mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities

<div><p>Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in <em>Escherichia coli</em> and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in <em>E. coli</em>. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct.</p> </div

(f-)PALM/STORM comparison of mMaple, mClavGR2 and mEos2.

<p>(<b>A–C</b>) Images of Δ<i>cheW E.coli</i> expressing CheW fusion proteins at L-arabinose concentrations optimal for swarming. Images contain (<b>A</b>) 1086 mMaple-CheW localizations (<b>B</b>) 694 mClavGR2-CheW localizations and (<b>C</b>) 229 mEos2-CheW localizations. (<b>D</b>) The mean number of photons emitted by each construct per photoconversion event (error is standard error, <i>N</i> = 3 independent measurements from distributions consisting of 4,000–32,000 localizations). Scale bars are 500 nm. (<b>E</b>) Distribution of the number of localizations observed for Δ<i>cheW E. coli</i> cells containing CheW fusions to mMaple, mClavGR2, and mEos2. Greater than 96% of cells expressing either mEos2- or mClavGR2-CheW fusions have less than 500 localizations (boxed region), whereas greater than 50% of cells expressing mMaple-CheW fusions have more than 500 localizations.</p

mMaple has improved <i>in vivo</i> brightness and enables 3D-SIM reconstructions.

<p>(<b>A</b>) Δ<i>cheW E.coli</i> expressing mMaple-CheW and a zoom (<b>B</b>) of the polar region of the cell denoted by the boxed region in (A). (<b>C</b>) One 125 nm slice of the 3D-SIM reconstruction of a Δ<i>cheW E. coli</i> expressing GFP-CheW. Red represents fluorescence of the membrane-specific dye FM4-64 and green represents FP fluorescence. Scale bars are 500 nm (A, C) and 100 nm (B).</p

Advantages and limitations of mMaple.

<p>Advantages and limitations of mMaple.</p

(f-)PALM/STORM characterization of the number of observed localizations and proteins per cell.

<p>(<b>A–B</b>) (f-)PALM/STORM images of fixed <i>E. coli</i> expressing cytoplasmic (<b>A</b>) mMaple (<i>N</i> = 1696 localizations), or (<b>B</b>) mEos2 (<i>N</i> = 472 localizations). Localizations are represented as normalized 2D Gaussian peaks with widths given by their theoretical localization precisions (left panels) and plotted as small markers grouped into clusters with adjacent spacing of 30 nm or less (right panels). Individual protein localizations are shown in grey whereas closely spaced localizations (<30 nm) are grouped into clusters of the same color (right panels). The bright field and conventional fluorescence images are shown for comparison (left panels, left and right inset respectively). Scale bars are 500 nm. (<b>C</b>) Average number of localizations per cell for each cytoplasmically expressed pcFP. (<b>D</b>) The distribution of cluster sizes (<30 nm interlocalization spacing) for cytoplasmically expressed pcFPs. (<b>E</b>) Average number of cytoplasmically expressed proteins per cell. Rather than counting each localization as a single molecule, we count each cluster of localizations (localizations spaced <30 nm) as a single protein. The dotted lines in (A–B) denote the <i>E. coli</i> cell boundary. Scale bars are 500 nm and 50 nm (zooms). Zooms in (A–B) show possible reversible photoswitching events of single proteins. Error is the standard deviation (N = 20 cells (mMaple), N = 17 cells (mClavGR2), N = 16 cells (mEos2)). The large error bars are primarily due to variation in protein expression between cells.</p

pcFP fusions expressed in mammalian cells.

<p>(<b>A</b>–<b>C</b>) Representative widefield fluorescence images of HeLa cells transfected with plasmids encoding either mMaple-actin (<b>A</b>), mEos2-actin (<b>B</b>), or mClavGR2-actin (<b>C</b>). (<b>D</b>) Flow cytometric analysis of the green fluorescence for HeLa cells transfected with the same plasmids used in A–C. A total of 7000 fluorescent cells were analyzed in each experiment. Relative median fluorescence intensity after excitation and emission correction is: 0.68 (mMaple), 0.77(mClavGR2) and 1.00 (mEos2). (<b>E</b>–<b>G</b>) Representative images of HeLa cells transfected with actinin fusions of mMaple (<b>E</b>), mEos2 (<b>F</b>), and mClavGR2 (<b>G</b>). (<b>H</b>) Flow cytometric analysis of the green fluorescence from HeLa cells transfected with the plasmids used in E–G. Relative corrected median fluorescence intensity is: 1.03 (mMaple), 1.25 (mClavGR2) and 1.00 (mEos2).</p

Properties of mMaple and related variants.

a<p>Extinction coefficent (mM⁻¹ cm⁻¹) at peak absorbance wavelength in PBS (pH 7.4). Value in parentheses was determined at pH 4 and pH 10, respectively.</p>b<p>Product of ε and Φ in mM⁻¹ cm⁻¹. For comparison, the brightness of EGFP and mCherry are 34 mM⁻¹ cm⁻¹ and 16 mM⁻¹ cm⁻¹, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051314#pone.0051314-Shaner2" target="_blank">[53]</a>.</p>c<p>Data from McKinney <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051314#pone.0051314-Mckinney1" target="_blank">[30]</a>.</p