Robust homeostatic plasticity is essential for survival, enabling neuronal circuits to withstand destabilizing forces and restore function during critical challenges such as predator evasion or toxin exposure. Semaphorin/Plexin signaling is central to presynaptic homeostatic potentiation at central synapses and at neuromuscular junctions across species. However, our understanding of this pathway has remained incomplete, as secreted Semaphorins bind weakly to their Plexin receptors and require additional coreceptors for efficient signaling. Here, we identify Neuropilin and Tolloid-like protein (Neto-α), an auxiliary subunit for ionotropic glutamate receptors (iGluRs), and the tyrosine kinase Abelson (Abl) as essential components of the Sema2b/PlexB signaling pathway that drives rapid homeostatic potentiation and stabilizes synaptic strength at the neuromuscular junction. Neto-α functions as a Neuropilin-like coreceptor that cooperates with Sema2b to relieve PlexB autoinhibition and initiate signaling. In parallel, Neto-α recruits Abl, which functions as a cytosolic amplifier to enhance pathway output. We demonstrate that both pathway activation and amplification are required for a rapid and effective homeostatic response. By integrating these two functions within a single molecular assembly, Neto-α ensures fast and efficient compensatory responses to perturbations. This evolutionarily conserved signaling module, Neto (or Neuropilin)/Sema/Plex/Abl, likely operates in cellular contexts beyond neural function, including in tumorigenesis.

In the wild, a mouse must flexibly perform multiple computations at once, rapidly navigating, sampling its environment, forming and using memories, and balancing internal needs. Capturing the self-paced, low-repetition, complex nature of decision making in the wild while preserving the experimental control needed to interpret its process remains a challenge. Here we show that lab mice, without food restriction, learn to forage for hidden resources cued by ambiguous sounds in a dynamic environment by spatiotemporally reorganizing their existing behavioral repertoire. Using a novel closed-loop assay, we required mice to sit still to receive informative but hard-to-localize sound cues emanating from one correct location they must find, out of 157 possible locations in a large arena. With experience, mice become efficient hunters. Efficient hunting cannot be explained by an increase in a particular behavioral module, but instead, mice reorganize existing behaviors into clustered bouts of high-quality sampling and site-checking. Looking forward, this work establishes an approach for studying the neural, molecular, and evolutionary basis of naturalistic decision-making in mice.

Whether recovering after a gust of wind, or rapidly saccading away from an oncoming predator, fruit flies show remarkable aerial dexterity about their body roll axis. Here, we investigated the detailed wing kinematic changes during free-flight roll motion and probed the neuromuscular basis for such changes. Consistent with previous work, we observed that flies manipulated the stroke amplitude difference between their wings to control their roll angle. Here, we show that flies are capable of achieving such changes by altering the stroke amplitude of either or both of their wings. Further we found that during corrections flies can also take advantage of an aerodynamically significant change in the angle of attack of their uppermost wing. Curiously, these corrective wing changes cannot be eliminated when motor neurons hypothesized to be used during roll maneuvers (i1, i2, b1, b2, and b3) are individually inhibited. However, free-flight optogenetic manipulations and quasi-steady aerodynamic calculations show that each of these motor neurons individually can effect kinematic changes consistent with a roll correction. Combining this evidence with an analysis of haltere inputs found in the BANC connectome, we propose that the observed robustness could be the result of two sets of muscular redundancies that receive shared inputs from haltere sensory afferents: one set, containing b1 and b2, is able to increase the stroke amplitude of the lower wing; while the other set, containing i1, i2, and b3, is able to decrease the stroke amplitude and wing pitch angle of the upper wing. Because of the redundancy in the input sensory information and output wing motion in the muscles in each cluster, the fly is able to perform roll stability maneuvers even when one of the constituent motor neurons is inhibited. This framework proposes new ways fast aerial maneuverability can be implemented when dealing with the fly’s most unstable rotational degree of freedom.

Yeast two hybrid (Y2H) assays serve as powerful platforms for the detection and engineering of protein-protein interactions. Here, we describe a strategy for using Y2H-based screening to assess and develop optogenetic tools based on light-controlled protein-protein interactions.

Sex differences in behavior exist across all animals, typically under strong genetic regulation. In Drosophila, fruitless/doublesex transcription factors identify dimorphic neurons, but their organization into functional circuits remains unclear. We present the connectome of the entire Drosophila male central nervous system. This contains 166,700 neurons spanning the brain and nerve cord, fully proofread and annotated, including fruitless/doublesex expression and 11,710 neuron types. We provide the first comprehensive comparison between male and female brain connectomes to synaptic resolution, finding 8,069 isomorphic, 138 dimorphic, 289 male-specific, and 71 female-specific types. This resource enables analysis of full sensory-to-motor circuits underlying complex behaviors and the impact of dimorphic elements. Sex-specific/dimorphic neurons are concentrated in higher brain centers, while the sensory and motor periphery is largely isomorphic. Within higher centers, male-specific connections are organized into hotspots defined by male-specific neurons or arbors. Dimorphic neurons reroute information across sexes.

