Biology subjects

Ruijtenberg, S.

Publications and source records attributed to Ruijtenberg, S..

5 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↗

bioRxiv2025

The First of Us: Ophiocordyceps use a novel scramblase-binding peptide to manipulate zombie ants

Parasite-adaptive manipulation of behavior is a widespread natural phenomenon. While Ophiocordyceps zombie fungi are well-known for hijacking ant behavior to increase their fitness, functionally characterizing the biomolecules involved in behavior manipulation remains difficult in these non-model organisms. To circumvent this problem, we have adopted the powerful genetics toolbox of Caenorhabditis elegans to identify molecular targets and neurophysiological effects of candidate Ophiocordyceps effectors. With this approach, we discovered a novel cysteine-rich, small secreted fungal peptide that binds to well-conserved calcium-dependent scramblase channels in neurons, particularly those associated with sensory tissues. This binding suppressed nematode motor coordination and dampened ant olfactory systems vital for communication. Our findings are the first to directly connect an Ophiocordyceps effector with its extended phenotype, while demonstrating the neuroethological role of Scramblase-1.

molecular biology↗

bioRxiv2024

Localized translation of erm-1 contributes to ERM-1 function in the C. elegans embryo

Translation of mRNAs into proteins is a key step in decoding the information stored in the genome. Localized translation ensures that proteins are expressed where needed, which is important for cell-specific protein expression, the establishment of cellular protein gradients, and the creation of protein hotspots within different cellular compartments. Although localized translation is believed to be important for cell fate determination and organismal development, our understanding of localized translation in the context of living animals is limited, as few methods that allow direct visualization and measurement of translation exist. We adapted the SunTag-based single-molecule translation imaging system for use in Caenorhabditis elegans and showed the dynamics and importance of localized erm-1 translation during development. We found erm-1 translation to be enriched at the plasma membrane, overlapping with the localization and function of the encoded membrane-cytoskeleton linker ERM-1. Re-localizing erm-1 translation to nuclear pores disrupts the function of ERM-1 protein, particularly its role in linking the actin cytoskeleton to the membrane. Our work demonstrates the power of translation imaging and highlights the importance of localized translation in C. elegans development.

developmental biology↗

bioRxiv2022

The mIAA7 degron improves auxin-mediated degradation in C. elegans

Auxin-inducible degradation (AID) is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the AID system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in C. elegans, as previously reported in human cells. We tested depletion of a series of proteins with various sub-cellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for five out of six proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for two of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system (C.e.AID2), which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the AID system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

molecular biology↗

bioRxiv2019

mRNA structural dynamics shape Argonaute-target interactions

Small RNAs (such as miRNAs, siRNAs and piRNAs) regulate protein expression in a wide variety of biological processes and play an important role in cellular function, development and disease. Association of small RNAs with Argonaute (AGO) family proteins guide AGO to target RNAs, generally resulting in target silencing through transcriptional silencing, translational repression or mRNA degradation. Here we develop a live-cell single-molecule imaging assay to simultaneously visualize translation of individual mRNA molecules and their silencing by human AGO2-siRNA complexes. We find that siRNA target sites are commonly masked in vivo by RNA secondary structures, which inhibit AGO2-target interactions. Translating ribosomes unmask AGO2 binding sites, stimulating AGO2-target interactions and promoting mRNA degradation. Using a combination of mathematical modeling and experiments, we find that mRNA structures are highly heterogeneous and continuously refolding. We show that structural dynamics of mRNAs shape AGO2-target recognition, which may be a common feature controlling mRNA-protein interactions.

molecular biology↗