Visualization of the entire actin cytoskeleton in BY-2 cells by transient GFP-mTn expression
The C-terminus of a GFP version optimized for use as a marker in plant cells (see Experimental procedures) was fused in frame to the C-terminal 197 amino acids of mouse talin that constitute the protein’s f-actin binding domain (McCann & Craig 1997). Particle bombardment was employed to transiently express the resulting fusion protein (GFP-mTn) under the control of the CaMV 35S promoter in tobacco BY-2 suspension cells. Confocal imaging of GFP-fluorescence emitted from transformed cells revealed that GFP-mTn localized to a dense filamentous network (Fig. 1a,b), which very closely resembles the actin cytoskeleton in BY-2 cells (Hasezawa et al. 1989;Katsuta et al. 1990) and in other plant cells (Seagull et al. 1987), as visualized by rhodamine-phalloidin staining after permeabilization. GFP-mTn labeled filaments in living BY-2 cells were randomly arranged in the cytoplasm at the cell cortex and around the nucleus (Fig. 1a,b). They also followed trans-vacuolar cytoplasmic strands, which are known to be stabilized by actin filaments (Staiger et al. 1994). Weak, diffuse GFP-mTn fluorescence but no labeling of filaments was observed in the nucleus. GFP-mTn fluorescence was absent from vacuoles and nucleoli (Fig. 1b).
Figure 1. Transient expression of GFP-mTn (a–c) and of untagged GFP (d–f) in living tobacco BY-2 suspension cells.
(a and d) Projections of serial confocal optical sections (a, 4× line averaging; d, 2× line averaging) showing the distribution of GFP fluorescence in entire cells.
(b and e) Single confocal optical sections through nuclei. Note the presence of bright filamentous structures in trans-vacuolar cytoplasmic strands and around the nucleus in (b).
(c and f) Transmitted light reference images. Size and shape of individual BY-2 suspension cells as well as number and size of nucleoli contained in their nuclei can vary considerably.
V, vacuoles; N, nuclei; n, nucleoli; nv, nucleolar vacuole; CS, cytoplasmic strands; P, gold particle that carried DNA into the cell. Scale bars: 100 μm.
Download figure to PowerPoint
Control BY-2 cells that expressed GFP without the mouse talin tag showed evenly distributed fluorescence throughout the cytoplasm (Fig. 1d,e). No fluorescence was found in vacuoles. GFP, which is small enough to freely diffuse through nuclear pores (Grebenok et al. 1997), slightly accumulated in the nucleus, as it does in other cell types (Haseloff et al. 1997). GFP fluorescence was absent from the nucleolar matrix but could be observed in nucleolar vacuoles (Fig. 1e). Under the conditions used to image transient expression of GFP-mTn or GFP, untransformed BY-2 cells did not emit detectable fluorescence (Fig. 1; data not shown).
To confirm that GFP-mTn binds to and visualizes actin filaments, BY-2 cells expressing the fusion protein were permeabilized and co-stained with rhodamine-phalloidin. Phalloidin is a fungal toxin that acts by stabilizing actin filaments to which it binds very specifically. Fluorescent conjugates of phalloidin are commonly use to visualize the actin cytoskeleton in permeabilized cells. At saturating concentrations, fluorescent phalloidin is presumed to visualize all actin filaments present in stained cells (Cooper 1987). Patterns of green GFP-fluorescence and red rhodamine-phalloidin-fluorescence emitted from double-labeled BY-2 cells were essentially identical (Fig. 2), showing that GFP-mTn binds to the entire BY-2 cell actin cytoskeleton in a specific manner. Adjacent cells that did not express GFP-mTn showed rhodamine-phalloidin labeling similar to double-labeled cells (Fig. 2b,c; data not shown), indicating that GFP-mTn expression does not alter f-actin organization.
Figure 2. Co-localization of transiently expressed GFP-mTn and of rhodamine-phalloidin in a permeabilized BY-2 cell.