Gustatory systems drive critical survival behaviors such as feeding, foraging, and social interactions. However, gustation remains one of the least mapped sensory modalities at the connectome level. Here, we present the first complete wiring diagram of the male Drosophila adult gustatory system, comprehensively reconstructing gustatory receptor neurons (GRNs) from peripheral organs in a contiguous electron microscopy volume spanning brain, cervical connective, and ventral nerve cord. Integrating this with existing datasets, we generated a pan-central nervous system (CNS), cross-sex connectome that reveals GRN diversity through connectivity-based clustering, molecular identity mapping, and sexual dimorphism analysis. We mapped all feeding motor neurons and traced complete sensory-to-motor pathways to feeding, foraging, endocrine, and social behavior circuits. The emerging circuit architectures reveal distinct circuits for nutrient assessment, motor control, neuroendocrine regulation, and courtship. This work defines the gustatory system's organization at synaptic resolution and provides a framework for understanding how internal states modulate sensory-driven decisions across behavioral contexts.

Visual systems transform photoreceptor inputs into rich perceptual representations through hierarchical networks that extract features along parallel pathways. Although this architecture is conserved across species, how visual information is routed throughout an entire brain remains elusive in any animal. Using the male Drosophila connectome, we trace signals from photoreceptors through the optic lobes—layered, retinotopic regions containing two-thirds of the brain’s neurons—and onward into the central brain. Network-based analyses reveal a multilayered architecture of pathway classes with distinct input mixtures. Signals from visual-input channels spread broadly yet converge in focal regions apparently specialized for particular features and fine spatial sampling. Predictions of receptive-field structure and feature-related input biases are consistent with physiological data and extend to thousands of uncharacterized neuron types. These analyses provide a neuron-by-neuron account of how a visual system organizes and integrates information across an entire brain.

DNA double-strand breaks (DSBs) require rapid signalling and physical stabilisation of broken DNA ends to preserve genome integrity. Here, we identify myosin VI (MVI) as an ATM-regulated component of the DSB response. DNA damage induces rapid nuclear accumulation and nanoscale reorganisation of MVI across multiple cell models, in an ATM-dependent manner. Pharmacological or genetic perturbation of MVI attenuates γH2AX signalling and disrupts Ku80 organisation, while DNA damage persists. This leads to increased sensitivity to cisplatin and bleomycin. Super-resolution imaging reveals spatial association of MVI with Ku80-containing repair structures, implicating MVI in non-homologous end joining (NHEJ). In a minimal reconstituted system, MVI and actin enhance the proximity of Ku70/80-bound DNA ends. Together, our findings identify MVI as a regulator of DSB repair that links ATM signalling to Ku-associated DNA-end stabilisation and suggest that targeting MVI may sensitise tumour cells to genotoxic therapy.

Many insects manipulate plants by injecting effector proteins. In one extreme example of this molecular "hijacking," aphids inject bicycle proteins into , contributing to the development of novel organs called galls. Bicycle proteins share no amino acid sequence similarity with proteins of known function. Here, we report the crystal structures of two divergent bicycle proteins. Both proteins contain saposin-like folds: one with multiple disulfide bonds exhibits a swapped domain topology; the other has no disulfide bonds and possesses two distinct, tandem domains. To explore the structural evolution of bicycle proteins, we attempted to predict bicycle protein structures with Alphafold2 (AF2) and other deep learning programs. While AF2 did not recover the two experimental structures using existing databases, it succeeded when provided with multiple sequence alignments (MSAs) of protein sequences from newly sequenced closely related species. Using this approach, we generated 2,400 high-confidence bicycle protein predictions from seven aphid species. While all aphid bicycle proteins contain predicted saposin-like folds, they display a vast diversity of structural and physicochemical properties. While this diversity thwarts prediction of conserved functions encoded in structure, it suggests that bicycle proteins have evolved to target diverse plant processes and/or to evade plant immune surveillance. Our extension of AF2 with custom MSAs of proteins from closely related species provides a generalizable, powerful approach for predicting structures of rapidly evolving protein families.

Summary Fluorescent imaging in live cells is a cornerstone of life sciences. While natural fluorescent proteins have been engineered to enhance individual features, no existing tag combines ideal properties into a single system: high brightness, reversible binding, compact size, and stability across diverse conditions. Here, we achieve this through de novo design of rhodamine binders (Rhobin). To harness the broad repertoire of rhodamine fluorophores, we developed a generalizable design strategy for a pan-rhodamine binder compatible with diverse wavelengths and applications. Rhobin enables live- and fixed-cell imaging of various subcellular targets in mammalian cells, showing brightness surpassing existing tags. Its reversible fluorophore binding supports super-resolution stimulated emission depletion (STED) and live-cell single-molecule imaging for extended durations compared with HaloTag. Beyond conventional systems, Rhobin enables live imaging of the extremophile Sulfolobus acidocaldarius at 75°C, previously inaccessible with current tags. Together, these results establish Rhobin as a versatile platform for next-generation imaging and biosensor design.