Primary cilia are conserved sensory hubs essential for signaling transduction and embryonic development. Ciliary dysfunction causes a variety of developmental syndromes with neurological features and cognitive impairment whose basis mostly remains unknown. Despite connections to neural function, the primary cilium remains an overlooked organelle in the brain. Most neurons have a primary cilium; however, it is still unclear how this organelle modulates brain architecture and function, given the lack of any systemic dissection of neuronal ciliary signaling. Here, we present the first in vivo glance at the molecular composition of cilia in the mouse brain. We have adapted in vivo BioID (iBioID), targeting the biotin ligase BioID2 to primary cilia in neurons of male and female mice. We identified tissue-specific signaling networks residing in neuronal cilia, including Eph/Ephrin signaling. We also uncovered a novel connection between primary cilia and GABA signaling. Our iBioID ciliary network presents a wealth of new and neural-specific ciliary signaling proteins and yields new insights into neurological disorders. Our findings are a promising first step in defining the fundamentals of ciliary signaling and their roles in shaping neural circuits and behavior. In the future, this work can be extended to pathological conditions of the brain, with the goal of identifying ciliary signaling pathways disrupted in these disorders and the ultimate aim of finding novel therapeutic strategies.Significance statement Primary cilia are sensory hubs crucial for signal transduction and embryonic development. Mutations in ciliary genes can lead to developmental disorders characterized by a wide spectrum of neurological impairments, the molecular basis of which is unknown. Despite its importance, the cilium's functions in the brain remain poorly understood. In this manuscript, we have adapted the in vivo proximity-dependent biotin identification (BioID) to identify the signaling outputs of cilia in neurons. We uncovered novel protein networks in neuronal cilia, including Eph/Ephrin and GABA receptor pathways. We also generated the first ciliary protein network in neurons and shared a wealth of neural hits that can help uncover how cilia mediate neural function and can become perturbed in neurological disorders.
Copyright © 2025 Loukil et al.

Mammalian oocytes undergo a long growth phase in the ovary, during which transcriptional levels gradually decrease. Growing oocytes must therefore accumulate maternal stores and regulate their translation to achieve successful divisions and early embryo development. Using immunofluorescence, mass spectrometry and electron microscopy, we identified a novel and transient compartment, the Zollo Body, in late growing mouse oocytes, constituted of RNPs and organelles. Morphologically, this structure resembles the Balbiani body found in most vertebrate species but it stains positively for nascent translation and active phospho-mTOR. RNAseq analysis and dry mass measurements of growing oocytes with or without this compartment further support its key role in boosting translation, allowing growing oocytes to avoid cytoplasmic dilution despite their rapid size increase, ultimately ensuring their developmental potential.

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The auxin-inducible degron (AID) system is a powerful tool for analyzing inducible protein loss in vitro, but it is toxic to mice. Here, we use an optimized second-generation AID system to achieve the conditional and reversible loss of the essential centrosomal protein CEP192 in live mice. We show that the auxin derivative 5-phenyl-indole-3-acetic acid is well tolerated over 2 weeks and drives near-complete CEP192 degradation in less than 1 hour in vivo. CEP192 loss did not affect centriole duplication but decreased γ-tubulin recruitment to centrosomes impairing mitotic spindle assembly. Sustained CEP192 loss in vivo led to cell division failure and cell death in proliferative tissues. Thus, the second-generation AID system is well suited for rapid and/or sustained protein depletion in live mice to study essential functions in vivo.

Numerous pathogenic variants causing human oocyte maturation arrest have been reported on the primate-specific TUBB8 gene. The main etiology is the dramatic reduction of tubulin α/β dimer, but still large numbers of variants remain unexplained.
Using microinjection mRNA and genome engineering to reintroduce the conserved pathogenic missense variants into oocytes or in generating TUBB8 variant knock-in mouse models, we investigated that the human deleterious variants alter microtubule nucleation and spindle assembly during meiosis. Live-cell imaging and immunofluorescence were utilised to track the dynamic expression of microtubule plus end-tracking proteins in vivo and analysed microtubule nucleation or spindle assembly in vitro, respectively. Immunoprecipitation-mass spectrometry and ultramicro-quantitative proteomics were performed to identify the differential abundance proteins and affected interactome of TUBB8 protein.
First, we observed a significant depletion of the EB1 signal upon microinjection of mutated TUBB8 mRNA (including R262Q, M300I, and D417N missense variants), indicating disruption of microtubule nucleation caused by these introduced TUBB8 missense variants. Mechanically, we demonstrated that the in vivo TUBB8-D417N missense variant diminished the affinity of EB1 and microtubules. It also harmed the interaction between microtubules and CKAP5/TACC3, which are crucial for initiating microtubule nucleation. Attenuated Ran-GTP pathway was also found in TUBB8-D417N oocytes, leading to disrupted spindle assembly. Stable microtubule was largely abolished on the spindle of TUBB8-D417N oocytes, reflected by reduced tubulin acetylation and accumulated HDAC6. More importantly, selective inhibition of HDAC6 by culturing TUBB8-D417N oocytes with Tubacin or Tubastatin A showed morphologically normal spindle and drastically recovered polar-body extrusion rate. These rescue results shed light on the strategy to treat meiotic defects in a certain group of TUBB8 mutated patients.
Our study provides a comprehensive mechanism elucidating how TUBB8 missense variants cause oocyte maturation arrest and offers new therapeutic avenues for treating female infertility in the clinic.
© 2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.

The Aurora Kinases (AURKs) are a family of serine-threonine protein kinases critical for cell division. Somatic cells express only AURKA and AURKB. However, mammalian germ cells and some cancer cells express all three isoforms. A major question in the field has been determining the molecular and cellular changes when cells express three instead of two aurora kinases. Using a systematic genetic approach involving different Aurora kinase oocyte-specific knockout combinations, we completed an oocyte-AURK genetic interaction map and show that one genomic copy of Aurka is necessary and sufficient to support female fertility and oocyte meiosis. We further confirm that AURKB and AURKC alone cannot compensate for AURKA. These results highlight the importance of AURKA in mouse oocytes, demonstrating that it is required for spindle formation and proper chromosome segregation. Surprisingly, a percentage of oocytes that lack AURKB can complete meiosis I, but the quality of those eggs is compromised, suggesting a role in AURKB to regulate spindle assembly checkpoint or control the cell cycle. Together with our previous studies, we wholly define the genetic interplay among the Aurora kinases and reinforce the importance of AURKA expression in oocyte meiosis.
Copyright © 2024 Blengini and Schindler.