Artificially colored projections of serial confocal optical sections (4× line averaging) are shown.
(a) GFP-mTn fluorescence (excitation, 488 nm; emission, 525–565 nm [green]).
(b) Rhodamine-phalloidin fluorescence (excitation, 543 nm; emission, ≥ 570 nm [red]).
(c) Superimposition of (a) and (b). Yellow color indicates areas where the two images overlap and where the two markers co-localize.
(d) Transmitted light reference image. Scale bar: 25 μm.
Download figure to PowerPoint
Transient expression of GFP-mTn had no apparent effect on the morphology or viability of BY-2 cells. Cells appeared unaffected (Fig. 1c; data not shown) even after confocal imaging of GFP-mTn labeled actin filaments, which could potentially cause phototoxic effects (Hepler & Gunning 1998). GFP-mTn levels were high enough to allow visualization of the actin cytoskeleton in single cells as early as 3 h after particle bombardment. Several hours later, pairs of GFP-mTn expressing cells were regularly observed, which obviously originated from targeted cells that had divided (see cover photograph). This indicates that GFP-mTn does not inhibit mitosis or cytokinesis, both processes which are presumed to depend on f-actin function (Hepler & Gunning 1998;Lloyd 1989;Nagata et al. 1992).
Constitutive, non-invasive labeling of actin filaments by GFP-mTn in transgenic Arabidopsis thaliana plants
Transgenic A. thaliana plants that constitutively expressed GFP-mTn under the control of the 35S promoter were generated. GFP-mTn labeling of the actin cytoskeleton was observed in all examined tissues of etiolated transgenic T2 seedlings, including epidermal as well as cortical cell layers of roots, hypocotyls and cotyledons (Fig. 3). Control seedlings expressing untagged GFP from the 35S promoter showed evenly distributed cytoplasmic and nuclear fluorescence (data not shown). No fluorescence emission was detected from untransformed seedlings under the conditions used for GFP-imaging (data not shown).
Figure 3. The actin cytoskeleton in cells of different tissues of etiolated transgenic T2 Arabidopsis thaliana seedlings as visualized by stable, constitutive expression of GFP-mTn and confocal microscopy.
Projections of serial confocal optical sections are shown.
(a) Epidermal cells of a cotyledon (8× line averaging). Scale bar: 100 μm.
(b) Stoma guard cells in the epidermal cell layer of a cotyledon (4× line averaging). Scale bar: 10 μm.
(c) Epidermal and cortical hypocotyl cells (8× line averaging). Scale bar: 100 μm.
(d) Epidermal and cortical root cells (4× line averaging). Scale bar: 50 μm.
Download figure to PowerPoint
At the T2 stage, transgenic lines have gone through each phase of the plant life cycle at least once. GFP-mTn expression did not have any apparent effects on plant morphology or development. These results demonstrate that GFP-mTn expression can visualize the actin cytoskeleton in different types of plant cells in a completely non-invasive manner. The transgenic GFP-mTn expressing A. thaliana lines we have established allow in vivo examination of the dynamic behavior and function of the actin cytoskeleton during a variety of actin-dependent processes in different tissues throughout plant development.
