August 2010
Q & A: Single-molecule localization microscopy for biological imaging
BMC Biology 2010, 8:106 (11 August 2010)
McEvoy AL, Greenfield D, Bates M, Liphardt J
McEvoy AL, Greenfield D, Bates M, Liphardt J
Biophysics Graduate Group, University of California Berkeley, Berkeley, CA 94720, USA
No Abstract: Text reads as - Prokaryotic and eukaryotic cells possess a complex internal structure, including protein networks, genetic material, internal and external membranes and organ elles. These elements provide physical structure to cells, and a means to localize particular biochemical processes to specific cellular regions. The structure of the cell is intimately linked to its biological functions, and hence the study of the physical structure and organization of the cell is a valuable means of gaining insight into cell biology............
July 2010
Bimolecular fluorescence complementation (BiFC) assay for protein-protein interaction in onion cells using the helios gene gun.
J Vis Exp. 2010 Jun 12;(40). pii: 1963. doi: 10.3791/1963.
Hollender CA, Liu Z.
Hollender CA, Liu Z.
Dept. Of Cell Biology and Molecular Genetics, University of Maryland, MD, USA.
Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay(1) allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)(2) and the visualization of their interactions mediated by BiFC in onion epidermal cells.
June 2010
H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca(2+)-dependent scavenging system.
Plant J. 2010 Jun 1;62(5):760-72.
Costa A, Drago I, Behera S, Zottini M, Pizzo P, Schroeder JI, Pozzan T, Schiavo FL.
Dipartimento di Biologia, Università degli Studi di Padova, Via U. Bassi 58/B, 35131 Padova, Italy.
Oxidative stress is a major challenge for all cells living in an oxygen-based world. Among reactive oxygen species, H2O2, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H2O2 in plant cells, it is necessary to have a reliable, specific and non-invasive methodology for its in vivo detection. Hence, the genetically encoded H2O2 sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)-based Cameleon Ca(2+) indicator, D3cpv-KVK-SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca(2+) increase is followed by Ca(2+) rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre- and post-bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca(2+) rise in vivo stimulates catalase activity, increasing peroxisomal H2O2 scavenging efficiency.
May 2010
Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll.
New Phytologist (2010) 186: 1018–1025.
Littlejohn GR, Gouveia JD, Edner C, Smirnoff N, Love J.
School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK.
Air spaces in the leaf mesophyll generate deleterious optical effects that compromise confocal microscopy. *Leaves were mounted in the nontoxic, nonfluorescent perfluorocarbon, perfluorodecalin (PFD), and optical enhancement and physiological effect were assessed using confocal microscopy and chlorophyll fluorescence. *Mounting leaves of Arabidopsis thaliana in PFD significantly improved the optical qualities of the leaf, thereby enabling high-resolution laser scanning confocal imaging over twofold deeper into the mesophyll, compared with using water. Incubation in PFD had less physiological impact on the mounted specimen than water. *We conclude that the application of PFD as a mounting medium substantially increases confocal image resolution of living mesophyll and vascular bundle cells, with minimal physiological impact.
April 2010
Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells.
Proc Natl Acad Sci U S A. 2010 Mar 29.
Ueda H, Yokota E, Kutsuna N, Shimada T, Tamura K, Shimmen T, Hasezawa S, Dolja VV, Hara-Nishimura I.
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to approximately 3.5 mum/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.
March 2010
Probing plant membranes with FM dyes: tracking, dragging or blocking?
Plant J. 2009 Dec 10.
Jelínková A, Malínská K, Simon S, Kleine-Vehn J, Pařezová M, Pejchar P, Kubeš M, Martinec J, Friml J, Zažímalová E, Petrášek J.
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic.
Remarkable progress in various techniques of in vivo fluorescence microscopy has brought an urgent need for reliable markers for tracking cellular structures and processes. The goal of this manuscript is to describe unexplored effects of the FM (Fei Mao) styryl dyes, which are widely used probes that label processes of endocytosis and vesicle trafficking in eukaryotic cells. Although there are few reports on the effect of styryl dyes on membrane fluidity and the activity of mammalian receptors, FM dyes have been considered as reliable tools for tracking of plant endocytosis. Using plasma membrane-localized transporters for the plant hormone auxin in tobacco BY-2 and Arabidopsis thaliana cell suspensions, we show that routinely used concentrations of FM 4-64 and FM 5-95 trigger transient re-localization of these proteins, and FM 1-43 affects their activity. The active process of re-localization is blocked neither by inhibitors of endocytosis nor by cytoskeletal drugs. It does not occur in A. thaliana roots and depends on the degree of hydrophobicity (lipophilicity) of a particular FM dye. Our results emphasize the need for circumspection during in vivo studies of membrane proteins performed using simultaneous labelling with FM dyes.
