Bob Goldstein

Genetically encoded F-actin probes enable visualization of actin organization and dynamics in living cells, but their performance and effects on cellular function can vary by probe, fluorescent protein, and biological context. Here, we systematically compared three F-actin probes – F-tractin, LifeAct, and Utrophin – fused to mScarlet-I, mNeonGreen, or mStayGold in vivo , using Caenorhabditis elegans . We used single-copy transgenic strains and evaluated fluorescence intensity, photostability, developmental and reproductive outcomes, cytokinetic ring closure, and probe localization across diverse cellular contexts. mStayGold provided the greatest brightness and photostability, making it well suited for long-term time-lapse imaging, whereas mNeonGreen offers an alternative for applications requiring photobleaching. Most of the strains exhibited minimal detectable effects on development and cellular function and outperformed previously generated strains tested. Probe performance varied across tissues and developmental stages. F-tractin generally exhibited lower fluorescence intensity than LifeAct and Utrophin, particularly in embryos. Although the probes largely exhibited subcellular localization patterns consistent with phalloidin staining, differences in their labeling of specific F-actin structures were observed. Together, these results establish a practical framework for selecting genetically encoded F-actin probes for C. elegans , they suggest best candidates for other in vivo systems, and they highlight the importance of considering both fluorescent protein and actin-binding probe when designing live-cell imaging experiments. Significance Statement: This collection of genetically encoded F-actin probe strains in C. elegans , together with modular plasmids for generating customized probes, provides accessible resources for a broad range of imaging applications. All tested probes support improved retention of normal biological function, and vary in fluorescence intensity, photostability, and the labeling of some actin structures, revealing trade-offs that inform probe selection. Evaluating probes across developmental stages and cellular processes demonstrates that the optimal probe depends on the biological context and experimental goals.

Summary: Apical constriction shapes tissues in fundamental events including gastrulation and neural tube formation. Diverse actin regulators play key roles in shaping cells, but the spatial distribution of most actin regulators in apical constriction is unknown. Here, we describe a systematic approach revealing the endogenous localization of 49 actin regulators in C. elegans gastrulation – nearly every actin-binding protein (ABP) expressed in the early embryo – and we use this atlas to test mechanistic hypotheses. We found that many actin regulators localized basolaterally, dependent on the Arp2/3 complex; that linear actin assembly factors enriched apically; and that apically constricting cells had an extra requirement for membrane-cortical linkers present in all cells. These findings answer some key questions about how ABPs function in apical constriction, and they establish a rich resource for investigating actin organization throughout early C. elegans development and a systematic in vivo protein localization reference for the actin cytoskeleton community.

The simple nematode Caenorhabditis elegans has long served as a powerful genetic model system for studying muscle structure and function, with sarcomeres of the striated body wall muscle (BWM) that are considered largely homologous to those in vertebrates. However, we have observed a population of F‐actin in BWM separate from I‐bands and centered at the sarcomere M‐line, something not seen in vertebrate sarcomeres. Here, we investigate whether M‐line‐associated F‐actin is a true feature of worm muscle, or an artifact of sample preparation. Using thin filament components tagged with fluorescent proteins, we confirm that living C. elegans BWM has a traditional sarcomere organization with actin restricted to I‐bands, whereas two methods of fixation resulted in varying degrees of mislocalization of actin as well as barbed end capping protein/CapZ and tropomodulin. Additionally, we observe in live animals that α‐actinin‐rich dense bodies are flanked by discrete patches of CapZ, something we cannot observe in fixed animals, but which provides some support for a model in which dense bodies anchor thin filaments in a manner similar to vertebrate Z‐discs. Overall, our work highlights the susceptibility of thin filaments in worm BWM to fixation artifacts and the potential utility in studying sarcomere structure in live animals.

In asexual reproduction, meiosis must be bypassed or altered to maintain ploidy from mother to daughter without fertilization. Most of the ways meiosis can be modified to this end are expected to reduce heterozygosity within individuals; however, many asexual species are highly heterozygous. Asexual reproduction is especially common among species of microscopic, desiccation-tolerant animals such as rotifers, nematodes, and tardigrades, but the cellular and genetic mechanisms underlying asexual reproduction have not been definitively documented in any species of tardigrade. Here, we show that the asexual tardigrade Hypsibius exemplaris fails to complete the cell division of meiosis I, followed by a complete meiosis II-like division, and reproduction proceeds without detectable loss of heterozygosity. We used a combined cytological and genomic approach to characterize the mechanism of reproduction and pattern of allele inheritance in this species. Furthermore, we identified heterozygous variants in a subset of transcriptionally active genes consistent with loss of function in one allele, suggesting that maintained heterozygosity in this species allowed divergence between alleles over time. This work establishes the meiotic mechanism and inheritance pattern of reproduction in H. exemplaris, which provides a framework for interpreting genetic variation in this organism as a laboratory model. Additionally, our finding that meiosis is modified in H. exemplaris via a mechanism that maintains heterozygosity across the genome adds to a growing list of asexual animals that have evolved to reproduce clonally despite the expected long-term costs.