Non-invasive visualization of the tobacco pollen tube actin cytoskeleton by transient GFP-mTn expression
GFP-mTn was also employed to visualize the actin cytoskeleton in growing tobacco pollen tubes, which is difficult to observe using conventional techniques (Cai et al. 1997;Taylor & Hepler 1997). Tobacco pollen was plated on a solid culture medium and bombarded with a vector containing the GFP-mTn sequence fused to the LAT52 promoter which, unlike the 35S promoter, confers strong expression in pollen (Twell et al. 1989;Twell et al. 1990). The culture medium used had been carefully optimized to allow normal pollen tube growth in vitro (Read et al. 1993a, 1993b). On this medium, tobacco pollen tubes elongate for more than 48 h, grow to a final length of at least 15 mm, show normal tip morphology (Fig. 4 m) and cytoplasmic streaming, form callose plugs at regular intervals, and support mitotic division of the generative cell into two sperm cells (Read et al. 1993a, 1993b; B. Kost, P. Spielhofer and N.-H. Chua, unpublished observations). Labeling of actin filaments in growing pollen tubes derived from successfully bombarded pollen grains was observed after only 2 h and remained visible for at least 16 h. Very brightly fluorescent tubes that apparently expressed GFP-mTn at far above average levels contained thick actin cables, showed slow cytoplasmic streaming, and tended to cease growth prematurely. In contrast to BY-2 cells and A. thaliana plants, growing tobacco pollen tubes were apparently affected by very high levels of GFP-mTn expression. Pollen tube growth is not only extraordinarily sensitive to GFP-mTn expression, but also to actin depolymerizing drugs (Cai et al. 1997;Taylor & Hepler 1997). CytochalasinD and LatrunculinB completely inhibit tobacco pollen germination and tube growth at low concentrations (5 μm and 10 nm, respectively; B. Kost, P. Spielhofer and N.-H. Chua, unpublished observations). These findings suggest that pollen tube elongation is strictly dependent on actin function.
Figure 4. The actin cytoskeleton in the tip of a growing tobacco pollen tube as visualized by transient expression of GFP-mTn and confocal microscopy.
This particular pollen tube was growing at an average rate (3.25 μm min–1) after confocal imaging.
(a–l) Serial confocal optical sections at a step size of 1 μm (8× line averaging) giving an overview over the entire pollen tube actin cytoskeleton. (a) Cell cortex facing the microscope lens. (l) Cell cortex facing the opposite direction.
(m) Transmitted light reference image.
VG, vegetative nucleus (generative cell is not visible); OEZ, organelle exclusion zone; arrow heads, actin ring around the organelle exclusion zone. Scale bar: 25 μm.
Download figure to PowerPoint
However, tip morphology (Fig. 4m) and cytoplasmic streaming (data not shown) of more moderately fluorescent tubes, such as the one shown in Fig. 4, were absolutely normal, even after imaging the actin cytoskeleton by serial confocal optical sectioning. The growth rate after confocal imaging of a number of such pollen tubes, which all displayed actin labeling similar to the one shown in Fig. 4, was determined. These pollen tubes elongated at the same average rate as untransformed pollen tubes (Fig. 5), which demonstrates that transient expression of GFP-mTn and confocal imaging also allows non-invasive visualization of the actin cytoskeleton in growing pollen tubes. Untagged GFP transiently expressed under the control of the LAT52 promoter was evenly distributed in the pollen tube cytoplasm, accumulated slightly in the nucleus and was excluded from the generative cell as well as from vacuoles (data not shown). Pollen tubes that did not express GFP were non-fluorescent (data not shown).
Figure 5. GFP-mTn expression and confocal imaging have no effect on tobacco pollen tube growth.
GFP-mTn, average growth rate of GFP-mTn expressing pollen tubes after confocal imaging. The growth rate of 21 tubes with actin labeling similar to the one shown in Fig. 4 was measured. Control, average growth rate of 21 untransformed pollen tubes in different areas of the same plates. Error bars: 95% confidence interval.
Download figure to PowerPoint
Confocal imaging of GFP-mTn fluorescence resulted in clear images of the in vivo organization of the tobacco pollen tube actin cytoskeleton (Fig. 4), which revealed a number of details that were not observed in rhodamine-phalloidin microinjected lily pollen tubes (Miller et al. 1996). Serial confocal optical sections at a step size of 1 μm through a representative GFP-mTn expressing tobacco pollen tube are shown in Fig. 4. Long and relatively thick actin bundles were abundant in the pollen tube cytoplasm but did not extend into a 20 μm long region at the very tube tip. In the cell cortex, parallel actin bundles were arranged in a helical pattern (Fig. 4a,b,k,l), whereas actin cables in central regions were generally straight and longitudinally oriented (Fig. 4d–h). Pollen tube actin bundles appeared to be relatively immobile. During the time it took to complete a serial confocal scan (about 90 sec), actin filaments did not detectably change position. Some actin filaments were found to be associated with the vegetative nucleus (Fig. 4e–h) and the generative cell (data not shown). However, we did not detect a dense F-actin network around these structures as was occasionally observed following antibody or phalloidin staining of actin in permeabilized pollen tubes. F-actin networks have been proposed to be involved in maintaining shape as well as positioning in the tube cytoplasm of vegetative nuclei and generative cells (Derksen et al. 1995;Pierson & Cresti 1992).
