Duarte, V. N. | bioRxiv Science

bioRxiv2026

Opposing Roles for the Spectraplakin Short Stop in Stable and Dynamic Dendrites Reveal Divergent DLK Signaling and a Role in Dendrite Regeneration

Dendrites are vital to neuronal function, and dendrite injury occurs after neurological traumas such as stroke, traumatic brain injury, or neurodegenerative diseases. Despite their importance, the mechanisms underlying dendrite maintenance or regeneration remain poorly understood. The Drosophila gene short stop (shot), orthologous to ACF7/MACF1 in mammals, functions as an actin-microtubule crosslinker during neuronal development. Here, we investigate shot's role in dendrite stability and repair using Drosophila sensory neurons. We find that shot plays opposing, cell type-specific roles in dendrite maintenance: it restricts excessive branch growth in simple, stable neurons, while being required for dendrite coverage in complex, dynamic neurons. These opposing functions are reflected in distinct localization patterns in stable versus dynamic dendrite arbors. Loss of shot destabilizes the microtubule cytoskeleton in stable neurons, and activates Wallenda/DLK signaling in both stable and dynamic neurons. Downstream of Wallenda/DLK, JNK signaling diverges between neuron types, with canonical basket/JNK activation occurring only in neurons with stable dendritic arbors. After injury, shot promotes dendrite regeneration in both neuron types and accumulates in distinct shapes in regenerated dendrites, with specific domains critical for proper Shot accumulation patterns. Collectively, these findings establish shot as a context-dependent regulator of dendrite maintenance and repair, and demonstrate that the structural identity of a dendritic arbor shapes how neurons sense and respond to cytoskeletal perturbation.

cell biology↗

bioRxiv2025

Optimizing CRISPR/Cas9 mutagenesis in Drosophila da neurons to avoid cytotoxicity

Genetic perturbations are one of the great strengths of the model organism Drosophila melanogaster, with approaches such as classical mutagenesis and RNA interference enabling a wealth of biological discoveries. A more recent approach for altering gene expression is CRISPR/Cas9-based mutagenesis, but as with any new tool, its use must be optimized. High expression of Cas9 has been shown to cause cytotoxicity in some cell types. Here, we show that Cas9 expression alone causes cytotoxicity in the dendritic arborization (da) neurons that are widely used to study neuronal development and regeneration. We then systematically evaluate alternative Cas9 transgenes designed to lower total Cas9 expression, called uCas9 transgenes. We show that expression of these uCas9 transgenes results in little to no cytotoxicity to da neurons. Lastly, we demonstrate the ability of uCas9 transgenes to effectively and specifically gene edit in da neuro ns. Thus, we expand the toolkit of genetic perturbations available to researchers working with Drosophila da neurons or other cell types suceptible to cytotoxicity due to high expression of Cas9.

genetics↗