Paper of the Month

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.

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.edu

Homotypic 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.

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/>

" ...... 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.
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.

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.

" 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.