Biology subjects

Griffin, E. E.

Publications and source records attributed to Griffin, E. E..

3 recordsLinked to original sources

bioRxiv2026

Epitope-based labeling for improved live-imaging of endogenous proteins in C. elegans

Visualizing protein expression dynamics with high temporal resolution is essential for understanding how cells acquire specific fates and functions during development, where key decisions can occur within minutes. Conventional direct fluorescent tagging often fails to capture these rapid changes in protein expression due to the relatively slow fluorophore maturation time. Indirect epitope-based labeling strategies offer a promising alternative, yet only a limited number of these systems have been developed and used in the context of multicellular organisms. Here, we evaluate and combine four epitope-based indirect labeling systems for live-imaging of proteins in C. elegans: the SunTag, Frankenbody, MoonTag and AlfaTag systems. Each system uses a fluorescently labeled high-affinity single-chain antibody or nanobody to recognize short peptide epitopes fused to a protein of interest, enabling immediate visualization of newly synthesized proteins. We demonstrate that all four systems specifically label epitope-tagged endogenous proteins and show no detectable cross-reactivity when used in dual-color combinations, enabling simultaneous visualization of distinct proteins within the same embryo. In addition, we show that the SunTag system offers three major advantages over direct labeling: earlier detection of proteins, enhanced sensitivity through signal amplification (as illustrated by CAM-1) and less impact on the function (as demonstrated for ERM-1). Together, this expanded toolkit of epitope-based labeling systems offers many new opportunities for visualizing rapid protein dynamics and for dissecting how their dynamics drive cell fate decisions during development. SUMMARYThe development of epitope-labeling systems has improved live-imaging quality of proteins. Unfortunately, limited systems exist for multicellular organisms to study protein expression in the context of development. Here, we expand the epitope-labeling toolbox for C. elegans by combining SunTag or Frankenbody with MoonTag or AlfaTag. Our data indicates that these systems simultaneously visualize different endogenous proteins without cross-reactivity. Moreover, the SunTag system shows advantages over direct labeling: earlier detection, enhanced sensitivity through signal amplification and less impact on protein function. This expanded epitope-labeling toolbox in C. elegans provides opportunities for accurate visualization of different proteins that drive cell fate decisions. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/703904v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@449fe0org.highwire.dtl.DTLVardef@15c68cforg.highwire.dtl.DTLVardef@1e51ff8org.highwire.dtl.DTLVardef@196114d_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

PLK-1 regulates MEX-1 polarization in the C. elegans zygote

The one-cell C. elegans embryo undergoes an asymmetric cell division during which germline factors such as the RNA-binding proteins POS-1 and MEX-1 segregate to the posterior cytoplasm, leading to their asymmetric inheritance to the posterior germline daughter cell. Previous studies found that the RNA-binding protein MEX-5 recruits polo-like kinase PLK-1 to the anterior cytoplasm where PLK-1 inhibits the retention of its substrate POS-1, leading to POS-1 segregation to the posterior. In this study, we tested whether PLK-1 similarly regulates MEX-1 polarization. We find that both the retention of MEX-1 in the anterior and the segregation of MEX-1 to the posterior depend on PLK kinase activity and on the interaction between MEX-5 and PLK-1. Human PLK1 directly phosphorylates recombinant MEX-1 on 9 predicted PLK-1 sites in vitro, four of which were identified in previous phosphoproteomic analysis of C. elegans embryos. The introduction of alanine substitutions at these four PLK-1 phosphorylation sites (MEX-1(4A)) significantly weakened the inhibition of MEX-1 retention in the anterior, thereby weakening MEX-1 segregation to the posterior. In contrast, mutation of a predicted CDK1 phosphorylation site had no effect on MEX-1 retention or on MEX-1 segregation. MEX-1(4A) mutants are viable and fertile but display significant sterility and fecundity defects at elevated temperatures. Taken together with our previous findings, these findings suggest PLK-1 phosphorylation drives both MEX-1 and POS-1 polarization during the asymmetric division of the zygote.

developmental biology↗

bioRxiv2019

SapTrap assembly of C. elegans MosSCI transgene vectors

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans (Schwartz and Jorgensen 2016 Genetics, 202:1277-1288). Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

genetics↗