In asexual reproduction, meiosis must be bypassed or altered to maintain ploidy from mother to daughter without fertilization. Most of the ways meiosis can be modified to this end are expected to reduce heterozygosity within individuals; however, many asexual species are highly heterozygous. Asexual reproduction is especially common among species of microscopic, desiccation-tolerant animals such as rotifers, nematodes, and tardigrades, but the cellular and genetic mechanisms underlying asexual reproduction have not been definitively documented in any species of tardigrade. Here, we show that the asexual tardigrade Hypsibius exemplaris fails to complete the cell division of meiosis I, followed by a complete meiosis II-like division, and reproduction proceeds without detectable loss of heterozygosity. We used a combined cytological and genomic approach to characterize the mechanism of reproduction and pattern of allele inheritance in this species. Furthermore, we identified heterozygous variants in a subset of transcriptionally active genes consistent with loss of function in one allele, suggesting that maintained heterozygosity in this species allowed divergence between alleles over time. This work establishes the meiotic mechanism and inheritance pattern of reproduction in H. exemplaris , which provides a framework for interpreting genetic variation in this organism as a laboratory model. Additionally, our finding that meiosis is modified in H. exemplaris via a mechanism that maintains heterozygosity across the genome adds to a growing list of asexual animals that have evolved to reproduce clonally despite the expected long-term costs.

Tardigrades are microscopic animals that can survive exceptional levels of ionizing radiation or desiccation—DNA-damaging conditions that would kill most animals. Irradiation or radiomimetic drug treatment of the tardigrade Hypsibius exemplaris can induce remarkably high expression levels of DNA repair genes, primarily those in the base excision repair and nonhomologous end joining pathways. How tardigrades can repair widespread DNA damage without producing frequent, large-scale chromosome structural abnormalities, like chromosome translocations and fusions, is unknown. Here, we report the results of examining chromosome and nuclear architecture throughout the cell cycle in early embryos of H. exemplaris. We found that H. exemplaris chromosomes are maintained in an individualized form throughout the cell cycle. We were surprised to also find that each chromosome is housed in a fully or partially separate lamin-lined compartment, instead of all chromosomes being housed in a single, nearly spherical nuclear lamina and envelope. Our results reveal unusual chromosomal and nuclear organization in a tardigrade. We speculate that these unexpected features might limit chromosomal rearrangements during DNA damage repair in extreme conditions.

Tardigrades are microscopic animals that can survive exceptional levels of ionizing radiation or desiccation – DNA-damaging conditions that would kill most animals. Irradiation or radiomimetic drug treatment of the tardigrade Hypsibius exemplaris can induce remarkably high expression levels of DNA repair genes, primarily those in the base excision repair and nonhomologous end joining pathways. How tardigrades can repair widespread DNA damage without producing frequent, large-scale chromosome structural abnormalities, like chromosome translocations and fusions, is unknown. Here, we report the results of examining chromosome and nuclear architecture throughout the cell cycle in early embryos of H. exemplaris . We found that H. exemplaris chromosomes are maintained in an individualized form throughout the cell cycle. We were surprised to also find that each chromosome is housed in a fully or partially separate lamin-lined compartment, instead of all chromosomes being housed in a single, nearly spherical nuclear lamina and envelope. Our results reveal unusual chromosomal and nuclear organization in a tardigrade. We speculate that these unexpected features might limit chromosomal rearrangements during DNA damage repair in extreme conditions. SIGNIFICANCE STATEMENT: We have investigated unusual chromosome organization in an organism that survives extremely DNA-damaging environments, a tardigrade, through early embryonic cell cycles. Chromosomes in fixed and stained embryos appeared more condensed through interphase than is typical for animal cells. Chromosomes remained individualized, in fully or partially separate lamin-lined compartments, through interphase. The results reveal a unique nuclear and chromosomal organization in tardigrades, which we speculate might contribute to limiting chromosomal structure abnormalities under DNA damaging conditions in nature.

Apical constriction is a critical cell shape change that drives cell internalization and tissue bending. How precisely localized actomyosin regulators drive apical constriction remains poorly understood. Caenorhabditis elegans gastrulation provides a valuable model to address this question. The Arp2/3 complex is essential in C. elegans gastrulation. To understand how Arp2/3 is locally regulated, we imaged embryos with endogenously tagged Arp2/3 and its nucleation-promoting factors (NPFs). The three NPFs—WAVE, WASP, and WASH—controlled Arp2/3 localization at distinct subcellular locations. We exploited this finding to study distinct populations of Arp2/3 and found that only WAVE depletion caused penetrant gastrulation defects. WAVE localized basolaterally with Arp2/3 and controlled F-actin levels near cell–cell contacts. WAVE and Arp2/3 localization depended on CED-10/Rac. Establishing ectopic cell contacts recruited WAVE and Arp2/3, identifying the contact as a symmetry-breaking cue for localization of these proteins. These results suggest that cell–cell signaling via Rac activates WAVE and Arp2/3 basolaterally and that basolateral Arp2/3 makes an important contribution to apical constriction.

Apical constriction is a critical cell shape change that bends tissues. How precisely-localized actomyosin regulators drive apical constriction remains poorly understood. C. elegans gastrulation provides a valuable model to address this question. The Arp2/3 complex is essential in C. elegans gastrulation. To understand how Arp2/3 is locally regulated, we imaged embryos with endogenously-tagged Arp2/3 and its nucleation-promoting factors (NPFs). The three NPFs – WAVE, WASP, and WASH – colocalized with Arp2/3 and controlled Arp2/3 localization at distinct subcellular locations. We exploited this finding to study distinct populations of Arp2/3 and found that only WAVE depletion caused penetrant gastrulation defects. WAVE localized basolaterally with Arp2/3 at cell-cell contacts, dependent on CED-10/Rac. Establishing ectopic cell contacts recruited WAVE and Arp2/3, identifying contact as a symmetry-breaking cue for localization of these proteins. These results suggest that cell-cell signaling via Rac activates WAVE and Arp2/3 basolaterally, and that basolateral Arp2/3 is important for apical constriction.