February 2010
Movement and remodeling of the endoplasmic reticulum in nondividing cells of tobacco leaves.
Plant Cell. 2009 Dec;21(12):3937-49.
Sparkes I, Runions J, Hawes C, Griffing L.
School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
Using a novel analytical tool, this study investigates the relative roles of actin, microtubules, myosin, and Golgi bodies on form and movement of the endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) leaf epidermal cells. Expression of a subset of truncated class XI myosins, which interfere with the activity of native class XI myosins, and drug-induced actin depolymerization produce a more persistent network of ER tubules and larger persistent cisternae. The treatments differentially affect two persistent size classes of cortical ER cisternae, those >0.3 microm(2) and those smaller, called punctae. The punctae are not Golgi, and ER remodeling occurs in the absence of Golgi bodies. The treatments diminish the mobile fraction of ER membrane proteins but not the diffusive flow of mobile membrane proteins. The results support a model whereby ER network remodeling is coupled to the directionality but not the magnitude of membrane surface flow, and the punctae are network nodes that act as foci of actin polymerization, regulating network remodeling through exploratory tubule growth and myosin-mediated shrinkage.
January 2010
Seeing is believing: on the use of image databases for visually exploring plant organelle dynamics.
Plant Cell Physiol. 2009 Dec;50(12):2000-14.
Mano S, Miwa T, Nishikawa S, Mimura T, Nishimura M.
Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585, Japan.
Organelle dynamics vary dramatically depending on cell type, developmental stage and environmental stimuli, so that various parameters, such as size, number and behavior, are required for the description of the dynamics of each organelle. Imaging techniques are superior to other techniques for describing organelle dynamics because these parameters are visually exhibited. Therefore, as the results can be seen immediately, investigators can more easily grasp organelle dynamics. At present, imaging techniques are emerging as fundamental tools in plant organelle research, and the development of new methodologies to visualize organelles and the improvement of analytical tools and equipment have allowed the large-scale generation of image and movie data. Accordingly, image databases that accumulate information on organelle dynamics are an increasingly indispensable part of modern plant organelle research. In addition, image databases are potentially rich data sources for computational analyses, as image and movie data reposited in the databases contain valuable and significant information, such as size, number, length and velocity. Computational analytical tools support image-based data mining, such as segmentation, quantification and statistical analyses, to extract biologically meaningful information from each database and combine them to construct models. In this review, we outline the image databases that are dedicated to plant organelle research and present their potential as resources for image-based computational analyses.
December 2009
In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots.
BMC Plant Biol. 2009 Nov 18;9:133.
Gattolin S, Sorieul M, Hunter PR, Khonsari RH, Frigerio L.
Department of Biological Sciences, University of Warwick, Coventry, UK. s.gattolin@warwick.ac.uk
BACKGROUND: Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known. RESULTS: We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures. CONCLUSION: We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs.
November 2009
Optical microscopy in photosynthesisOptical microscopy in photosynthesis.
Photosynth Res (2009) 102:111–141.
Richard Cisek • Leigh Spencer • Nicole Prent • Donatas Zigmantas • George S. Espie • Virginijus Barzda.
Department of Chemical and Physical Sciences, Department of Physics, and Institute for Optical Sciences, University of Toronto, 3359 Mississauga Road, Mississauga,ON L5L 1C6, Canada: DZ- Department of Chemical Physics, Lund University, Lund, Sweden: GSE- Department of Biology, University of Toronto,Mississauga, Canada.
Department of Chemical and Physical Sciences, Department of Physics, and Institute for Optical Sciences, University of Toronto, 3359 Mississauga Road, Mississauga,ON L5L 1C6, Canada: DZ- Department of Chemical Physics, Lund University, Lund, Sweden: GSE- Department of Biology, University of Toronto,Mississauga, Canada.
Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented,focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.
October 2009
Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis.
Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):13106-11.
Kadota A, Yamada N, Suetsugu N, Hirose M, Saito C, Shoda K, Ichikawa S, Kagawa T, Nakano A, Wada M.
Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement.
September 2009
Oryzalin bodies: in addition to its anti-microtubule properties, the dinitroaniline herbicide oryzalin causes nodulation of the endoplasmic reticulum.
Protoplasma. 2009 Jul;236(1-4):73-84.
Langhans M, Niemes S, Pimpl P, Robinson DG.
Dept. Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, Heidelberg, Germany.
Dept. Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, Heidelberg, Germany.
Oryzalin is a much-used pre-emergence herbicide which causes microtubules (Mt) to depolymerize. Here, we document that this dinitroaniline herbicide also leads to characteristic changes in the morphology of the endoplasmic reticulum (ER) and Golgi apparatus. These effects, which are reversible upon washing out the herbicide, are already elicited at low concentrations (2 microM) and become most pronounced at 20 microM. For our studies, we have employed roots of Arabidopsis thaliana, tobacco leaf epidermal cells, and BY-2 suspension cultures, all expressing the luminal ER marker GFP::HDEL. In all cell types, the typical cortical network of the ER assumed a pronounced nodulated morphology with increasing oryzalin concentrations. This effect was enhanced through subsequent application of brefeldin A (BFA). Thin sections of Arabidopsis roots observed in the electron microscope revealed the nodules to consist of a mass of anastomosing ER tubules. Oryzalin also caused the cisternae in Golgi stacks to increase in number but reduced their diameter. Oryzalin retarded ER mobility but did not prevent latrunculin B-induced clustering of Golgi stacks on islands of cisternal ER. While the mechanism underlying these changes in endomembranes remains unknown, it is specific for oryzalin since these effects were not elicited with other Mt-depolymerizing herbicides, e.g., trifluralin, amiprophosmethyl, or colchicine.
August 2009
Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.
PLoS One. 2009 May 27;4(5):e5716.
Elgass K, Caesar K, Schleifenbaum F, Stierhof YD, Meixner AJ, Harter K.
Institute for Physical and Theoretical Chemistry, University of Tübingen, Tübingen, Germany.
Institute for Physical and Theoretical Chemistry, University of Tübingen, Tübingen, Germany.
BACKGROUND: Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences. PRINCIPAL FINDINGS: Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes. SIGNIFICANCE: One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.
July 2009
Accuracy and precision in quantitative fluorescence microscopy.
J Cell Biol. 2009 Jun 29;185(7):1135-48.
Waters JC.
Harvard Medical School, Department of Cell Biology, Boston, MA 02115, USA.
Harvard Medical School, Department of Cell Biology, Boston, MA 02115, USA.
The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.
June 2009
Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.
PLoS One. 2009 May 29;4(5):e5744
Vidali L, Rounds CM, Hepler PK, Bezanilla M.
Department of Biology, University of Massachusetts, Amherst, Massachusetts, USA.
Department of Biology, University of Massachusetts, Amherst, Massachusetts, USA.
Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. METHODOLOGY/PRINCIPAL FINDINGS: In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. CONCLUSIONS/SIGNIFICANCE: Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.
Application of Lifeact reveals F-actin dynamics in Arabidopsis thaliana and the liverwort, Marchantia polymorpha.
Plant Cell Physiol. 2009 Jun;50(6):1041-8.
Era A, Tominaga M, Ebine K, Awai C, Saito C, Ishizaki K, Yamato KT, Kohchi T, Nakano A, Ueda T.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Application of Lifeact reveals F-actin dynamics in Arabidopsis thaliana and the liverwort, Marchantia polymorpha.
Plant Cell Physiol. 2009 Jun;50(6):1041-8.
Era A, Tominaga M, Ebine K, Awai C, Saito C, Ishizaki K, Yamato KT, Kohchi T, Nakano A, Ueda T.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact-Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact-Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.
May 2009
Peroxule extension over ER-defined paths constitutes a rapid subcellular response to hydroxyl stress.
