Brighter reporter genes from multimerized fluorescent proteins.

If low signal-to-noise ratios in GFP assays have you singing a sad tune, Genové et al. (p. 814) can offer a solution. Recognizing that GFP fluorescence can be almost undetectable when reporters are used downstream of relatively weak promoters or during detection in problematic tissues such as the CNS, the authors investigated whether tandem repeats of fluorescent proteins could yield quantitative data in reporter gene assays. In search of the best readout, Genové et al. compared fluorescence intensity of one, two, or three copies of EGFP, EYFP, and Venus. Although a significant improvement in detection was evidenced with the trimeric EGFP, six tandem copies revealed fluorescence levels similar to the one-copy construct. Trimeric EYFP showed a similar improvement in signal, as both an IRES-translated protein and as a protein fusion with Fos. However, the brightest results involved Venus, a highly optimized EYFP variant. The trimeric Venus construct displayed a 12-fold higher fluorescence output than a single EFYP, and even after 5 minutes of constant illumination remains severalfold brighter. Based on this work, the prospects of previously problematic reporter assays should brighten considerably.</p

Requirement of the C-terminal portion of PpSec16 for tER localization of PpSec12 in both <i>P. pastoris</i> and <i>S. cerevisiae</i>.

<p>(A) As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g001" target="_blank">Fig. 1</a>, <i>P. pastoris</i> cells expressed PpSec12-GG from the endogenous promoter plus untagged Sec12 from the <i>AOX1</i> promoter, resulting in a high total level of PpSec12 expression. In the same cells, a truncated version of PpSec16 lacking residues 1967–2550 was tagged with GFP and overexpressed as a second copy using the <i>AOX1</i> promoter. PpSec16(Δ1967–2550)-GFP was found in punctate tER sites. By contrast to the result obtained when full-length PpSec16 was expressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g001" target="_blank">Fig. 1</a>), PpSec12-GG was found in the general ER. (B) <i>S. cerevisiae</i> cells expressed the same truncated version of PpSec16 as in (A), except that the protein was tagged with YFP and was expressed under control of the <i>GAL10</i> promoter. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g002" target="_blank">Fig. 2B</a>, ScSec13-CFP and PpSec12-GG were also expressed in these cells. By contrast to the result obtained when full-length PpSec16 was expressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g002" target="_blank">Fig. 2B</a>), PpSec12-GG was found in the general ER. Scale bars, 2 µm.</p

Viability of <i>S. cerevisiae</i> cells carrying <i>PpSEC12</i> as the only gene from the <i>SEC12</i> family.

<p>A plasmid shuffle was performed in <i>sed4</i>Δ <i>sec12</i>Δ cells, with <i>SED4</i> in a <i>URA3</i> plasmid plus either <i>ScSEC12</i> (top row) or <i>PpSEC12</i> (middle row) in a <i>LEU2</i> plasmid. Both strains grew on rich media (YPD) and also on media containing 5-FOA, indicating that <i>PpSEC12</i> could replace <i>ScSEC12</i> even in the absence of <i>SED4</i>. As a control, <i>sec12</i>Δ cells carrying <i>ScSEC12</i> on a <i>URA3</i> plasmid were plated on the same media, and no growth was seen in the presence of 5-FOA.</p

Colocalization of mammalian Sec12 with Sec16A at tER sites.

<p>(A) U2OS human osteosarcoma cells were subjected to immunofluorescence as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone.0031156-Bhattacharyya2" target="_blank">[28]</a> using commercial antibodies against human Sec12 and Sec16A. Scale bar, 2 µm. (B) Plasmids encoding YFP-tagged full-length human Sec12 and CFP-tagged full-length Sec16B were co-transfected into U2OS cells. The cells were imaged at 16 h post-transfection, a time point that yielded relatively low expression levels. Scale bar, 2 µm. (C) HeLa cells were transfected where indicated with plasmids encoding either monomeric GFP fused to a C-terminal region (“CTR”) of human Sec16A (residues 1909–2332), or an N-terminally triple-FLAG-tagged cytosolic domain (“Cyt”) of human Sec12 (residues 1–386). At 24 h post-transfection, the cells were lysed and the lysate was subjected to immunoprecipitation (“IP”) with anti-FLAG antibody. The immunoprecipitated material and 5% of the lysate (“5% Input”) was subjected to SDS-PAGE followed by immunoblotting with either anti-FLAG or anti-GFP antibody.</p

Effect of deleting nonessential PpSec16 regions on PpSec12 localization in <i>P. pastoris</i>.

