The development of the root apex is determined by progress of cells from the meristematic region to the successive post-mitotic developmental zones for transition, cell elongation and final cell differentiation. We addressed root development, tissue architecture and root developmental zonation by means of light-sheet microscopic imaging of Arabidopsis thaliana seedlings expressing END BINDING protein 1c (EB1c) fused to green fluorescent protein (GFP) under control of native EB1c promoter. Unlike the other two members of the EB1 family, plant-specific EB1c shows prominent nuclear localization in non-dividing cells in all developmental zones of the root apex. The nuclear localization of EB1c was previously mentioned solely in meristematic cells, but not further addressed. With the help of advanced light-sheet microscopy, we report quantitative evaluations of developmentally-regulated nuclear levels of the EB1c protein tagged with GFP relatively to the nuclear size in diverse root tissues (epidermis, cortex, and endodermis) and root developmental zones (meristem, transition, and elongation zones). Our results demonstrate a high potential of light-sheet microscopy for 4D live imaging of fluorescently-labeled nuclei in complex samples such as developing roots, showing capacity to quantify parameters at deeper cell layers (e.g., endodermis) with minimal aberrations. The data presented herein further signify the unique role of developmental cell reprogramming in the transition from cell proliferation to cell differentiation in developing root apex.

• Klíčová slova
• development, end-binding 1c (EB1c), light-sheet microscopy, nucleus, root apex, transition zone,
• Publikační typ
• časopisecké články MeSH

Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro. It was the active form of MAP kinase that was enriched with microtubules and followed similar dynamics to γ-tubulin, moving from poles to midzone during the anaphase-to-telophase transition. We found a novel substrate for MPK6, the microtubule plus end protein, EB1c. The mpk6-2 mutant was sensitive to 3-nitro-l-tyrosine (NO2 -Tyr) treatment with respect to mitotic abnormalities, and root cells overexpressing AP2C3 showed defects in chromosome segregation and spindle orientation. Our data suggest that the active form of MAP kinase interacts with γ-tubulin on specific subsets of mitotic microtubules during late mitosis. MPK6 phosphorylates EB1c, but not EB1a, and has a role in maintaining regular planes of cell division under stress conditions.

• Klíčová slova
• Arabidopsis, EB1c, MPK6, cell division, microtubules, mitogen-activated protein kinase, nitrosative stress, γ-tubulin,
• MeSH
• anafáze účinky léků   MeSH
• aparát dělícího vřeténka účinky léků   metabolismus   MeSH
• Arabidopsis cytologie   účinky léků   enzymologie   MeSH
• butadieny farmakologie   MeSH
• cytokineze účinky léků   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• fyziologický stres * účinky léků   MeSH
• kinetochory účinky léků   metabolismus   MeSH
• meristém cytologie   účinky léků   metabolismus   MeSH
• mikrotubuly účinky léků   metabolismus   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• nitrily farmakologie   MeSH
• nitrosace účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• rostlinné buňky účinky léků   metabolismus   MeSH
• segregace chromozomů účinky léků   MeSH
• telofáze účinky léků   MeSH
• tubulin metabolismus   MeSH
• tyrosin analogy a deriváty   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-nitrotyrosine MeSH Prohlížeč
• butadieny MeSH
• EB1c protein, Arabidopsis MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy MeSH
• mitogenem aktivované proteinkinasy MeSH
• MPK6 protein, Arabidopsis MeSH Prohlížeč
• nitrily MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny huseníčku MeSH
• tubulin MeSH
• tyrosin MeSH
• U 0126 MeSH Prohlížeč

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
• aktiny metabolismus   MeSH
• Arabidopsis účinky léků   genetika   fyziologie   MeSH
• cytokineze účinky léků   genetika   MeSH
• cytoskelet účinky léků   metabolismus   MeSH
• forminy genetika   metabolismus   MeSH
• mikrotubuly účinky léků   metabolismus   MeSH
• tabák účinky léků   genetika   fyziologie   MeSH
• thioketony farmakologie   MeSH
• tubulin metabolismus   MeSH
• uracil analogy a deriváty   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• aktiny MeSH
• forminy MeSH
• SMIFH2 compound MeSH Prohlížeč
• thioketony MeSH
• tubulin MeSH
• uracil MeSH