GFP-TUA6 Dotaz Zobrazit nápovědu
Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole "overrules" the stabilization by cortical cytoskeletal elements.
- Klíčová slova
- Arabidopsis hypocotyl, GFP-ABD, GFP-MAP4, GFP-TUA6, actin microfilaments, cytoskeleton, deplasmolysis, microtubules, plasmolysis,
- Publikační typ
- časopisecké články MeSH
Pattern formation, cell proliferation, and directional cell growth, are driving factors of plant organ shape, size, and overall vegetative development. The establishment of vegetative morphogenesis strongly depends on spatiotemporal control and synchronization of formative and proliferative cell division patterns. In this context, the progression of cell division and the regulation of cell division plane orientation are defined by molecular mechanisms converging to the proper positioning and temporal reorganization of microtubule arrays such as the preprophase microtubule band, the mitotic spindle and the cytokinetic phragmoplast. By focusing on the tractable example of primary root development and lateral root emergence in Arabidopsis thaliana, genetic studies have highlighted the importance of mechanisms underlying microtubule reorganization in the establishment of the root system. In this regard, severe alterations of root growth, and development found in extensively studied katanin1 mutants of A. thaliana (fra2, lue1, and ktn1-2), were previously attributed to defective rearrangements of cortical microtubules and aberrant cell division plane reorientation. How KATANIN1-mediated microtubule severing contributes to tissue patterning and organ morphogenesis, ultimately leading to anisotropy in microtubule organization is a trending topic under vigorous investigation. Here we addressed this issue during root development, using advanced light-sheet fluorescence microscopy (LSFM) and long-term imaging of ktn1-2 mutant expressing the GFP-TUA6 microtubule marker. This method allowed spatial and temporal monitoring of cell division patterns in growing roots. Analysis of acquired multidimensional data sets revealed the occurrence of ectopic cell divisions in various tissues including the calyptrogen and the protoxylem of the main root, as well as in lateral root primordia. Notably the ktn1-2 mutant exhibited excessive longitudinal cell divisions (parallel to the root axis) at ectopic positions. This suggested that changes in the cell division pattern and the occurrence of ectopic cell divisions contributed significantly to pleiotropic root phenotypes of ktn1-2 mutant. LSFM provided evidence that KATANIN1 is required for the spatiotemporal control of cell divisions and establishment of tissue patterns in living A. thaliana roots.
- Klíčová slova
- Arabidopsis, ectopic cell division, katanin, light-sheet fluorescence microscopy, live cell imaging, microtubules, root development,
- Publikační typ
- časopisecké články MeSH
Microtubule bundling is an essential mechanism underlying the biased organization of interphase and mitotic microtubular systems of eukaryotes in ordered arrays. Microtubule bundle formation can be exemplified in plants, where the formation of parallel microtubule systems in the cell cortex or the spindle midzone is largely owing to the microtubule crosslinking activity of a family of microtubule associated proteins, designated as MAP65s. Among the nine members of this family in Arabidopsis thaliana, MAP65-1 and MAP65-2 are ubiquitous and functionally redundant. Crosslinked microtubules can form high-order arrays, which are difficult to track using widefield or confocal laser scanning microscopy approaches. Here, we followed spatiotemporal patterns of MAP65-2 localization in hypocotyl cells of Arabidopsis stably expressing fluorescent protein fusions of MAP65-2 and tubulin. To circumvent imaging difficulties arising from the density of cortical microtubule bundles, we use different superresolution approaches including Airyscan confocal laser scanning microscopy (ACLSM), structured illumination microscopy (SIM), total internal reflection SIM (TIRF-SIM), and photoactivation localization microscopy (PALM). We provide insights into spatiotemporal relations between microtubules and MAP65-2 crossbridges by combining SIM and ACLSM. We obtain further details on MAP65-2 distribution by single molecule localization microscopy (SMLM) imaging of either mEos3.2-MAP65-2 stochastic photoconversion, or eGFP-MAP65-2 stochastic emission fluctuations under specific illumination conditions. Time-dependent dynamics of MAP65-2 were tracked at variable time resolution using SIM, TIRF-SIM, and ACLSM and post-acquisition kymograph analysis. ACLSM imaging further allowed to track end-wise dynamics of microtubules labeled with TUA6-GFP and to correlate them with concomitant fluctuations of MAP65-2 tagged with tagRFP. All different microscopy modules examined herein are accompanied by restrictions in either the spatial resolution achieved, or in the frame rates of image acquisition. PALM imaging is compromised by speed of acquisition. This limitation was partially compensated by exploiting emission fluctuations of eGFP which allowed much higher photon counts at substantially smaller time series compared to mEos3.2. SIM, TIRF-SIM, and ACLSM were the methods of choice to follow the dynamics of MAP65-2 in bundles of different complexity. Conclusively, the combination of different superresolution methods allowed for inferences on the distribution and dynamics of MAP65-2 within microtubule bundles of living A. thaliana cells.