<p>(A) Diagram of the domain organization of PpSec16. Shading indicates conserved regions while hatch marks indicate an essential region. CCD, central conserved domain; Q, glutamine-rich region; CTR, C-terminal conserved region. Deletions introduced by gene replacement are indicated. None of these deletions affected PpSec12 localization. (B) Representative images of PpSec12-GG localization in <i>P. pastoris</i> cells carrying the indicated deletions in PpSec16. Scale bar, 2 µm.</p

Biochemical interaction of the C-terminal portion of PpSec16 with the cytosolic domain of PpSec12.

<p>Glutathione-agarose beads were incubated with a bacterial lysate from cells expressing either GST alone, or GST fused to the C-terminal residues 1960–2550 of PpSec16. Sufficient lysate was used to saturate the binding sites on the glutathione-agarose. A second incubation was then performed with sub-saturating amounts of a bacterial lysate from cells expressing a hexahistidine-tagged version of the cytosolic domain of PpSec12 (PpSec12(cyto)-His6). The beads were centrifuged, and the unbound material in the supernatant was collected. Bound protein was eluted from the beads with 100 mM glutathione. I, input (100% relative to other lanes); U, unbound; B, bound. PpSec12(cyto)-His6 bound to the beads carrying GST-PpSec16(1960–2550) but not to the beads carrying GST alone.</p

Requirement of the C-terminal portion of PpSec16 for recruiting overexpressed PpSec12 to tER sites.

<p>As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g001" target="_blank">Fig. 1</a>, PpSec16-GFP was overexpressed in <i>P. pastoris</i> cells overexpressing PpSec12, except that deletions were introduced as indicated near the C-terminus of PpSec16. Two hundred randomly chosen cells from each of the indicated <i>P. pastoris</i> strains were examined by immunofluorescence and scored for colocalization of PpSec12-GG with PpSec16-GFP. Cells in which nearly all of the PpSec12-GG overlapped with PpSec16-GFP were scored as having strong colocalization (+). Cells in which PpSec12-GG showed clear concentration in the PpSec16-GFP puncta but also showed prominent staining outside of these puncta were scored as having partial colocalization (+/−). Cells showing no visible concentration of PpSec12-GG in the PpSec16-GFP puncta were scored as having no colocalization (−). Colocalization was virtually abolished by deleting the entire C-terminal portion of PpSec16, and was strongly reduced by deleting only the C-terminal conserved region (CTR).</p

Recruitment of overexpressed PpSec12 to tER sites in <i>P. pastoris</i> by simultaneous overexpression of PpSec16.

<p>PpSec12 was tagged with the Glu-Glu epitope (PpSec12-GG) by gene replacement, and a second untagged copy of PpSec12 was expressed in the same cells using the methanol-inducible <i>AOX1</i> promoter, resulting in a high total level of PpSec12 expression. Top row: in a strain overexpressing PpSec12, PpSec16 was expressed at normal levels after being tagged by gene replacement with GFP. A small fraction of the PpSec12-GG colocalized with PpSec16-GFP, but most of the PpSec12-GG was in the general ER as indicated by the prominent nuclear envelope signal. Bottom row: in a strain overexpressing PpSec12, PpSec16-GFP was overexpressed as a second copy using the <i>AOX1</i> promoter. Most of the PpSec12-GG colocalized with PpSec16-GFP in exaggerated tER sites. Scale bar, 2 µm.</p

A Rapidly Maturing Far-Red Derivative of DsRed-Express2 for Whole-Cell Labeling

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy

A Rapidly Maturing Far-Red Derivative of DsRed-Express2 for Whole-Cell Labeling

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy