The study, published in the journal Stress Biology, focuses on a long-standing puzzle in plant immunity: how the actin cytoskeleton, a dynamic meshwork of protein filaments just beneath the cell membrane, communicates with the signaling pathways that mobilize defense. Scientists have known for years that when a plant detects pathogen-associated molecular patterns, or PAMPs, through cell-surface receptors, the actin network rapidly reorganizes. Filament density, orientation and bundling all shift within minutes. Disrupting this network, whether chemically or genetically, typically leaves plants more vulnerable to infection. What remained unclear was which molecular players orchestrate the remodeling and how it connects to downstream immune outputs such as reactive oxygen species production.

To answer these questions, the team turned to the rice-Magnaporthe oryzae pathosystem, a classic model in which fungal spores land on the leaf surface, germinate, and build dome-shaped infection structures called appressoria. These structures generate enormous turgor pressure through glycerol accumulation, allowing the fungus to mechanically puncture the plant cuticle. The host cytoskeleton and cell wall at the infection site are critical barriers to this penetration, making them natural focal points for studying basal immunity.

The researchers first confirmed the importance of actin polymerization using latrunculin B, a macrolide compound derived from marine sponges that binds monomeric actin and prevents filament elongation. When seedlings of the japonica cultivars TP309 and Nipponbare were pretreated with the drug before spray inoculation with a virulent M. oryzae isolate, they became markedly more susceptible to blast, with higher concentrations producing stronger effects. Quantitative PCR revealed greater fungal growth in treated plants. The drug also broadly suppressed defense hormone pathways: genes in the salicylic acid pathway, including OsPAL1, OsICS1 and PR4, and genes in the jasmonic acid and ethylene pathways, including OsAOS2 and OsERF1, were all downregulated. In other words, a structurally intact actin network is not merely a passive barrier but an active prerequisite for full immune signaling.

That result pointed the investigators toward BUI1, also known as RMD or OsFH5, a class II formin protein previously famous for its role in rice development. Loss of BUI1 function causes bent uppermost internodes, dwarfism, wavy panicles and abnormal seeds, all traced to impaired cell elongation and disrupted cytoskeletal arrays. The protein carries three conserved domains: a PTEN domain that targets it to the chloroplast surface, a proline-rich FH1 domain that interacts with profilin to promote actin polymerization, and an FH2 domain that binds the growing ends of actin filaments. Notably, the team found that latrunculin B treatment downregulated BUI1 expression, hinting that the gene sits within the very regulatory circuit the drug perturbs.

Testing the bui1 mutant, derived from the moderately resistant cultivar Zhejing 22, confirmed the suspicion. After punch inoculation with M. oryzae, the mutant developed significantly larger lesions than wild type, and field trials in a blast nursery showed dramatically reduced resistance under natural infection pressure. Using a GFP-tagged fungal strain, the researchers tracked infection in real time and found that appressorium formation and maturation were both more efficient on bui1 leaf sheath cells, suggesting that BUI1-mediated actin organization actively impedes this critical early step of fungal invasion. BUI1 expression itself was upregulated upon fungal inoculation, consistent with a positive role in defense.

The most striking finding came when the team visualized actin filaments directly. Using fluorescent phalloidin staining and confocal microscopy, they showed that treatment with the PAMPs chitin or flg22 triggered a rapid, transient increase in actin filament bundling in wild-type leaf sheath cells within five minutes. In the bui1 mutant, this response was severely attenuated, and the baseline cytoskeleton appeared disorganized. During actual fungal infection, wild-type cells accumulated dense actin bundles around infection sites by twelve hours post-inoculation, while the mutant failed to mount this defense-associated remodeling. BUI1, the data showed, is essential for PAMP-triggered actin reorganization, placing a formin protein squarely within the earliest tier of the plant immune response.

Broad-spectrum resistance is the holy grail of crop protection, and BUI1 delivered on that front too. The mutant proved more susceptible not only to blast but also to bacterial blight, developing longer lesions after inoculation with the Xoo strain PXO99A, and to sheath blight when tested in CRISPR-generated knockout lines in the Nipponbare background. Conversely, plants overexpressing BUI1 under its native promoter showed enhanced bacterial blight resistance proportional to expression level. Time-course transcriptome analysis after Xoo infection revealed extensive transcriptional reprogramming in wild-type plants that was largely absent in the mutant, with key defense pathways, including phenylpropanoid biosynthesis, MAPK signaling and plant-pathogen interaction networks, failing to activate without BUI1.

Perhaps most intriguingly, BUI1 turned out to act downstream of ROD1, a calcium sensor previously shown to suppress rice immunity by activating catalase-mediated ROS scavenging, whose loss confers broad-spectrum resistance. When the researchers knocked out BUI1 in the rod1 background, the strong resistance of rod1 plants was significantly compromised against blast, bacterial blight and sheath blight alike, though the double mutants remained less susceptible than fully wild-type plants. Yeast two-hybrid assays detected no direct physical interaction between BUI1 and ROD1 or the ROD1-interacting catalase OsCatB, indicating the relationship is genetic rather than structural. DAB staining and hydrogen peroxide measurements showed that the double mutants accumulated lower ROS levels than rod1 alone, pointing to ROS homeostasis as the mechanistic bridge.

RNA sequencing after M. oryzae infection explained why. In the bui1 knockout background, genes encoding peroxidases and metallothioneins, including OsPOD, POXA, OsCatB, OsMT1d and OsMT1f, were strongly upregulated, while ROS-producing genes such as RbohA and RbohB were unchanged. The mutant exhibited higher peroxidase activity and correspondingly lower hydrogen peroxide accumulation. BUI1, it appears, maintains immune competence primarily by restraining ROS-scavenging pathways, allowing the oxidative burst that hallmark of pattern-triggered immunity to reach effective levels. The protein also contains intrinsically disordered regions predicted to mediate liquid-liquid phase separation, and truncated variants lacking these regions failed to undergo phase separation in vitro, suggesting that BUI1 may form biomolecular condensates that serve as organizational hubs integrating cytoskeletal dynamics with defense signaling. For breeders, the message is clear: a single formin gene links the cell’s structural skeleton, its oxidative chemistry and its hormone signaling into one coordinated defense program, and tuning that gene could yield rice lines that shrug off multiple diseases without yield penalties.

Subject of Research: The role of the rice formin gene BUI1 in coordinating actin cytoskeleton remodeling and reactive oxygen species homeostasis during broad-spectrum plant disease resistance.

Article Title: BUI1 coordinates actin cytoskeleton remodeling and ROS homeostasis to confer broad-spectrum disease resistance in rice

Article References: BUI1 coordinates actin cytoskeleton remodeling and ROS homeostasis to confer broad-spectrum disease resistance in rice. (n.d.). https://doi.org/10.1007/s44154-026-00321-5

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00321-5

Keywords: BUI1, rice immunity, actin cytoskeleton, ROS homeostasis, Magnaporthe oryzae, pattern-triggered immunity, formin, ROD1, broad-spectrum resistance, Xanthomonas oryzae, peroxidase, plant pathology

The research, led by Dora Gjirlić, Anja Weber, Guido Posern, and Anurag Kumar Singh of the Institute for Physiological Chemistry, focuses on the actin-depolymerizing factor/cofilin family, a group of proteins that sever and depolymerize actin filaments, thereby controlling the constant turnover of the cytoskeleton. Vertebrates possess two major isoforms: Cofilin-1, which is broadly expressed in many cell types, and Cofilin-2, which is characteristic of muscle tissue. Although both proteins perform the same core biochemical task — cutting filamentous actin into globular subunits — their roles during myogenesis, the process by which muscle cells form, have remained incompletely understood. The new work provides the most detailed picture yet of how these isoforms are swapped during muscle differentiation and why that swap matters.

Using the immortalized mouse myoblast cell line C2C12, a workhorse of muscle biology, the team tracked the expression of both cofilin isoforms as cells transitioned from growth medium into differentiation medium. What emerged was a pronounced isoform switch. As differentiation proceeded, Cofilin-1 and the related Actin-Depolymerizing Factor (ADF) were progressively downregulated, while Cofilin-2 expression rose. Analyses of messenger RNA and protein stability indicated that this switching is controlled primarily at the level of gene expression rather than through altered degradation of the proteins themselves, suggesting that transcriptional reprogramming sits at the heart of the isoform transition.

To probe function rather than mere correlation, the researchers deployed an impressive arsenal of techniques. They used CRISPR/Cas9 genome editing to knock out the Cofilin-1 gene, shRNA-mediated knockdown to deplete it partially, immunofluorescence microscopy to visualize cellular morphology, quantitative gene expression analyses to monitor myogenic marker genes, MRTF reporter assays to measure the activity of the myocardin-related transcription factor A, and pharmacological inhibition of LIM kinase to block a key regulatory phosphorylation pathway. Each approach interrogated a different facet of the same question: what happens to differentiating muscle cells when the actin-severing machinery is perturbed?

The answer, for complete loss of Cofilin-1, was dramatic. Cells lacking the protein developed marked morphological abnormalities, failed to exit the cell cycle properly, showed elevated MRTF activity, and formed far fewer myotubes — the multinucleated structures that represent successful differentiation. This constellation of defects makes mechanistic sense. MRTF-A is a transcriptional coactivator whose nuclear activity is restrained by binding to globular actin; when actin polymerization dynamics are disturbed and the monomeric actin pool shifts, MRTF signaling can be unleashed inappropriately. Cofilin-1, by regulating the filament-monomer balance, normally helps keep this signaling pathway calibrated during the transition from proliferation to differentiation. Remove it entirely, and the cell’s transcriptional program falls out of register with its cytoskeletal state.

