Abstract
BackgroundThe 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.ResultsA 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.ConclusionThe 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.
Highlights
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
Two fusion proteins have become widely accepted for visualizing F-actin in living cells: These are the Green Fluorescent Protein (GFP)::mTn [13] and the GFP::ABD2-FIMBRIN1 [14] constructs
Biolistic bombardment of onion epidermal cells with 1 μm diameter gold particles coated with the mEosFP-cytosolic DNA resulted in cells that fluoresced green upon illumination through an Endow GFP-LP filter cube (Figure 1A)
Summary
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. Two fusion proteins have become widely accepted for visualizing F-actin in living cells: These are the GFP::mTn [13] and the GFP::ABD2-FIMBRIN1 [14] constructs The former probe is composed of a modified GFP fused to the F-actin binding C-terminal 197 amino acids of the Talin gene from mouse [13]. While recent trends appear to prefer the xFP::FIM1-ABD2 fusion protein over the xFP::mTn probe, in our hands both probes can perform well provided care is taken to maintain certain baseline growth and development conditions Transgenic lines for both probes when stressed do display many of the artifacts highlighted in the studies of Ketelaar et al [19] and Holweg [21] and underscore the importance of rigorous observations in reporting changes in the actin cytoskeleton. Their perceived limitations and advantages aside, the availability of two independent probes highlighting F-actin is a useful development for plant biologists because it provides cross-checks on observations and lends greater credibility to studies on the actin cytoskeleton in plant cells
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