Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

• Keywords
• auxin carriers, correlative microscopy, nanodomains, plasma membrane,
• MeSH
• Arabidopsis genetics   growth & development   MeSH
• Cell Membrane genetics   metabolism   ultrastructure   MeSH
• Microscopy, Confocal MeSH
• Metal Nanoparticles chemistry   MeSH
• Indoleacetic Acids metabolism   MeSH
• Microscopy, Electron, Scanning * MeSH
• Image Processing, Computer-Assisted MeSH
• Protoplasts metabolism   ultrastructure   MeSH
• Plant Growth Regulators genetics   metabolism   MeSH
• Nicotiana genetics   metabolism   ultrastructure   MeSH
• Gold chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH
• Gold MeSH

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

• Keywords
• Hordeum vulgare, chromatin, deconvolution microscopy, metaphase chromosome, nanoscopy, photoactivated localization microscopy, stimulated emission depletion microscopy, structured illumination microscopy, topoisomerase II, wide-field microscopy,
• MeSH
• Chromosomes, Plant chemistry   genetics   metabolism   MeSH
• DNA Topoisomerases, Type II metabolism   MeSH
• Fluorescent Dyes chemistry   MeSH
• Microscopy, Fluorescence methods   MeSH
• Indoles chemistry   MeSH
• Hordeum cytology   genetics   MeSH
• Microscopy, Confocal methods   MeSH
• Metaphase genetics   MeSH
• Reproducibility of Results MeSH
• Single Molecule Imaging methods   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• DAPI MeSH Browser
• DNA Topoisomerases, Type II MeSH
• Fluorescent Dyes MeSH
• Indoles MeSH

Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.

• Keywords
• Chromatin, Expansion microscopy, Hordeum vulgare, Nucleus, Structured illumination microscopy,
• MeSH
• Cell Nucleus ultrastructure   MeSH
• Chromatin ultrastructure   MeSH
• Fluorescent Antibody Technique MeSH
• In Situ Hybridization, Fluorescence MeSH
• Hordeum genetics   MeSH
• Microscopy methods   standards   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

• MeSH
• Arabidopsis cytology   genetics   metabolism   MeSH
• Brefeldin A pharmacology   MeSH
• Cell Membrane metabolism   MeSH
• Cytokinins chemistry   metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Fluorescent Dyes chemistry   metabolism   MeSH
• Plants, Genetically Modified MeSH
• Meristem cytology   metabolism   MeSH
• Protein Kinases genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Receptors, Cell Surface genetics   metabolism   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Brefeldin A MeSH
• Cytokinins MeSH
• Fluorescent Dyes MeSH
• Protein Kinases MeSH
• Arabidopsis Proteins MeSH
• Receptors, Cell Surface MeSH
• Recombinant Fusion Proteins MeSH
• WOL protein, Arabidopsis MeSH Browser
• Green Fluorescent Proteins MeSH

Centromeres are essential for proper chromosome segregation to the daughter cells during mitosis and meiosis. Chromosomes of most eukaryotes studied so far have regional centromeres that form primary constrictions on metaphase chromosomes. These monocentric chromosomes vary from point centromeres to so-called "meta-polycentromeres", with multiple centromere domains in an extended primary constriction, as identified in Pisum and Lathyrus species. However, in various animal and plant lineages centromeres are distributed along almost the entire chromosome length. Therefore, they are called holocentromeres. In holocentric plants, centromere-specific proteins, at which spindle fibers usually attach, are arranged contiguously (line-like), in clusters along the chromosomes or in bands. Here, we summarize findings of ultrastructural investigations using immunolabeling with centromere-specific antibodies and super-resolution microscopy to demonstrate the structural diversity of plant centromeres. A classification of the different centromere types has been suggested based on the distribution of spindle attachment sites. Based on these findings we discuss the possible evolution and advantages of holocentricity, and potential strategies to segregate holocentric chromosomes correctly.

• Keywords
• CENH3, CENP-A, Cuscuta, Lathyrus, Luzula, Pisum, Rhynchospora, clustered centromere, holocentromere, microtubule, monocentromere, structured illumination microscopy,
• MeSH
• Cell Cycle MeSH
• Centromere metabolism   MeSH
• Chromosomes, Plant metabolism   MeSH
• Microscopy * MeSH
• Evolution, Molecular MeSH
• Plants metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND: In the present work, we provide an account of structured illumination microscopy (SIM) imaging of fixed and immunolabeled plant probes. We take advantage of SIM, to superresolve intracellular structures at a considerable z-range and circumvent its low temporal resolution capacity during the study of living samples. Further, we validate the protocol for the imaging of fixed transgenic material expressing fluorescent protein-based markers of different subcellular structures. RESULTS: Focus is given on 3D imaging of bulky subcellular structures, such as mitotic and cytokinetic microtubule arrays as well as on the performance of SIM using multichannel imaging and the quantitative correlations that can be deduced. As a proof of concept, we provide a superresolution output on the organization of cortical microtubules in wild-type and mutant Arabidopsis cells, including aberrant preprophase microtubule bands and phragmoplasts in a cytoskeletal mutant devoid of the p60 subunit of the microtubule severing protein KATANIN and refined details of cytoskeletal aberrations in the mitogen activated protein kinase (MAPK) mutant mpk4. We further demonstrate, in a qualitative and quantitative manner, colocalizations between MPK6 and unknown dually phosphorylated and activated MAPK species and we follow the localization of the microtubule associated protein 65-3 (MAP65-3) in telophase and cytokinetic microtubular arrays. CONCLUSIONS: 3D SIM is a powerful, versatile and adaptable microscopy method for elucidating spatial relationships between subcellular compartments. Improved methods of sample preparation aiming to the compensation of refractive index mismatches, allow the use of 3D SIM in the documentation of complex plant cell structures, such as microtubule arrays and the elucidation of their interactions with microtubule associated proteins.

• Keywords
• Immunofluorescence, Microtubule associated proteins, Microtubules, Structured illumination microscopy,
• Publication type
• Journal Article MeSH