The most striking discovery, however, came from the knockdown experiments. When the researchers reduced Cofilin-1 only partially, something unexpected happened: myoblast fusion was enhanced. Cells with modestly lowered Cofilin-1 fused with their neighbors more readily than controls. But when depletion was pushed harder, the opposite occurred — differentiation collapsed and myotube formation was impaired. This bidirectional response demonstrates a dosage-sensitive requirement for Cofilin-1, a Goldilocks principle in which the protein must be present at precisely the right level. Too much Cofilin-1 appears to restrain fusion, while too little disrupts the cytoskeletal remodeling that fusion itself requires. The finding adds muscle formation to a growing list of biological processes in which gene dosage, rather than simple presence or absence, determines the outcome.

Cofilin-2 deficiency, by contrast, produced comparatively mild effects during early differentiation. This asymmetry is intriguing because Cofilin-2 is the isoform ultimately enriched in mature muscle. The results suggest that the two isoforms, despite their biochemical similarity, are not interchangeable in function or timing: Cofilin-1 is the critical regulator of the early, decision-making phase of myogenesis, whereas Cofilin-2 likely assumes importance later, in the context of mature contractile fibers. The progressive switch from one isoform to the other may therefore represent a carefully staged handover of cytoskeletal control, timed to the changing needs of the cell as it commits to the muscle lineage.

The study also illuminates how Cofilin-1’s activity, not just its abundance, is regulated during myogenesis. The researchers found that Cofilin-1 undergoes transient phosphorylation by LIM kinase during early differentiation. Phosphorylation by LIM kinase — which itself sits downstream of the Rho-associated kinase ROCK — inhibits cofilin’s actin-severing capacity, stabilizing actin filaments at specific moments. When the team blocked this pathway with the LIM kinase inhibitor LIMKi3 (BMS-5), myogenic progression was disrupted. The picture that emerges is one of dynamic, moment-to-moment control: the cell not only dials down Cofilin-1 expression over the course of differentiation but also rapidly toggles the remaining protein on and off through phosphorylation, fine-tuning actin turnover as the cell cycle exits and fusion machinery engages.

These findings connect several threads of muscle biology that had previously run in parallel. The actin cytoskeleton must be dismantled and rebuilt for a myoblast to elongate and fuse; the MRTF and serum response factor (SRF) transcriptional pathway reads actin dynamics and translates them into gene expression; and the myogenic regulatory factors MyoD and myogenin drive the differentiation program, with myomaker and myomixer/myomerger executing the fusion step itself. The new work positions Cofilin-1 as a molecular link between the physical and the transcriptional layers of this process — a dosage-sensitive node where cytoskeletal remodeling is converted into signals that govern proliferation, differentiation, and ultimately cell fusion. Caveolin-3, myosin heavy chain, and other differentiation markers tracked in the study provided the readouts confirming that these signaling changes translate into real changes in myogenic identity.

Beyond its immediate significance for understanding how skeletal muscle forms, the research carries potential implications for regenerative medicine and muscle disease. Muscle regeneration after injury recapitulates many steps of embryonic myogenesis, including the fusion of satellite-cell-derived myoblasts onto damaged fibers. If Cofilin-1 dosage and LIM kinase signaling set the efficiency of that fusion, they become candidate levers for improving muscle repair — or potential culprits in conditions where repair fails. The Halle team, whose work was supported by internal faculty core funding from Martin Luther University Halle-Wittenberg and enabled by open-access funding through Projekt DEAL, emphasizes that coordinated regulation of Cofilin-1 expression and activity, together with the timely transition to Cofilin-2, is required for efficient myoblast fusion and muscle formation. In revealing that a humble actin-severing protein must be tuned like an instrument rather than simply switched on or off, the study adds a subtle but essential rule to the growing rulebook of how cells build tissue.

Subject of Research: Dosage-sensitive regulation of Cofilin-1 and LIM kinase signaling in myoblast differentiation and fusion

Article Title: Dosage-sensitive regulation of Cofilin-1 and LIMK signaling controls myoblast differentiation and fusion

Article References: Gjirlić, D., Weber, A., Posern, G., & Singh, A. K. (2026). Dosage-sensitive regulation of Cofilin-1 and LIMK signaling controls myoblast differentiation and fusion. Cellular and Molecular Life Sciences. https://doi.org/10.1007/s00018-026-06437-1

Image Credits: AI Generated

DOI: 10.1007/s00018-026-06437-1

Keywords: myogenesis, Cofilin-1, Cofilin-2, actin cytoskeleton, LIM kinase, MRTF signaling, myoblast differentiation, cell fusion, cytoskeletal regulation, skeletal muscle, Dosage-sensitive, regulation

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