NDE1 Localizes to the Subdistal Appendages to Maintain Centrosome Integrity and Microtubule Organization

Abstract

Centrosomes organize microtubules, coordinate ciliogenesis, and support accurate cell division. At the mature mother centriole, distal and subdistal appendages confer specialized functions in ciliary docking, microtubule organization, and intracellular trafficking. Nuclear distribution element 1 (NDE1) is a centrosome-associated regulator of dynein-dependent processes and ciliogenesis, but its nanoscale organization and contribution to centrosome architecture are poorly understood. Here, using expansion microscopy and STED super-resolution imaging, we show that endogenous NDE1 forms a ring-like structure at the subdistal appendages in mouse embryonic fibroblasts and human RPE-1 cells. NDE1 occupies an intermediate radial position between the more centriole-proximal CEP128 layer and the more peripheral NIN (ninein) layer. Depletion of ODF2 or CEP128 reduces centrosomal NDE1, whereas CEP170 depletion has little effect. Conversely, NDE1 depletion impairs NIN centrosomal enrichment, positioning NDE1 downstream of ODF2 and CEP128 and upstream of NIN within the subdistal appendage assembly hierarchy. NDE1 depletion compromises centrosome integrity, reduces the centrosomal enrichment of core centriolar proteins, increases the separation between paired centrioles, and generates ectopic foci containing multiple centriolar markers. Loss of NDE1 also disrupts pericentriolar material organization and impairs the establishment of focused, centrosome-associated microtubule arrays. Together, our findings establish NDE1 as a subdistal appendage-associated factor that supports centrosome architecture and microtubule-organizing activity. More broadly, they support an emerging view of subdistal appendages as a molecularly layered platform in which distinct but cooperating components connect mother centriole maturation to microtubule organization, ciliary regulation, and intracellular trafficking.