Small-molecule probes are transformative for cell biology, offering unprecedented insights into subcellular structures, including in systems without molecular genetic tools. Centrioles are fundamental for generating the axoneme of cilia and flagella, as well as centrosomes, but a generic small-molecule probe allowing selective visualization of centriolar and axonemal microtubules is lacking. We engineered CenSpark as a cell-permeable dual-ligand fluorescent probe exploiting the juxtaposition of inner and outer microtubule-binding sites of microtubule triplets and doublets present exclusively in centriolar and axonemal microtubules. This design endows CenSpark high selectivity in live and fixed specimen analysis of centrioles, cilia and flagella across systems. We deployed CenSpark to uncover the rate of primary cilium formation and track centrioles in chimeric antigen receptor T cells during polarization at the immunological synapse with unprecedented resolution. Overall, CenSpark is a novel versatile small-molecule fluorescent probe to monitor centrioles, cilia and flagella without the need for genetic manipulation.

Multicellular development is tightly coupled to the polarization of individual cells, which partitions polar proteins along the cell cortex and can control asymmetric cell division, anisotropic growth, local differentiation or physiology. Mechanisms driving cell polarization have been described in fungi and animals, but these lack counterparts in plants. While several polarized proteins have been identified in plants, the overall mechanisms guiding their polar localization are poorly characterized. Through iterative affinity proteomics on the recently identified SOSEKI polar proteins, we discovered a network of polar proteins that is conserved in the flowering plant Arabidopsis and the liverwort Marchantia. We next used this collection of novel polarized proteins for systematic proximity ligation proteomics in these two species to map their global polar proteome. We identified a subfamily of polarized Protein S-acyl transferase (PAT) enzymes that are required for membrane targeting of Arabidopsis SOSEKI proteins. Using human and fruit fly models, we showed that PAT19 is sufficient for membrane targeting of SOSEKI1, likely through direct palmitoylation. This work demonstrates a conserved mechanism for polar protein targeting in plants and offers a resource for studying polar protein localization.

Abstract Animal microtubule-organizing centers, including the centrosome and the Golgi apparatus, regulate microtubule nucleation and anchoring through the γ-tubulin ring complex (γ-TuRC) and CAMSAP-mediated minus-end stabilization. However, functional redundancy between these pathways has impeded dissection of their contributions to controlling microtubule organization and density. Here, we addressed this problem using combinatorial gene knockouts, protein depletions and Expansion Microscopy. By simultaneously eliminating CAMSAP2 and the γ-TuRC-targeting proteins AKAP450, pericentrin, CDK5RAP2, myomegalin, ninein and AKNA, we generated viable RPE1 cells that lack both Golgi-derived microtubules and γ-TuRC localization within the pericentriolar material and at subdistal appendages. Despite the disruption of these major microtubule-organizing pathways, overall microtubule density was only partially reduced. The remaining microtubules depended on CEP192 and NEDD1, which, together with ch-TOG, can activate γ-TuRC at the centriole wall, in acentriolar cells, and in biochemical reconstitution assays. Our results demonstrate that in the absence of CAMSAP-mediated stabilization, interphase microtubule formation strongly relies on γ-TuRC activation, which occurs through several redundant pathways.

Kinesin tails are structurally diverse and mediate a range of functions, including autoinhibition, cargo binding, and microtubule regulation. Within the kinesin-4 family, KIF21A and KIF21B have emerged as key regulators of microtubule network organization and dynamics in neurons and immune cells, yet the molecular basis of this activity has remained unclear. Here, we combined single-particle cryo-electron microscopy (cryo-EM) and cryo- electron tomography (cryo-ET) to examine how the KIF21B tail engages microtubules. We find that conserved residues in the WD40 β-propeller and an adjacent N-terminal linker contact successive tubulin dimers along a single protofilament, forming an extended longitudinal binding mode that spans both the intradimer and interdimer interfaces. Cryo-EM 3D classification further revealed two distinct engagement states, a tilted state, in which the β- propeller makes partial contacts with the microtubule while the linker remains anchored, and a flat state, in which the β-propeller lies flush with the lattice surface. Cryo-ET of full-length KIF21B reveals multiple binding configurations on the microtubule lattice, including orientations consistent with crosslinking adjacent microtubules. Together, these findings provide a structural framework for tail-mediated microtubule attachment by a neuronal kinesin-4 and suggest how its distinctive WD40 domain may contribute to microtubule regulation and organization of microtubule arrays.

