1.2 Comparison
of FP Variants in Yeast
Many FP variants now exist, and we tested a few promising ones to
see whether this would improve the signal: sfGFP, mNG2, eGFP,
muGFP, and Envy. sfGFP is the superfolder GFP and muGFP is
one of its ultrastable monomeric variants [74]. mNG is a bright and
stable variant derived from a Branchiostoma lanceolatum fluorescent protein [75], and Envy is an FP that performs particularly well
in S. cerevisiae [76]. The cDNA coding for these fluorescent proteins was cloned into pET296, replacing the yeGFP tag, such that
they are expressed as monomeric variants lacking NLS
(MCP-1xFP). The plasmids were then transformed into yeast cells
expressing the DOA1 gene tagged with 24xMBSV6 as described in
[58]. Cells were then grown, fixed, and observed under the microscope (Fig. 2a–c). We found that mRNAs were undetectable with
MCP-1xeGFP, MCP-1xsfGFP, and MCP-1xmuGFP, while they
were nicely visible with the MCP-1xEnvy and MCP-1xmNG2
(with best results for MCP-1xEnvy).
1.3 Comparison
of Lowand High-Affinity MCP
Variants in Yeast
The original MS2 system was designed with high-affinity variants of
both the MBS and the MCP [40]. Indeed, this MCP variant carried
the V29I mutation that enhances binding stability by 5–10-fold
[77]. Since a too high affinity of the MCP-MBS interaction was
found to cause artifacts in yeast [58], we tested the effect of
reverting the V29I mutation. A plasmid expressing MCP-I29V2xyeGFP was generated and transformed in yeast cells expressing
the DOA1 gene tagged with MBV6. No signals corresponding to
single mRNAs could be detected, suggesting that the affinity of the
MS2V6/MCP-I29V is probably too low for an efficient mRNA
detection. Overall, MBSV6/MCP-V29I appears to have the best
affinity compromise to allow for an artifact-free mRNA detection in
yeast.
MCP-NLS-GFPEnvy
MCP-NLS-2xyeGFP
MCP-NLS-eGFP
a
b
c
Fig. 2 Comparison of MCP-FP variants in yeast. Images are from yeast cells expressing the DOA1 gene tagged
with 24xMBSV6 transformed with (a) MCP-NLS-2xyeGFP plasmid, (b) MCP-NLS-GFPEnvy plasmid, (c) MCPNLS-eGFP plasmid. Panels are maximum intensity projections of Z-stacks. Scale bar 5 μm
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of FP Variants in Yeast
Many FP variants now exist, and we tested a few promising ones to
see whether this would improve the signal: sfGFP, mNG2, eGFP,
muGFP, and Envy. sfGFP is the superfolder GFP and muGFP is
one of its ultrastable monomeric variants [74]. mNG is a bright and
stable variant derived from a Branchiostoma lanceolatum fluorescent protein [75], and Envy is an FP that performs particularly well
in S. cerevisiae [76]. The cDNA coding for these fluorescent proteins was cloned into pET296, replacing the yeGFP tag, such that
they are expressed as monomeric variants lacking NLS
(MCP-1xFP). The plasmids were then transformed into yeast cells
expressing the DOA1 gene tagged with 24xMBSV6 as described in
[58]. Cells were then grown, fixed, and observed under the microscope (Fig. 2a–c). We found that mRNAs were undetectable with
MCP-1xeGFP, MCP-1xsfGFP, and MCP-1xmuGFP, while they
were nicely visible with the MCP-1xEnvy and MCP-1xmNG2
(with best results for MCP-1xEnvy).
1.3 Comparison
of Lowand High-Affinity MCP
Variants in Yeast
The original MS2 system was designed with high-affinity variants of
both the MBS and the MCP [40]. Indeed, this MCP variant carried
the V29I mutation that enhances binding stability by 5–10-fold
[77]. Since a too high affinity of the MCP-MBS interaction was
found to cause artifacts in yeast [58], we tested the effect of
reverting the V29I mutation. A plasmid expressing MCP-I29V2xyeGFP was generated and transformed in yeast cells expressing
the DOA1 gene tagged with MBV6. No signals corresponding to
single mRNAs could be detected, suggesting that the affinity of the
MS2V6/MCP-I29V is probably too low for an efficient mRNA
detection. Overall, MBSV6/MCP-V29I appears to have the best
affinity compromise to allow for an artifact-free mRNA detection in
yeast.
MCP-NLS-GFPEnvy
MCP-NLS-2xyeGFP
MCP-NLS-eGFP
a
b
c
Fig. 2 Comparison of MCP-FP variants in yeast. Images are from yeast cells expressing the DOA1 gene tagged
with 24xMBSV6 transformed with (a) MCP-NLS-2xyeGFP plasmid, (b) MCP-NLS-GFPEnvy plasmid, (c) MCPNLS-eGFP plasmid. Panels are maximum intensity projections of Z-stacks. Scale bar 5 μm
126
Xavier Pichon et al.