To test whether the number of expressing cells depends on the construct used for transformation, we compared the number of cells expressing the talin—YFP fusion with those expressing the unfused YFP-construct, both driven by a 35S-promotor Fig. This result indicates that the number of expressing cells is indeed affected by the nature of the construct.
For a successful transformation event two conditions have to be met: first, the foreign DNA has to be delivered into the cell; second, the cell has to express the introduced gene. Actin is organized in two major arrays in epidermal cells of Graminean coleoptiles Waller et al.
Depending on the developmental stage of the cell these arrays are manifest to a different degree. When the coleoptiles are transfected during the final stages of elongation growth when growth rates are below 0. To check whether the association with talin—YFP leads to a stabilization of actin microfilaments that would impair their dynamic organization, individual cells were followed over time in intact coleoptiles. It is therefore possible to distinguish between bundles of microfilaments and to locate them through several frames white arrows.
A closer look reveals, however, that orientation, thickness, and fine structure of individual strands differ between frames. New strands appear, others disappear, neighbouring strands merge or seem to deviate see white arrows. This indicates a considerable degree of fluctuation on the level of individual microfilament strands, despite an apparently stable organization of the array as an entity.
To test whether talin-labeled actin filaments remain sensitive to actin depolymerising drugs, cells were treated with latrunculin B and cytochalasin B. Stable longitudinal bundles of actin typical for a cell with arrested elongation Fig. By bright-field microscopy data not shown this center could be identified as the site where the gold particle had entered. Since talin—YFP was found to truly label both microfilament arrays characteristic for these cells and to preserve their innate dynamics, we asked whether it was possible to follow the signal-dependent responses of microfilaments in this system.
The stimuli of major biological relevance in these coleoptiles are auxin and light.
Whereas auxin triggers a stimulation of growth, light causes growth cessation. The long, dense and apparently stable bundles remained more or less unchanged before auxin was added Fig. In addition to these bundles, only a few, mostly longitudinal cortical strands were visible that only rarely reached into the cell pole. After addition of auxin, the bundles were rapidly replaced by finer strands and the cortical fine microfilaments became more prominent with an increased tendency to deviate more from the long axis of the cell Fig.
Moreover, the newly formed alignment appeared to be much more dynamic with microfilaments changing their orientation rapidly between subsequent frames Fig. This observation is also true for strands appearing at cell poles after addition of auxin that continuously changed their position, created new interconnections within the cell pole or simply disappeared. When the fine cortical microfilaments characteristic for rapidly elongating coleoptiles are followed during exposure to white light, they display a reorientation from initially transverse to oblique into a clearly longitudinal direction Fig.
Interestingly, these microfilaments did not form bundles comparable to those seen in auxin-depleted cells compare Fig.
Additionally, microfilaments were still observed to reach into the cell pole. Thus, although both auxin depletion and light cause a cessation of cell elongation, the microfilament organization observed under these conditions differs considerably.
The goal of these experiments was to assess the potential of a transient transfection system to observe in vivo responses of the actin cytoskeleton in Graminean coleoptiles. The Graminean coleoptile was chosen as experimental plant material because of several advantages. Firstly, in contrast to other systems commonly used for transient assays, such as onion epidermis, the epidermal cell layer of coleoptiles has a central function in active growth and exhibits fast growth responses to various stimuli such as gravity, light and auxin.
Furthermore, as growth is based exclusively on cell expansion, not on cell division, this allows correlations between growth rate and cellular events such as reorientations of microtubules and actin filaments. Finally, the coleoptile as intact organ is preserved in its entirety during all experimental procedures including treatments and microscopic observation.
Several preceding studies using immunolabeling or phalloidin staining have demonstrated altered cytoskeletal arrangements after the onset of diverse stimuli [for review see Nick ].
However, since these techniques are highly invasive and in their final consequence lethal, information about the dynamics of the cytoskeleton could only be inferred by analyzing large population of fixed cells. To get a more detailed view of the dynamics in a single cell it is necessary to observe reorientations of microtubules and actin filaments in the very same cell before and after stimulation.
We achieved this by combining the advantages of the coleoptile system with biolistic delivery of fluorescent fusion proteins labeling the cytoskeleton. The use of an in vivo marker for actin allows following the stimuli-induced rearrangements of actin microfilaments continuously in the same cell at a high temporal and spatial resolution.
Even very discrete positional changes of individual actin filaments can be observed providing additional indications on functions of the actin cytoskeleton. From the technical point of view the number of hit cells in biolistic transfection systems is generally low. As the morphology of the actin cytoskeleton can vary considerably among the different cells of a tissue, the question of efficiency is very important.
Statistical studies were therefore conducted to estimate success rates with either the non-fused or the fused FP vectors or with double transfections. As seen from the results for talin—YFP, single transformation was found to be highly efficient. Interestingly, double transformation of talin—YFP with an additional cyano fluorescent protein CFP vector also resulted in high transfection rates.
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With respect to the ability of our in vivo system to reflect the actin cytoskeleton in its natural structure and behaviour, several observations can be made. Firstly, fine transversally orientated actin strands at the cell cortex can be distinguished from thick longitudinal bundles of actin microfilaments. In addition to the fact that fine bundles of actin microfilaments are well preserved throughout the whole cell cortex this is also true for their functionality and responsiveness during exposure to certain stimuli.
Exogenous auxin induces a loosening of thick longitudinal bundles and a formation of a fine polar meshwork which can be now in advantage to previous studies continuously and directly observed. Following light treatment the actin cytoskeleton showed a dramatic realignment from transverse to loose longitudinal arrays. This was quite surprising considering the cytoskeletal responses known from other studies of growth under light conditions: coleoptile growth in rice, similar to other Graminean seedlings, is controlled by the phytochrome system Pjon and Furuya Whereas elongation of individual cells is promoted in the dark, elongation ceases rapidly in response to light, allowing the primary leaf to pierce through the coleoptile tip.
In addition to light, elongation in coleoptiles is regulated by auxin as has been shown in detail in dose-response experiments [for rice see Wang and Nick ]. While the application of exogenous auxin promotes cell growth and induces loose arrays of actin microfilaments, growth halts in response to auxin depletion, actin filaments are bundled and the polar mesh disappears Waller et al.
Assuming that light acts by lowering auxin levels, it would be expected that this cross-talk would be also mirrored by a corresponding organization of actin. This would mean a bundling response in the case of auxin deficiency or light and a loose array formation in the case of auxin promoted growth in dark. However, we observed actin, in response to light, to realign into fine and dispersed longitudinal strands not exhibiting any bundling effect Fig.
The fact that the actin response to light is totally different from that to auxin depletion might indicate signal-specific and independent mechanisms. This could mean that light controls actin via components that are either independent of auxin or act upstream of those events that modulate the actin cytoskeleton in response to auxin depletion. On the other hand, the longitudinal reorientation of actin induced by light is reminiscent of the characteristic behaviour of microtubules under light conditions. Cortical microtubules are found to be transverse in cells where elongation is promoted.
In contrast, they rapidly reorientate longitudinally under light conditions which inhibit growth Nick , Waller et al. Thus, the light-induced reorientation of microfilaments is in parallel to that of microtubules and is therefore consistent with a mutual control of both cytoskeletal components Collings and Allen , Tominaga et al. Parallel rearrangements were also recently observed in embryogenic cells of maize where redistributions of microtubules and actin filaments to the cell cortex occurred during the developmental switch from unpolarized to polarized cells in response to auxin deprivation Samaj et al.
In addition to the realignment of actin filaments another observation was made in respect of the cell poles. The amount of actin filaments appears to be elevated at pole regions of cells when transverse microfilaments predominate along the cell axis Fig. Enrichment or deprivation of actin in end-poles of cells might therefore be linked to a certain developmental stage of the cells within tissues like the epidermal layer of higher plants. From the present observation one might therefore conclude that actin extensions at outermost regions of cell-poles in parallel to a transversal orientation of actin filaments at lateral domains does not only reflect a stage where cell-growth is promoted but also represents a manifestation of cell polarity.
Myosin VIII localizes especially at the plasmodesmata in the non-growing cross walls. In cells of the transition zone, unique AF bundles develop which are proposed to participate in the onset of rapid cell elongation. These AF bundles are initiated at the nuclear peripheries and are organized via myosin VIII-enriched cross-walls, these two sites obviously act as the major AFOCs of postmitotic root-body cells.
Treatment of roots with latrunculin B reveals that dynamic AFs are essential for the vacuome-driven cell elongation and for the root hair formation. In the transition zone and elongation region, cells of the inner cortex localize plant myosin VIII molecules abundantly at pit-fields.
Ana Paez-Garcia 2 ,. Cheol-Min Yoo 2 ,. Karuppaiah Palanichelvam 1 ,. Elison B.
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