Abstract
<jats:p>Axon collateral branching is a fundamental determinant of neuronal connectivity, enabling individual neurons to innervate multiple targets and establish functional neural circuits. Although branch initiation requires local actin remodelling within the axon shaft, the mechanisms that restrict actin assembly to prevent excessive branching remain poorly understood. Here, we identify Kaptin (Kptn) as a conserved negative regulator of axon collateral formation that limits the maturation of axonal actin patches. Using primary neuronal cultures, quantitative live-cell imaging, and zebrafish genetics, we show that loss of Kptn enhances the conversion of actin patches into filopodial protrusions, leading to excessive collateral branching. Kptn-deficient zebrafish exhibit increased motor axon arborisation, elevated neuromuscular junction density and impaired motor behaviour. Mechanistically, Kptn antagonises the actin elongation factor Formin-2 (Fmn2) to regulate actin filament barbed-end dynamics, thereby controlling the threshold for productive branch initiation. Importantly, these branching defects occur independently of Kptn's established role in mTORC1 signalling, revealing a distinct physiological function during neuronal development. Our findings identify Kptn as a key molecular brake that constrains axon collateral branching and establish negative regulation of actin patch maturation as a fundamental mechanism controlling neuronal circuit assembly. This work provides a mechanistic framework for understanding how KPTN mutations associated with intellectual disability and epilepsy disrupt neuronal connectivity.</jats:p>