Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

Abstract

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 wildtype 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.

Combined Bimolecular Fluorescence Complementation and Förster Resonance Energy Transfer Reveals Ternary SNARE Complex Formation in Living Plant Cells

“…The drawbacks of using the BiFC system to monitor protein-protein interactions were discussed above and elsewhere (Hu et al, 2002;Held et al, 2008;Kerppola, 2008) and include, among others, the irreversibility of complex formation, which may stabilize specific as well as unspecific protein-protein interactions. Another implication of the irreversibility of BiFC complex formation is that, with respect to two of the partners, ternary protein complexes can only be studied statically by BiFC-FRET analysis.…”

“…A documented weakness of the BiFC assay is that the complemented YFP, once formed, does not disassemble, which stabilizes the entire protein complex (Hu et al, 2002;Held et al, 2008;Kerppola 2008). To exclude the possibility that protein complex stabilization by the BiFC system caused the observed clustering phenomenon, we coexpressed the three SNARE partners tagged with distinct full-size monomeric fluorophores (CrFP-AtVAMP722, mYFP-AtPEN1, and AtSNAP33-mCherry; for mYFP, Zacharias et al, 2002; for mCherry, Shu et al, 2006) in Arabidopsis epidermal cells.…”

“…Fluorescence microscopy allows imaging and tracing of these proteins in real time in living cells. However, the resolution of a conventional epifluorescence microscope or a confocal laser scanning microscope is insufficient to resolve distances smaller than approximately 200 nm between biological macromolecules and to demonstrate their vicinity at the molecular scale (Bhat et al, 2006;Held et al, 2008).…”

Membrane Trafficking: Intracellular Highways and Country Roads

“…The Staehelin and Kang (2008) Update reviews state-of-the-art, highresolution capabilities in obtaining structural definitions in three dimensions for individual organelles and, in particular, reconstruction of organelles that are undergoing dynamic changes. This is followed by the Held et al (2008) Update on the prowess of what combinations of fluorescence-based microscope and live cell imaging can achieve for spatial and functional assignments for specific molecules within the entire endomembrane system. The Rojo and Denecke (2008) and Robinson et al (2008) Updates examine the most recently defined players in the secretory and endosomal pathways and how these may be used to interpret, reconcile, or extend prior results.…”

mEosFP-Based Green-to-Red Photoconvertible Subcellular Probes for Plants

“…Numerous subcellular targeted FP probes have been created for live imaging of plants at the organ, tissue, cell, subcellular, and suborganeller levels. Several dedicated Web-based educational resources have been developed to provide comprehensive and frequently updated information on subcellular targeted FP probes for plants (Mathur, 2007;Held et al, 2008;Mano et al, 2008Mano et al, , 2009). …”