Actin exists in a dynamic equilibrium between monomeric G-actin and polymerized filamentous F-actin, and dozens of actin-binding proteins govern that balance. Formins nucleate and elongate long, unbranched filaments; the Arp2/3 complex builds branched networks; tropomyosin stabilizes mature filaments; cofilin severs filaments and recycles monomers. Because actin underpins muscle contraction, immune cell migration, synaptic plasticity, and cell division, its dysregulation has been tied to sarcopenia, neurodegeneration, immunodeficiency, and cancer. In Alzheimer’s disease brains, for example, cofilin-rich actin rods accumulate, and in Drosophila, F-actin buildup in the brain has been shown to drive brain aging and limit healthspan.

Genetic work in worms had already established actin as a central modulator of longevity. Overexpression of the stress-responsive transcription factor HSF-1 extends lifespan partly by improving actin stability, and the bromodomain protein BET-1 promotes actin maintenance and longevity when overexpressed. The same laboratory previously used RNA interference to knock down genes encoding actin-binding proteins and found that targeted disruption of actin generally shortened lifespan and exacerbated hallmarks of aging, with some tissue-specific nuances. But RNAi has well-known drawbacks: incomplete or transient silencing, off-target effects, inefficient delivery to certain tissues, and compensation by redundant paralogs. Chemical perturbation, by contrast, offers rapid, tunable, dose-dependent control and works across model systems where genetic tools are unavailable.

To probe that promise, the researchers treated wild-type N2 worms from the first larval stage with a panel of small molecules dissolved into the agar of their culture plates. CK666 was used to block Arp2/3-mediated actin branching; SMIFH2 to inhibit formin-driven nucleation and elongation; TR100, an anticancer compound originally developed against the tropomyosin isoform Tpm3.1, to target tropomyosin-dependent regulation; SZ-3, a first-in-class cofilin inhibitor, to interfere with filament severing; and phalloidin, a mushroom toxin that binds F-actin with high affinity, to stabilize existing filaments. Concentrations ranged from 1 to 100 micromolar, with dimethyl sulfoxide as the vehicle control, and lifespan assays were scored every other day from day 5 of adulthood across three independent biological replicates.

The results were strikingly asymmetric. CK666 produced a clear, dose-dependent reduction in lifespan: concentrations below 5 micromolar had minimal effects, while higher concentrations significantly shortened survival. None of the other compounds had major effects on longevity, although SZ-3 showed a modest but statistically significant lifespan reduction in most replicates. Locomotor assays added further nuance. CK666-treated worms actually thrashed slightly more on days 5 and 9 of adulthood, while TR100 reduced motility on day 9, indicating that changes in movement do not track neatly with lifespan outcomes when actin is perturbed pharmacologically.

To determine whether the compounds were actually engaging their targets in vivo, the team imaged actin organization directly using transgenic worms expressing the fluorescent actin reporter LifeAct::mRuby in either body-wall muscle or the hypodermis. Muscle actin in C. elegans forms highly ordered striations running in parallel with myosin filaments, and these striations progressively fragment, lose alignment, and become wavy with age. CK666 treatment clearly exacerbated this age-associated muscle actin disorganization, with severity increasing up to 10 micromolar. Interestingly, the percentage of muscle cells classified as disrupted did not change significantly; rather, the severity of disorganization within affected cells worsened, suggesting Arp2/3 inhibition deepens existing damage rather than spreading it to new cells. CK666 also transiently reduced measurable hypodermal actin structures at day 1 of adulthood, an effect that vanished by mid-life.

The other four compounds told a different story. SMIFH2, SZ-3, phalloidin, and TR100 produced no detectable changes in muscle actin organization at any age examined, and SZ-3 likewise left hypodermal actin untouched despite its modest lifespan effect. This contrasted sharply with earlier genetic work, in which RNAi knockdown of the tropomyosin gene lev-11 or the cofilin gene unc-60 produced robust muscle actin disruption, and in which the actin stabilizer jasplakinolide extended lifespan at low concentrations. The discrepancy is not necessarily evidence that these pathways are unimportant. TR100 selectively disrupts filaments containing the mammalian Tpm3.1 isoform while sparing those containing alphaTmfast, and sequence alignment shows that only some C. elegans LEV-11 isoforms resemble Tm3.1, so isoform expression patterns may blunt the drug’s activity in worms.

Several technical factors likely explain why most of the molecules failed to reproduce genetic phenotypes. The impermeable, negatively charged cuticle of C. elegans limits compound penetration, potentially keeping intracellular drug concentrations below effective thresholds. Drug stability over the several weeks required for lifespan assays is another concern, since temperature, light exposure, and degradation can erode effective doses even when plates are freshly prepared. Systemic delivery is also a fundamental limitation: small molecules cannot be targeted to specific tissues, whereas actin-related aging phenotypes may depend on perturbation within particular cell types. And target engagement was not directly measured, so the absence of a phenotype should not be read as proof that a pathway is dispensable for aging.

The authors conclude that pharmacological perturbation, while attractive for its translational relevance, may offer limited advantages in C. elegans, where the genetic toolkit, the low cost of RNAi, and the robustness of genetic phenotypes make RNAi-based approaches substantially more practical. Small molecules remain invaluable in systems where genetic manipulation is impractical, and they more closely resemble the therapeutic strategies used in mammalian medicine, but the compounds tested here were expensive, required high concentrations, and carried uncertainties in stability and delivery. The study’s most durable contribution may be its framework: a systematic, dose-controlled, tissue-resolved protocol for evaluating cytoskeletal drugs during aging, anchored by the finding that CK666-mediated Arp2/3 inhibition is the most robust pharmacological perturbation of actin yet demonstrated in this model. That result reinforces a growing consensus that Arp2/3-mediated actin branching is a critical guardian of cytoskeletal integrity as animals grow old, and it cautions the field that negative drug results must be interpreted only after target engagement is verified.

Subject of Research: Pharmacological targeting of actin regulatory pathways during aging in C. elegans

Article Title: Evaluating pharmacological targeting of actin regulatory pathways during aging in C. elegans

Article References: Wang, T., Alcala, A., Berdan, D., Kuo, V., Garcia, G., Higuchi-Sanabria, R., & Averbukh, M. (2026). Evaluating pharmacological targeting of actin regulatory pathways during aging in C. elegans. Biogerontology, 27(5), Article 156. https://doi.org/10.1007/s10522-026-10503-3

Image Credits: AI Generated

DOI: 10.1007/s10522-026-10503-3

Keywords: actin, aging, C. elegans, Arp2/3 complex, CK666, lifespan, cytoskeleton, muscle actin disorganization, tropomyosin, cofilin, formins, pharmacological perturbation