Verwei, H. N. | bioRxiv Science

bioRxiv2026

In situ structures of the ciliary tip show the multi-step mechanism of axoneme assembly

Motile cilia are highly complex, ordered organelles which extend from the cell surface to generate fluid flow. During motile cilia assembly, components rapidly self-organize at the ciliary tip. Here, over two hundred different components bind microtubules to form an intricate, repeating structure called the axoneme. Despite in-depth structural characterization of axonemal complexes, we have limited understanding of how these structures are generated. Here, we directly visualize the steps of axonemal assembly in situ by performing cryo-electron tomography of Chlamydomonas reinhardtii cilia tips. Using subtomogram averaging and classification, we identified conserved microtubule luminal proteins that establish periodicity and show that controlled microtubule lattice perforation can facilitate incorporation of late-binding components. By uncovering unexpected assembly intermediates, we explain how structural integrity and the emergence of motility are coordinated within the axoneme. Overall, we demonstrate that self-assembly proceeds through regulated, sequential construction steps and provide a molecular framework for understanding axoneme biogenesis.

molecular biology↗

bioRxiv2025

Polarity reversal of stable microtubules during neuronal development

Neurons critically depend on long-distance transport orchestrated by motor proteins walking over their highly asymmetric microtubule cytoskeleton. These microtubules are organized uniformly in axons with their plus-end pointing away from the soma. In contrast, in the dendrites of vertebrate neurons, microtubules are of mixed polarity, but organized into bundles of uniform polarity with stable, long-lived microtubules preferentially oriented minus-end-out and dynamic microtubules oriented plus-end-out. This organization is thought to be essential for guiding selective transport into dendrites, yet how this organization is established is unclear. Here we use a combination of single molecule localization microscopy, expansion microscopy, and live-cell imaging to examine how the microtubule cytoskeleton is reorganized during neuronal development of cultured rat hippocampal neurons. We find that, while the youngest neurites contain microtubules of mixed polarity, stable microtubules are initially preferentially oriented plus-end-out. At this stage of development many stable microtubules are connected to the centrioles, providing an explanation for their plus-end out orientation in emerging neurites. In later stages, these microtubules are released from the centrioles and reorient by sliding between or within neurites to become progressively more minus-end-out. Moreover, prior to axon specification, we commonly observed already one or two minor neurites with an almost uniformly plus-end-out microtubule network, indicative of transient polarization. Together, our findings reveal how stable microtubules are reorganized to help establish the stereotypical microtubule networks seen in the axon and dendrites of mature vertebrate neurons.

cell biology↗