Actin labeling in permeabilized cells has also provided evidence suggesting the presence of a dense actin network at the very pollen tube tip (Derksen et al. 1995). We did not find any indication of such a network, which is in accordance with what was observed in rhodaminephalloidin microinjected, living lily pollen tubes (Miller et al. 1996). Rather, imaging of GFP-mTn fluorescence revealed that the very pollen tube tip (a 20 μm long region at the end of the pollen tube) contains only sparse and fine actin filaments (Fig. 4c–h). No fluorescence was detected in the organelle exclusion zone at the extreme tube apex (Fig. 4d), which is thought to exclusively contain secretory vesicles (Derksen et al. 1995;Taylor & Hepler 1997), indicating that this region is devoid of actin bundles. This does not exclude, however, that individual, very fine actin filaments are occasionally present in the organelle exclusion zone, as was observed using sophisticated electron microscopical techniques (Miller et al. 1996). Interestingly, we regularly observed a ring of bright fluorescence around the apical organelle exclusion zone (Fig. 4b–h, arrow heads). This ring may correspond to a site where actin bundles are attached to the plasma membrane. Possible membrane attachment sites have been found close to the tip in rhodamine-phalloidin microinjected lily pollen tubes (Miller et al. 1996). However, since most actin bundles in tobacco pollen tubes do not extend into the proximity of the observed actin ring, other explanations for its function appear more likely. The ring actually marks the boundary between the regular cytoplasm and the organelle exclusion zone. It could mediate the maintenance of the distinction between the two different cytoplasmic domains by forming a physical barrier for all organelles that are larger than secretory vesicles. A similar function has been proposed for the cortical actin network in chromaffin cells, which is thought to regulate exocytosis by physically blocking access of secretory granules to the plasma membrane in the uninduced state (Aunis 1998).
The actin cytoskeleton at the pollen tube tip has been suggested to guide secretory vesicles to apical membrane docking sites and to drive pollen tube elongation by a mechanism related to the actin-mediated movement of amoeboid cells (Derksen et al. 1995;Steer & Steer 1989). F-actin organization in pollen tubes, as observed by confocal imaging of GFP-mTn fluorescence, is not in agreement with these postulated actin functions, which would require the presence of a dense actin network immediately underlying the plasma membrane at the extreme pollen tube apex. Instead, filamentous actin was found to be relatively sparse in pollen tube tips and to be essentially absent from the very tube apex. We propose that filamentous actin is essential for pollen tube growth because it is required for the prominent cytoplasmic streaming observed in these cells (Cai et al. 1997;Derksen et al. 1995;Taylor & Hepler 1997). It is well established that what is perceived as cytoplasmic streaming, is actually myosin dependent movement of cell organelles along actin filaments (Williamson 1993). Conceivably, organelle movement is necessary for sustained transport of secretory vesicles, which deliver cell membrane and cell wall material required for growth, to the extending pollen tube tip. The abundance and the arrangement of filamentous actin observed in the cytoplasm of GFP-mTn expressing pollen tubes is consistent with a function of actin filaments as tracks for organelle movement. In addition to its role in cytoplasmic streaming, filamentous actin in the form of a ring around the organelle exclusion zone might be required to maintain proper cytoplasmic organization at the pollen tube tip.