes were stained, and vacuolar membranes began to be labelled as well (Fig. 3d). Between 3 and 10 h (depending on the experiment), all vacuolar membranes were stained. Ten to 30 h after dye loading, the vacuolar membranes remained stained, and dye fluorescence was even sometimes observed in the lumen of vacuoles (Kim et al., 2001; Kutsuna & Hasezawa, 2002; see also Fig. 5d).
FM4-64 staining in plant cells also highlights some specific plant features, such as the nascent cell plates in a dividing cell, between the two daughter cells. Forming cell plates were instantaneously stained but appearing only if their membranes were in contact with at least one side of the mother cell (Fig. 4a), suggesting that labelling of the cell plate is via its connection with the peripheral plasma membrane. This observation is consistent with the dye being able to diffuse laterally. Forming cell plates not yet anchored on mother cell membranes did not exhibit this immediate strong fluorescence. However, with longer incubation (30–60 min), the forming cell plate became strongly stained (Fig. 4b,c), even if there was no connection with the mother plasma membrane (as shown by collecting a series of optical sections at different optical planes down the z-axis). This forming plate was often surrounded by numerous micrometre- and submicrometre-sized fluorescent structures (Fig. 4d), which could be vesicles or larger organelles similar to those described in interphase cells after this time of staining, and suggests that organelle membranes pass to or come from the forming cell plate.
The fluorescence intensity of the stained plasma membrane may be far higher than that of organelle membranes. By reducing the gain of the photomultiplier detector of the CLSM, this feature can be used to image just the plasma membrane in living cells of, for example, the shoot apical meristem, which can be followed over several days (Grandjean et al., 2004). The instantaneous and strong labelling of the plasma membrane has also been used for morphometric studies involving root hair measurements (Procissi et al., 2003).
3.2. Do the kinetics of FM-dye internalization vary?
We have already noted that variations in experimental conditions or in dye concentration from one experiment to another may generate differences in the kinetics of internalization (see sections 1.2 and 1.3). Besides the influence of these physico-chemical parameters, it is clear that the kinetics of internalization also vary with physiological conditions, both in fungi and in plant cells. In fungal cells, fast growing hyphae of Neurospora internalize FM4-64 rapidly and dye can be detected in the cytoplasm 10 s after being added (Read & Hickey, 2001). Slow growing germ tubes of Neurospora internalize FM4-64 far more slowly (G. Wright & N. D. Read unpublished observations). Spores of Magnaporthe internalize FM4-64 2–3 min after being hydrated with the dye (Atkinson et al., 2002).
Similarly, in plant cells, Carroll et al. (1998) showed that actively secreting protoplasts from maize root cap cells have a much lower rate of FM1-43 internalization compared with that in nonsecreting protoplasts. Parton et al. (2001) also reported a quantitative relationship between FM4-64 staining and growth rate within an individual pollen tube. In our laboratory, BY-2 cells from a 3-day-old culture internalized FM4-64 at a slower rate than 2-day-old cultured cells. Interestingly, the observation was the same for protoplasts from the latter cultures, suggesting that the cell wall and shape had no impact on membrane internalization. Transgenic BY-2 cell lines like BY-2/ST-GFP (Saint-Jore et al., 2002; Brandizzi et al., 2004) also exhibited different rates of FM-dye internalization compared with wild-type cells, suggesting that these different cell lines internalize membranes at different rates. Last but not least, apparently conflicting reports exist concerning drug effects upon FM-dye internalization. Brefeldin A (BFA), when applied to maize root cap protoplasts, reduced the rate of internalization of FM1-43 (Carroll et al., 1998). In BY-2 cells, BFA stimulated the overall uptake of dye but blocked further transport to targeted organelles, creating an accumulation of large vesicles labelled with FM1-43 (Emans et al., 2002). BFA may also induce larger fluorescent patches of FM4-64 to form in other cell types (Geldner et al., 2003). However, artefactual effects of BFA on FM4-64 internalization should not be discarded, as both molecules may alter the physical environment of the plasma membrane. They might even compete with each other, modifying the kinetics of membrane exchange within cells.