The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

• MeSH
• Actins metabolism   MeSH
• Arabidopsis drug effects   genetics   physiology   MeSH
• Cytokinesis drug effects   genetics   MeSH
• Cytoskeleton drug effects   metabolism   MeSH
• Formins genetics   metabolism   MeSH
• Microtubules drug effects   metabolism   MeSH
• Nicotiana drug effects   genetics   physiology   MeSH
• Thiones pharmacology   MeSH
• Tubulin metabolism   MeSH
• Uracil analogs & derivatives   pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Actins MeSH
• Formins MeSH
• SMIFH2 compound MeSH Browser
• Thiones MeSH
• Tubulin MeSH
• Uracil 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.

• Keywords
• Arabidopsis, ectopic cell division, katanin, light-sheet fluorescence microscopy, live cell imaging, microtubules, root development,
• Publication type
• Journal Article MeSH

Advanced bioimaging uncovers insights into subcellular structures of plants.

• MeSH
• Microscopy, Fluorescence MeSH
• Microscopy, Confocal MeSH
• Plant Cells metabolism   MeSH
• Imaging, Three-Dimensional * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Cell division and expansion are two fundamental biological processes supporting indeterminate root growth and development of plants. Quantitative evaluations of cell divisions related to root growth analyses have been performed in several model crop and non-crop plant species, but not in important legume plant Medicago sativa. Light-sheet fluorescence microscopy (LSFM) is an advanced imaging technique widely used in animal developmental biology, providing efficient fast optical sectioning under physiological conditions with considerably reduced phototoxicity and photobleaching. Long-term 4D imaging of living plants offers advantages for developmental cell biology not available in other microscopy approaches. Recently, LSFM was implemented in plant developmental biology studies, however, it is largely restricted to the model plant Arabidopsis thaliana. Cellular and subcellular events in crop species and robust plant samples have not been studied by this method yet. Therefore we performed LSFM long-term live imaging of growing root tips of transgenic alfalfa plants expressing the fluorescent molecular marker for the microtubule-binding domain (GFP-MBD), in order to study dynamic patterns of microtubule arrays during mitotic cell division. Quantitative evaluations of cell division progress in the two root tissues (epidermis and cortex) clearly indicate that root growth rate is correlated with duration of cell division in alfalfa roots. Our results favor non-invasive environmental LSFM as one of the most suitable methods for qualitative and quantitative cellular and developmental imaging of living transgenic legume crops.

• Keywords
• Medicago sativa, cell division, developmental imaging, light-sheet microscopy, microtubules, root growth, transgenic crops,
• Publication type
• Journal Article MeSH