A disease-associated PPP2R3C-MAP3K1 phospho-regulatory module controls centrosome function.

Centrosomes have critical roles in microtubule organization, ciliogenesis, and cell signaling.1,2,3,4,5,6,7,8 Centrosomal alterations also contribute to diseases, including microcephaly, cancer, and ciliopathies.9,10,11,12,13 To date, over 150 centrosomal proteins have been identified, including several kinases and phosphatases that control centrosome biogenesis, function, and maintenance.2,3,4,5,14,15,16,17,18,19,20,21 However, the regulatory mechanisms that govern centrosome function are not fully defined, and thus how defects in centrosomal regulation contribute to disease is incompletely understood. Using a systems genetics approach, we find here that PPP2R3C, a poorly characterized PP2A phosphatase subunit, is a distal centriole protein and functional partner of centriolar proteins CEP350 and FOP. We further show that a key function of PPP2R3C is to counteract the kinase activity of MAP3K1. In support of this model, MAP3K1 knockout suppresses growth defects caused by PPP2R3C inactivation, and MAP3K1 and PPP2R3C have opposing effects on basal and microtubule stress-induced JNK signaling. Illustrating the importance of balanced MAP3K1 and PPP2R3C activities, acute overexpression of MAP3K1 severely inhibits centrosome function and triggers rapid centriole disintegration. Additionally, inactivating PPP2R3C mutations and activating MAP3K1 mutations both cause congenital syndromes characterized by gonadal dysgenesis.22,23,24,25,26,27,28 As a syndromic PPP2R3C variant is defective in centriolar localization and binding to centriolar protein FOP, we propose that imbalanced activity of this centrosomal kinase-phosphatase pair is the shared cause of these disorders. Thus, our findings reveal a new centrosomal phospho-regulatory module, shed light on disorders of gonadal development, and illustrate the power of systems genetics to identify previously unrecognized gene functions.

Heterozygous cis HYDIN mutations cause primary ciliary dyskinesia.

BACKGROUND: The product of ciliary gene HYDIN is an integral component for c2b projection within the motile cilia central pair (CP) apparatus. Biallelic mutations of this gene cause primary ciliary dyskinesia (PCD), an uncommon heterogeneous recessive disorder affecting motile cilia, resulting in defective mucociliary clearance that leads to chronic suppurative lung disease. METHODS: Nasal brushing samples were collected from two siblings attending the Victorian Diagnostic service for PCD. Nasal airway epithelial cells (NAECs) were cultured before cilia structure and function studies using high-speed video microscopy (HSVM), transmission electron microscopy, and immunofluorescence. FINDINGS: Cultured NAECs from both siblings showed defective cilia beating patterns under HSVM. A confirmatory PCD diagnosis was achieved through immunofluorescence, which showed the loss of HYDIN and the associated protein SPEF2 from the cilia axoneme. CONCLUSIONS: This case report details the diagnosis of two siblings who displayed similar defective cilia beating phenotypes seen in patients with PCD bearing recessive HYDIN mutations. Uniquely, both siblings carry two previously unreported HYDIN mutations, which are in the cis position, demonstrating the possibility for disease manifestation without biallelic mutations of ciliary genes. FUNDING: The authors declare no funding support for this study.

The effects of primary cilia-mediated mechanical stimulation on nestin+-BMSCs during bone-tendon healing.

INTRODUCTION: Mechanical stimulation has been proven to promote bone-tendon interface (BTI) healing, but the mechanism remains unclear. OBJECTIVE: To investigate the effects of mechanical stimulation on the biological behavior of nestin+-bone mesenchymal stem cells (BMSCs) during the BTI healing, and to reveal the mechanisms of mechanical stimulation affecting BTI healing by primary cilia on the nestin+-BMSCs. METHODS: Transgenic tracing mice (nestin creERT2:: IFT88fl/fl/ROSA26 YFP) with primary cilia on nestin+-BMSCs conditioned knocked out were constructed, and the littermates (nestin creERT2:: ROSA26 YFP) with normal cilia on nestin+-BMSCs were the control. After establishing mouse supraspinatus insertion injury models, samples were collected at week-2 (n = 5 per group), 4 and 8 (n = 15 per group, respectively). In vivo, the repair efficiency was evaluated by histology, imaging, biomechanics, and the migration of nestin+-BMSCs, detected by immunofluorescence staining. In vitro, nestin+ BMSCs were sorted and stimulated by tensile force to study the mechanisms of primary cilium-mediated mechanosensitive basis. RESULTS: Mechanical stimulation (MS) accelerated the recruitment of nestin+-BMSCs and promoted osteogenic and chondrogenic capacity. Histological, imaging and biomechanical results showed that the BTI healing quality of the IFT88+/+, MS group was better than that of the other groups. After the conditionally knockout IFT88 in nestin+-BMSCs, the repair ability of the BTI was obviously deteriorated, even though mechanical stimulation did not increase significantly (IFT88-/-, MS group). In vitro results showed the tensile loading enhanced the proliferation, migration and osteogenic or chondrogenic gene expression of nestin+-BMSCs with normal cilia. On the other hand, osteogenesis and chondrogenic expression were significantly decreased after inhibiting actin- Hippo/YAP pathway components. CONCLUSION: The primary cilia mediated mechanical stimulation regulated osteogenic and chondrogenic differentiation potential of nestin+-BMSCs through the actin- Hippo/YAP pathway, and then promoted the BTI healing process.

