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[Preprint]. 2024 Jun 13:2024.06.11.598534.
doi: 10.1101/2024.06.11.598534.

Comparative analysis of new, mScarlet-based red fluorescent tags in Caenorhabditis elegans

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Comparative analysis of new, mScarlet-based red fluorescent tags in Caenorhabditis elegans

Wen Xi Cao et al. bioRxiv. .

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Abstract

One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode C. elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and maturation time of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3 and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4, encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1, a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the panneuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP.

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Figures

Figure 1.
Figure 1.. Analogous CRISPR-mediated insertion of fluorophores at the N-terminus of the GOLG-4 locus.
A: WormBase genome browser snapshot of the golg-4 locus and the site of CRIPSR fluorophore insert. Codon-optimized fluorophores are schematized below: derivatives of mScarlet – mScarlet3 and mScarlet-I3 – show amino acid mutations relative to mScarlet in black. GFP cloned from pPD95.75 expression plasmid is also included for comparison. Introns are also annotated and were added as recommended by the MPI C. elegans codon adapter (Redemann et al. 2011). B: Representative expression of red fluorophore-tagged GOLG-4 in the head is shown. Average fluorescence intensity of the pharyngeal terminal bulb of individual animals is quantified and plotted on the right, and the median values are indicated.
Figure 2.
Figure 2.. Expression of fluorophore-tagged GOLG-4 through embryonic development.
Representative images of single focal plane through the middle of each embryo are shown, comparing the expression dynamics between the mScarlet variants and GFP. Strains are those shown in Figure 1. For comparison to GFP, imaging setting were chosen to match the fluorescence intensity of GFP::GOLG-4 to that of mScarlet-I3::GOLG-4 in the adult, quantified in Figure 1. Eight embryo developmental stages from the 4-cell stage to the 4-fold stage, spanning most the embryogenesis, are represented in columns and compared across fluorophores, in rows. Embryo stages were determined by DIC (not shown), and eggshells are traced in dashed lines.
Figure 3.
Figure 3.. Expression of pks-1::SL2::GFP::H2B and pks-1::SL2::mScarlet-I3::H2B during embryo development.
A: WormBase genome browser snapshot of the psk-1 locus and the site of CRISPR-mediated insertion at the C-terminus, after the stop codon. Schematic of fluorophore constructs are shown below. B: Expression of fluorophores in the L3 larval head and midbody. Both GFP and mScarlet-I3 reporters are expressed in the nuclei of CANL/R and intestinal (int) cells. Lower expression is also observed in several nuclei within the pharynx. An additional nucleus in the head, presumed to be the head mesodermal cell (HMC), is also labeled. Images are of a single plane containing one of the two CAN nuclei in focus. C: Expression of fluorophores in various stages of the embryo. Focal plane containing one of the two nuclei is shown for each embryo. Fluorescent signal can be visualized from the comma stage onwards, for both GFP and mScarlet-I3 constructs (red arrowheads). Note the pks-1 reporter constructs specifically label CAN nuclei at these embryonic stages shown.
Figure 4.
Figure 4.. Expression of fluorophore-tagged UNC-75, comparing GFP to mScarlet-I3.
A: WormBase genome browser snapshot of the unc-75 locus, showing the site of CRISPR-mediated insertion at the N-terminus. Fluorophore constructs are the same as in previous figures. The fluorophores were inserted at the N-terminus to avoid disruption of a C-terminal nuclear localization sequence (Loria et al. 2003). B: Expression of fluorophore-tagged UNC-75 in the adult worm. Images are maximum intensity projections through the head, midbody and tail sections, capturing dense regions of neuron nuclei. C: Expression of fluorophore-tagged UNC-75 at five stages in embryo development, determined by DIC. Single Z planes through the approximate midpoint of each embryo are shown, and eggshells are traced with dashed lines.

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