Plant J. 2009 March [Epub ahead of print]
Sinclair AM, Trobacher CP, Mathur N, Greenwood JS, Mathur J.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Plants survive against myriad environmental odds while remaining rooted to a single spot. The time scale over which plant cells can respond to environmental cues is seldom appreciated. Fluorescent protein-assisted live imaging of peroxisomes reveals that they respond within seconds of exposure to hydrogen peroxide and hydroxyl radicals by producing dynamic extensions called peroxules. Observations of the Arabidopsis flu mutant and treatments with xenobiotics eliciting singlet oxygen and superoxide reactive oxygen species suggest that the observed responses are specific for hydroxyl radicals. Prolonged exposure to hydroxyl radicals inhibits peroxule extension, and instead causes motile and spherical peroxisomes in a cell to become immotile and elongate several-fold. Expression of photo-convertible EosFP-PTS1 demonstrates that vermiform peroxisomes result from rapid stretching of individual peroxisomes, while the subsequent 'beads-on-a-string' morphology results from differential protein distribution within an elongated tubule. Over time, the beads in elongated peroxisomes also extend peroxules randomly before undergoing asynchronous, asymmetrical fission. Peroxule extension does not appear to involve cytoskeletal elements directly, but is closely aligned with and reflects the dynamics of ER tubules. Peroxisomal responses reveal a rapidly invoked subcellular machinery that is involved in recognition of hydroxyl stress thresholds, and its possible remediation locally through extension of peroxules or globally by increasing peroxisome numbers. A matrix protein retro-flow mechanism that supports peroxisome-ER connectivity in plant cells is suggested.
April 2009
Rapid, combinatorial analysis of membrane compartments in intact plants with a multi-color marker set.
Plant J. 2009 Feb 26.
Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J.
The Salk Institute, Plant Biology Laboratory, 10010 North Torrey Pines Road, CA 92037, USA.
The Salk Institute, Plant Biology Laboratory, 10010 North Torrey Pines Road, CA 92037, USA.
Plant membrane compartments and trafficking pathways are highly complex and often distinct from animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all these cases a central role is played by the endosomal membrane system which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series and generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns and stable, non-toxic expression. We demonstrate the power of this multi-color "Wave" marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immuno-electron microscopy. Among other findings, our systematic co-localisation analysis revealed that a class of plant Rab1-homologs has a much more extended localization than previously assumed and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available and will promote rapid progress in diverse areas of plant cell biology.
March 2009
The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion.
Plant Physiol. 2008 Nov;148(3):1380-93.
Seguí-Simarro JM, Coronado MJ, Staehelin LA.
Plant cells exhibit a high rate of mitochondrial DNA (mtDNA) recombination. This implies that before cytokinesis, the different mitochondrial compartments must fuse to allow for mtDNA intermixing. When and how the conditions for mtDNA intermixing are established are largely unknown. We have investigated the cell cycle-dependent changes in mitochondrial architecture in different Arabidopsis (Arabidopsis thaliana) cell types using confocal microscopy, conventional, and three-dimensional electron microscopy techniques. Whereas mitochondria of cells from most plant organs are always small and dispersed, shoot apical and leaf primordial meristematic cells contain small, discrete mitochondria in the cell periphery and one large, mitochondrial mass in the perinuclear region. Serial thin-section reconstructions of high-pressure-frozen shoot apical meristem cells demonstrate that during G1 through S phase, the large, central mitochondrion has a tentaculate morphology and wraps around one nuclear pole. In G2, both types of mitochondria double their volume, and the large mitochondrion extends around the nucleus to establish a second sheet-like domain at the opposite nuclear pole. During mitosis, approximately 60% of the smaller mitochondria fuse with the large mitochondrion, whose volume increases to 80% of the total mitochondrial volume, and reorganizes into a cage-like structure encompassing first the mitotic spindle and then the entire cytokinetic apparatus. During cytokinesis, the cage-like mitochondrion divides into two independent tentacular mitochondria from which new, small mitochondria arise by fission. These cell cycle-dependent changes in mitochondrial architecture explain how these meristematic cells can achieve a high rate of mtDNA recombination and ensure the even partitioning of mitochondria between daughter cells.
February 2009
Lifeact: a versatile marker to visualize F-actin.
Nat Methods. 2008 Jul;5(7):605-607.
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z, Sixt M, Wedlich-Soldner R.
Max Planck Institute of Biochemistry, Independent Junior Research Group Cellular Dynamics and Cell Patterning, Am Klopferspitz 18, 82152 Martinsried, Germany.
Max Planck Institute of Biochemistry, Independent Junior Research Group Cellular Dynamics and Cell Patterning, Am Klopferspitz 18, 82152 Martinsried, Germany.
Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
JANUARY 2009
Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations
Proc Natl Acad Sci U S A. 105(47):18343-18348.
Adam V, Lelimousin M, Boehme S, Desfonds G, Nienhaus K, Field MJ, Wiedenmann J, McSweeney S, Nienhaus GU, Bourgeois D.
European Synchrotron Radiation Facility, 6 Rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France. Germany.
European Synchrotron Radiation Facility, 6 Rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France. Germany.
Photoactivatable fluorescent proteins (FPs) are powerful fluorescent highlighters in live cell imaging and offer perspectives for optical nanoscopy and the development of biophotonic devices. Two types of photoactivation are currently being distinguished, reversible photoswitching between fluorescent and nonfluorescent forms and irreversible photoconversion. Here, we have combined crystallography and (in crystallo) spectroscopy to characterize the Phe-173-Ser mutant of the tetrameric variant of EosFP, named IrisFP, which incorporates both types of phototransformations. In its green fluorescent state, IrisFP displays reversible photoswitching, which involves cis-trans isomerization of the chromophore. Like its parent protein EosFP, IrisFP also photoconverts irreversibly to a red-emitting state under violet light because of an extension of the conjugated pi-electron system of the chromophore, accompanied by a cleavage of the polypeptide backbone. The red form of IrisFP exhibits a second reversible photoswitching process, which may also involve cis-trans isomerization of the chromophore. Therefore, IrisFP displays altogether 3 distinct photoactivation processes. The possibility to engineer and precisely control multiple phototransformations in photoactivatable FPs offers exciting perspectives for the extension of the fluorescent protein toolkit.
December 2008
A green fluorescent protein with photoswitchable emission from the deep sea.
PLoS ONE. 2008;3(11):e3766. Epub 2008 Nov 19.
Vogt A, D'Angelo C, Oswald F, Denzel A, Mazel CH, Matz MV, Ivanchenko S, Nienhaus GU, Wiedenmann J.
Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany.
Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany.
A colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.
November 2008
Reflection across plant cell boundaries in confocal laser scanning microscopy.
J Microsc. 2008 Aug;231(2):349-357.
Liu DY, Kuhlmey BT, Smith PM, Day DA, Faulkner CR, Overall RL.
School of Biological Sciences, University of Sydney, NSW 2006, Australia.
School of Biological Sciences, University of Sydney, NSW 2006, Australia.
The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.
October 2008
Plant Methods. 2008 Sep 19;4:21.
Schenkel M, Sinclair AM, Johnstone D, Bewley JD, Mathur J.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
The actin cytoskeleton responds quickly to diverse stimuli and plays numerous roles in cellular signalling, organelle motility and subcellular compartmentation during plant growth and development. Molecular and cell biological tools that can facilitate visualization of actin organization and dynamics in a minimally invasive manner are essential for understanding this fundamental component of the living cell. RESULTS: A novel, monomeric (m) Eos-fluorescent protein derived from the coral Lobophyllia hemprichii was assessed for its green to red photo-convertibility in plant cells by creating mEosFP-cytosolic. mEosFP was fused to the F-(filamentous)-Actin Binding Domain of the mammalian Talin gene to create mEosFP::FABDmTalin. Photo-conversion, visualization and colour quantification protocols were developed for EosFP targeted to the F-actin cytoskeleton. Rapid photo-conversion in the entire cell or in a region of interest was easily achieved upon illumination with an approximately 400 nm wavelength light beam using an epi-fluorescent microscope. Dual color imaging after photo-conversion was carried out using a confocal laser-scanning microscope. Time-lapse imaging revealed that although photo-conversion of single mEosFP molecules can be rapid in terms of live-cell imaging it involves a progressive enrichment of red fluorescent molecules over green species. The fluorescence of photo-converted cells thus progresses through intermediate shades ranging from green to red. The time taken for complete conversion to red fluorescence depends on protein expression level within a cell and the quality of the focusing lens used to deliver the illuminating beam. Three easily applicable methods for obtaining information on fluorescent intensity and colour are provided as a means of ensuring experimental repeatability and data quantification, when using mEosFP and similar photo-convertible proteins. CONCLUSION: The mEosFP::FABD-mTn probe retains all the imaging qualities associated with the well tested GFP::mTn probe while allowing for non-invasive, regional photo-conversion that allows colour based discrimination within a living cell. Whereas a number of precautions should be exercised in dealing with photo-convertible probes, mEosFP::FABD-mTn is a versatile live imaging tool for dissecting the organization and activity of the actin cytoskeleton in plants.
September 2008
Advances in fluorescent protein-based imaging for the analysis of plant endomembranes
Plant Physiol. 2008 Aug;147(4):1469-81.
Held MA, Boulaflous A, Brandizzi F.
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312, USA.
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312, USA.
An exciting new era for live cell imaging of plant endomembranes was ushered in just over 10 years ago when Jim Haseloff and colleagues reported on the removal of a cryptic intron in the sequence of wild-type GFP for successful expression in plant cells (Haseloff et al., 1997). Haseloff's success was quickly welcomed by labs around the globe; by targeting GFP to secretory organelles, scientists could now bring to light the secret life of plant endomembranes. The plant endomembranes comprise several organelles functionally interlinked for the production and transport of secretory compounds, such as proteins, lipids, and sugars. Most of the secretory compounds are synthesized in the endoplasmic reticulum (ER) and then transported to the Golgi apparatus to be sorted for transport to distal compartments such as the plasma membrane and vacuoles. A forward transport of secretory molecules from the ER is counterbalanced by a retrograde flow from distal compartments that allows membrane homeostasis and communication with the cell's surroundings. Fluorescent protein technology applied to live cell imaging of plant endomembranes has aided immensely in providing subcellular markers for the study of the complex spatial and temporal relationships among secretory organelles. Live cell imaging is now moving forward to novel challenges. With the integration of genomic, transcriptomic, and proteomic techniques, we begin to collect data on the putative functions of plant genes; we need to understand the intricate relationships among thousands of proteins. To complete the puzzle, we must understand how the pieces fit together; we must define the functions of gene products. Important questions include: When are our proteins synthesized? Where are they targeted? With what elements do they interact? Advances in the development of optical imaging at the cellular level now offer the exciting opportunity to answer these questions.
In this Update, we discuss several state-of-the-art applications of GFP imaging and how these techniques have been used to answer critical biological questions, with a focus on plant endomembrane trafficking.
In this Update, we discuss several state-of-the-art applications of GFP imaging and how these techniques have been used to answer critical biological questions, with a focus on plant endomembrane trafficking.
August 2008
Diversity and evolution of coral fluorescent proteins
PLoS ONE. 2008 Jul 16;3(7):e2680.
Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, Beltran-Ramirez V, Miller DJ, Wiedenmann J, Salih A, Matz MV.
Section of Integrative Biology, University of Texas at Austin, Austin, Texas, United States of America.
Section of Integrative Biology, University of Texas at Austin, Austin, Texas, United States of America.
GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.
May 2008
Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations
Plant J. 2008 Feb;53(4):610-35.
Lalonde S, Ehrhardt DW, Loqué D, Chen J, Rhee SY, Frommer WB.
Carnegie Institution, 260 Panama Street, Stanford, CA 94305, USA. slalonde@stanford.eduHomotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies. Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics. In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen (2008; this issue, pp. 597-609). In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces.
April 2008
Colocalization of fluorescent markers in confocal microscope images of plant cells
Nat Protoc. 2008;3(4):619-28
French AP, Mills S, Swarup R, Bennett MJ, Pridmore TP.
Centre for Plant Integrative Biology, Main Building, University of Nottingham, Sutton Bonington, LE12 5RD, UK.
Centre for Plant Integrative Biology, Main Building, University of Nottingham, Sutton Bonington, LE12 5RD, UK.
This protocol describes the steps needed to perform quantitative statistical colocalization on two-color confocal images, specifically of plant cells. The procedure includes a calibration test to check the chromatic alignment of the confocal microscope. A software tool is provided to calculate the Pearson and Spearman correlation coefficients ('Pearson-Spearman correlation colocalization' ImageJ plug-in) across regions of interest within the image. Steps are included to help the user practice using the software. The result is a quantitative estimate of the amount of colocalization in the images. Manual masking takes about 1-15 min per image, depending on the detail required, and calculating the correlation coefficients is almost instantaneous. Examples of suitable dyes for such two-color colocalization include Oregon Green or Alexa Fluor 488 dyes in the green range (excited with 488-nm laser line) and Alexa Fluor 555 dye in the red range (excited with 543-nm laser line).
March 2008
LEDs for Fluorescence Microscopy
Biophotonics International . 2008 Feb
James Beacher
CoolLED <http://www.coolled.com/precisexcite/>
CoolLED <http://www.coolled.com/precisexcite/>
" ...... recent advances driven by mass market applications for LED technology, such as domestic and automotive lighting, are making it possible for LEDs to replace lamps as an excitation source for fluorescence microscopy. Thus, biologists can benefit from the advantages of these light sources......"