Abstract The ciliary transition zone gates bidirectional protein trafficking to maintain the specialized ciliary proteome using microtubule doublets as a scaffold. While ciliary axonemal doublets are well-characterized, the molecular architecture of the transition zone doublet remains elusive. Here, we report the structure of the mammalian transition zone doublet from bovine tracheal cilia using cryo-electron tomography at 4.7-5.0 Å resolution. The transition zone doublet is a structurally independent segment defined by an 8 nm-periodic arrangement of unique microtubule-inner proteins (MIPs) and microtubule-associated proteins (MAPs). We identify the calcium-binding protein CAPSL as a lumenal MIP that forms a pseudo-helical spiral and stabilizes microtubules in vitro . Furthermore, a dense MAP network on the A-microtubule surface clashes with intraflagellar transport (IFT) motor binding sites, suggesting anterograde IFT is directed to the B-microtubule for potential regulation by MAP9. Our work provides a structural framework for understanding gated ciliary transport and transition zone-linked human ciliopathies.

Microtubules are cytoskeletal filaments that form dynamic cytoplasmic arrays and stable cores of centrioles and cilia. Microtubule dynamics depend on GTP hydrolysis by tubulin: addition of GTP-tubulin creates a stabilizing cap, whereas cap loss triggers depolymerization. In structures with very slowly growing microtubules, such as centrioles and cilia, long GTP caps cannot form; instead, specialized protein complexes stabilize microtubule plus ends and support their slow elongation. Recent studies showed that although centrioles and cilia use distinct proteins to control their microtubule plus ends, the underlying mechanisms are similar. Both rely on complexes combining microtubule growth inhibitors with polymerases that counteract inhibition, which jointly stabilize the plus ends and drive their gradual extension. Both centriolar and ciliary microtubule tip regulators form assemblies that span inner and outer microtubule surfaces and reduce protofilament peeling. Because many centriole and ciliary tip proteins are mutated in human disorders, including microcephaly and ciliopathies, these findings provide insight into the molecular basis of such diseases.

Microtubule organization depends on cell type and function. Microtubule networks of many differentiated cell types, such as epithelial cells, are poorly understood due to their complexity and high density. Here, we used expansion microscopy to visualize and quantitatively map the three-dimensional organization of the microtubule network in human airway multiciliated cells. In these cells, most apical and apicobasal microtubules nucleate and anchor at the basal foot, a part of the ciliary basal body. A small subset of stable microtubules is detached from basal bodies and forms an apical crescent. By combining expansion microscopy with a newly developed averaging tool for multichannel volumetric data, we generated a high-resolution 3D map of the basal body. We delineated the position of structural components and proteins involved in microtubule nucleation and anchoring, uncovering some interesting differences with centrioles of dividing cells. γ-TuRC, its binding partners NEDD1 and augmin/HAUS, and centriolar appendage proteins ninein and AKNA localize to the basal foot. Functional analyses demonstrated that NEDD1 is essential for basal foot-dependent microtubule organization. Our data reveal the distinct architecture of microtubule-organizing centers responsible for the formation of dense microtubule arrays in multiciliated cells.

The cortically anchored adaptor KANK1 organizes microtubules at focal adhesions through a long, intrinsically disordered linker (L2), yet how this linker spans the ~35-50 nm membrane-microtubule gap is unclear. Here, we combine in-cell, biochemical, and biophysical assays, predictions of motif interaction and multivalent assembly using AlphaFold, and structural analysis by electron microscopy to show that the hub protein LC8, which binds more than 100 clients, converts the intrinsically disordered 600 amino acid L2 into an elongated, multivalent, rod-like assembly. In contrast, isolated motif peptides fail to bind LC8 at physiologically relevant concentrations, indicating that strong complex formation arises from cooperativity among multiple weak sites. These results establish LC8 as a molecular switch that rigidifies and extends KANK1 L2 via distributed weak motifs and short linkers. This interaction produces compositionally homogeneous yet conformationally adaptable rods, long enough to bridge the membrane-microtubule gap, resolving the paradox. This work expands the LC8 binding repertoire, reveals design principles for multivalent assembly, and suggests a generalizable strategy for tuning length, rigidity, and flexibility in large protein architectures.

