Lv, Q. H. | bioRxiv Science

bioRxiv2025

Dynein-2 is tuned for the A-tubule of the ciliary doublet through tubulin tyrosination

Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia structures rely on intraflagellar transport (IFT), which involves dynein-2 for retrograde and kinesin-2 for anterograde movements along doublet microtubules. Unlike dynein-1, which works on singlet microtubules within the cytoplasm, dynein-2 specifically works on the doublet microtubules inside the cilia. Previous cryo-electron tomography studies have shown that retrograde IFT, driven by dynein-2, occurs on the A-tubule of the doublet, suggesting a specialized regulatory mechanism involving dynein-2. However, the molecular basis of this specificity remains unclear. Here, we investigated this mechanism using cryo-electron tomography (cryo-ET) with Volta Phase Plate (VPP), molecular dynamics (MD) simulations, and biochemical analysis. Our biochemical assay revealed that the microtubule-binding domain of dynein-2 exhibits a higher affinity for the ciliary doublet microtubule compared to dynein-1. Cryo-ET with VPP further visualized the preferential binding of dynein-2 to the A-tubule of the doublet microtubule. MD simulations suggest that the preferential binding of dynein-2 is attributed to the tyrosinated tubulin in the A-tubule. These findings uncover a tyrosination-dependent regulatory mechanism that governs the bidirectional transport of IFT on doublet microtubules, providing new insights into the spatial and functional specialization of ciliary transport systems.

biophysics↗

bioRxiv2024

Optical Control of Microtubule Accumulation and Dispersion by Tau-Derived Peptide-Fused Photo-Responsive Protein

Microtubules, a major component of the cytoskeleton consisting of tubulin dimers, are involved in various cellular functions, including forming axons and dendrites of neurons and retaining cell shapes by forming various accumulated superstructures such as bundles and doublets. Moreover, microtubule-accumulated structures like swarming microtubule assemblies are attractive components for dynamic materials, such as active matter and molecular robots. Thus, dynamic control of microtubule superstructures is an important topic. However, implementing stimulus-dependent control of superstructures remains challenging. This challenge can be resolved by developing designer protein approaches. We have previously developed a Tau-derived peptide (TP), which binds to the inner or outer surface of microtubules depending on the timing of the incubation. In this report, we designed the TP-fused photo-switchable protein Dronpa (TP-Dronpa) that reversibly photoconverts between monomeric and tetrameric states to photocontrol microtubule assemblies. The formation of microtubule superstructures, including bundles and doublets, was induced by tetrameric TP-Dronpa, whereas monomeric TP-Dronpa ensured that microtubules remained dispersed. Tetrameric TP-Dronpa also induced motile aster-like structures and swarming movement of microtubules on a kinesin-coated substrate. The formation/dissociation of these microtubule superstructures can be controlled by light irradiation. This system can generate and photocontrol various microtubule superstructures and provides an approach to facilitate the assembly of dynamic materials for various applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/614838v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@fdcc92org.highwire.dtl.DTLVardef@81bf32org.highwire.dtl.DTLVardef@2f7f5eorg.highwire.dtl.DTLVardef@52fc9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