Excerpt: Biophotonics International © Laurin Publishing Co. Inc.
Excerpt: Biophotonics International © Laurin Publishing Co. Inc.
Feb 2008
Cortical microtubule arrays in the Arabidopsis seedling
Curr Opin Plant Biol. 2008 Feb;11(1):94-8.
Lucas J, Shaw SL.
Department of Biology, Indiana University, Bloomington, IN 47405, United States.
Department of Biology, Indiana University, Bloomington, IN 47405, United States.
Advances in live-cell imaging technology have provided an unprecedented look at the dynamic behaviors of the plant microtubule cytoskeleton. Recent studies revisit the classic question of how plants create cell shape through the patterned construction of the cell wall. Visualization of the cellulose synthase complex traveling in the plasma membrane has brought a watershed of new information about cellulose deposition. Observation of the cellulose synthase complex tracking precisely over the underlying cortical microtubules has provided clear evidence that the microtubule array pattern serves as a spatial template for cellulose microfibril extrusion. Understanding how the microtubules are organized into specific array patterns remains a challenge, though new ideas are arising from genetic and cell biological studies. Long-term time-lapse observations of the microtubule arrays in light-grown hypocotyl cells have revealed a striking process of microtubule patterning possibly linked to the creation of polylamellate cell walls.
Jan 2008
The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.
Plant Physiol. 2007 Dec;145(4):1090-9.
Ohad N, Shichrur K, Yalovsky S.
Department of Plant Sciences, Tel-Aviv University, Tel-Aviv 69978, IsraelUSA.
Department of Plant Sciences, Tel-Aviv University, Tel-Aviv 69978, IsraelUSA.
" In this Update, we first discuss the principles of BiFC and its major advantages and disadvantages. We then describe the adaptation of BiFC to plant systems, provide practical suggestions for its use, and review protein-protein interactions that have been identified and confirmed in plants using this technique. Finally, additional potential exploitations of BiFC are discussed."
Dec 2007
Advances in fluorescent protein technology
J Cell Sci. 2007 Dec 15;120(Pt 24):4247-60.
Shaner NC, Patterson GH, Davidson MW. The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Current fluorescent protein (FP) development strategies are focused on fine-tuning the photophysical properties of blue to yellow variants derived from the Aequorea victoria jellyfish green fluorescent protein (GFP) and on the development of monomeric FPs from other organisms that emit in the yellow-orange to far-red regions of the visible light spectrum. Progress toward these goals has been substantial, and near-infrared emitting FPs may loom over the horizon. The latest efforts in jellyfish variants have resulted in new and improved monomeric BFP, CFP, GFP and YFP variants, and the relentless search for a bright, monomeric and fast-maturing red FP has yielded a host of excellent candidates, although none is yet optimal for all applications. Meanwhile, photoactivatable FPs are emerging as a powerful class of probes for intracellular dynamics and, unexpectedly, as useful tools for the development of superresolution microscopy applications.
Nov 2007
Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2
New Phytol. Nov 20; [Epub ahead of print]
Wang YS, Yoo CM, Blancaflor EB. Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA
* The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). * To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. * The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. * This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.
October 2007
Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex.
Plant J. 2007 Oct 11
Konopka CA, Bednarek SY. Program in Cell and Molecular Biology and Department of Biochemistry, University of Wisconsin – Madison, 433 Babcock Drive, Madison, WI 53706, USA.
Live-cell microscopy imaging of fluorescent-tagged fusion proteins is an essential tool for cell biologists. Total internal reflection fluorescence microscopy (TIRFM) has joined confocal microscopy as a complementary system for the imaging of cell surface protein dynamics in mammalian and yeast systems because of its high temporal and spatial resolution. Here we present an alternative to TIRFM, termed variable-angle epifluorescence microscopy (VAEM), for the visualization of protein dynamics at or near the plasma membrane of plant epidermal cells and root hairs in whole, intact seedlings that provides high-signal, low-background and near real-time imaging. VAEM uses highly oblique subcritical incident angles to decrease background fluorophore excitation. We discuss the utilities and advantages of VAEM for imaging of fluorescent fusion-tagged marker proteins in studying cortical cytoskeletal and membrane proteins. We believe that the application of VAEM will be an invaluable imaging tool for plant cell biologists.