Abstract The manchette is a transient microtubule (MT)-based structure that is vital for the correct shaping of sperm during spermiogenesis. Throughout spermiogenesis, the manchette retains structural integrity for several days, raising the question of how its MTs are regulated. Here, using cryo-electron tomography of manchettes isolated from rat testes, we find that manchette MT ends are structurally diverse. We show that the MT-binding protein CLASP2 is present throughout the manchette and likely regulates both MT ends. Using cryo-electron microscopy single particle analysis and super-resolution microscopy, we reveal that SPACA9 and MNMIP1 (SH3D21) bind to the seam of manchette MTs from the luminal side. SPACA9 binds to both α- and β-tubulin of protofilament 1 but does not interact directly with protofilament 13, while MNMIP1 binds directly to protofilament 13. MNMIP1 further extends and threads through the MT lattice at the seam. Our study reveals a novel seam MT inner protein complex with a unique binding mode, providing a plausible explanation for MT regulation that maintains manchette integrity over an extended period.

Microtubules are well-researched components of the cytoskeleton, yet we lack a holistic understanding that bridges molecular and cellular details with the broader functions of the microtubule cytoskeleton in development, ageing and disease. For example, how microtubule properties and functions are affected by tubulin post-translational modifications, disease-related mutations or variation of the microtubule lattice remains unexplored. In this Roadmap, we argue that integrating various experimental and theoretical approaches to bridge different spatial and temporal scales will offer new opportunities for gaining insights into essential cellular mechanisms and physiology, eventually revealing how microtubule dysfunction can lead to a broad spectrum of human diseases. Built on the current state of the art in the microtubule field, our Roadmap highlights future opportunities and challenges and proposes ways to tackle them. Given the many fundamental questions remaining to be answered, the microtubule cytoskeleton will continue to inspire scientists as it has been doing for decades.

In vitro air-liquid interface culture of airway epithelial cells is used as a model system to study respiratory diseases. This culture system not only overcomes the need for animal models or continuous biopsies from individuals but also enables studies of pathophysiology associated with the disease in a patient background. Human airway basal cells serve as progenitor cells for a functional pseudostratified airway epithelium composed mainly of multiciliated and secretory cells. However, due to the limited ability of basal cells to proliferate and differentiate, the long-term use of primary material in culture is restricted. This challenges research that requires genome editing. Here, we describe airway stem cells from nasal and bronchial origin immortalized by hTERT overexpression followed by polyclonal expansion. We demonstrate that this diverse panel of cell lines shows differentiation patterns similar to primary stem cells and can be used for lentiviral and CRISPR/Cas9 genome editing. These cell lines and optimized protocols facilitate airway biology research and disease phenotyping. ### Competing Interest Statement The authors have declared no competing interest. Netherlands Organization for Scientific Research, Gravitation programme IMAGINE! (project number 24.005.009) EindhovenWageningen-Utrecht Alliance, Centre for Living Technologies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, hijacks host cellular machinery to replicate and spread. Understanding how SARS-CoV-2 reorganizes host cell architecture to accommodate this is essential for elucidating its pathogenesis and identifying therapeutic targets. While the molecular mechanisms of SARS-CoV-2 entry are well characterized, the pathways governing viral egress remain incompletely understood. Conventional approaches such as transcriptomics and electron microscopy have provided valuable insights but lack the combined spatial and molecular resolution needed to map these processes within intact cells. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to visualize SARS-CoV-2–induced remodeling of the endolysosomal system in multiciliated cells of primary human airway epithelial tissue. This approach reveals Golgi fragmentation and the formation of enlarged virus-containing organelles. Analysis of endolysosomal markers in Vero E6 cells shows that these structures are positive for CD63, Rab7, and LC3, consistent with amphisome identity. Moreover, pharmacological inhibition of Rab27-dependent amphisome–plasma membrane fusion with Nexinhib20 reduces viral infection, implicating secretory autophagy as a pathway for SARS-CoV-2 egress. These findings establish expansion microscopy as a powerful tool for spatial virology and uncover a Rab27-mediated amphisome fusion mechanism as a druggable route for SARS-CoV-2 release.

Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome. We show that CEP104, a protein with a tubulin-binding TOG domain, and its luminal partner CSPP1 inhibit microtubule growth and shortening. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics but act as scaffolds for their partners. Cryo-electron tomography demonstrated that, together, ciliary tip module members form plus-end-specific cork-like structures that reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.

Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but it reduced cell stiffness. Mitochondria and endoplasmic reticulum (ER) sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.