Relan, M.; Mallampalli, V.; Barad, B. A.; Stavoe, A. K. H.; Waxham, M. N.

Axons are extended cellular compartments that mediate neuronal connectivity over extraordinary distances, placing unique demands on local organelle organization and trafficking. How these geometric constraints influence organelle architecture remains poorly understood. Here, we used cryoelectron tomography and quantitative morphometric analyses to define the three-dimensional ultrastructural organization of dorsal root ganglion axons in a near native state. We identify distinct vesicle populations with tightly versus broadly constrained size distributions and reveal a continuum of endolysosomal and autophagic intermediates that highlight the dynamic nature of membrane remodeling in growing axons. Unexpectedly, we observe vesicular structures enclosed within the lumen of the endoplasmic reticulum, suggesting a previously undescribed mechanism of ER membrane remodeling. Across multiple organelle classes, morphology and size are constrained by axonal geometry. This principle is most evident in mitochondria, which undergo dramatic narrowing and remodeling at varicosity to axon boundaries to traverse confined axonal segments. Together, these findings reveal spatial confinement as a fundamental organizing principle of axonal cell biology.