The role of cilia in the development, survival, and regeneration of hair cells.

Mutations impacting cilia genes lead to a class of human diseases known as ciliopathies. This is due to the role of cilia in the development, survival, and regeneration of many cell types. We investigated the extent to which disrupting cilia impacted these processes in lateral line hair cells of zebrafish. We found that mutations in two intraflagellar transport (IFT) genes, ift88 and dync2h1, which lead to the loss of kinocilia, caused increased hair cell apoptosis. IFT gene mutants also have a decreased mitochondrial membrane potential, and blocking the mitochondrial uniporter causes a loss of hair cells in wild-type zebrafish but not mutants, suggesting mitochondria dysfunction may contribute to the apoptosis seen in these mutants. These mutants also showed decreased proliferation during hair cell regeneration but did not show consistent changes in support cell number or proliferation during hair cell development. These results show that the loss of hair cells seen following disruption of cilia through either mutations in anterograde or retrograde IFT genes appears to be due to impacts on hair cell survival but not necessarily development in the zebrafish lateral line.

Protofilament-specific nanopatterns of tubulin post-translational modifications regulate the mechanics of ciliary beating.

Controlling ciliary beating is essential for motility and signaling in eukaryotes. This process relies on the regulation of various axonemal proteins that assemble in stereotyped patterns onto individual microtubules of the ciliary structure. Additionally, each axonemal protein interacts exclusively with determined tubulin protofilaments of the neighboring microtubule to carry out its function. While it is known that tubulin post-translational modifications (PTMs) are important for proper ciliary motility, the mode and extent to which they contribute to these interactions remain poorly understood. Currently, the prevailing understanding is that PTMs can confer functional specialization at the level of individual microtubules. However, this paradigm falls short of explaining how the tubulin code can manage the complexity of the axonemal structure where functional interactions happen in defined patterns at the sub-microtubular scale. Here, we combine immuno-cryo-electron tomography (cryo-ET), expansion microscopy, and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nanopatterns at a sub-microtubular scale. These nanopatterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are indispensable for their regulation during ciliary beating. Our findings offer a new paradigm for understanding how different tubulin PTMs, such as glycylation, glutamylation, acetylation, tyrosination, and detyrosination, can coexist within the ciliary structure and specialize individual protofilaments for the regulation of diverse protein complexes. The identification of a ciliary tubulin nanocode by cryo-ET suggests the need for high-resolution studies to better understand the molecular role of PTMs in other cellular compartments beyond the cilium.

Encoding spatiotemporal asymmetry in artificial cilia with a ctenophore-inspired soft-robotic platform.

A remarkable variety of organisms use metachronal coordination (i.e. numerous neighboring appendages beating sequentially with a fixed phase lag) to swim or pump fluid. This coordination strategy is used by microorganisms to break symmetry at small scales where viscous effects dominate and flow is time-reversible. Some larger organisms use this swimming strategy at intermediate scales, where viscosity and inertia both play important roles. However, the role of individual propulsor kinematics-especially across hydrodynamic scales-is not well-understood, though the details of propulsor motion can be crucial for the efficient generation of flow. To investigate this behavior, we developed a new soft robotic platform using magnetoactive silicone elastomers to mimic the metachronally coordinated propulsors found in swimming organisms. Furthermore, we present a method to passively encode spatially asymmetric beating patterns in our artificial propulsors. We investigated the kinematics and hydrodynamics of three propulsor types, with varying degrees of asymmetry, using Particle Image Velocimetry and high-speed videography. We find that asymmetric beating patterns can move considerably more fluid relative to symmetric beating at the same frequency and phase lag, and that asymmetry can be passively encoded into propulsors via the interplay between elastic and magnetic torques. Our results demonstrate that nuanced differences in propulsor kinematics can substantially impact fluid pumping performance. Our soft robotic platform also provides an avenue to explore metachronal coordination at the meso-scale, which in turn can inform the design of future bioinspired pumping devices and swimming robots.

Circadian cilia transcriptome in mouse brain across physiological and pathological states.

Primary cilia are dynamic sensory organelles that continuously undergo structural modifications in response to environmental and cellular signals, many of which exhibit rhythmic patterns. Building on our previous findings of rhythmic cilia-related gene expression in diurnal primates (baboon), this study extends the investigation to the nocturnal mouse brain to identify circadian patterns of cilia gene expression across brain regions. We used computational techniques and transcriptomic data from four publicly available databases, to examine the circadian expression of cilia-associated genes within six brain areas: brainstem, cerebellum, hippocampus, hypothalamus, striatum, and suprachiasmatic nucleus. Our analysis reveals that a substantial proportion of cilia transcripts exhibit circadian rhythmicity across the examined regions, with notable overrepresentation in the striatum, hippocampus, and cerebellum. We also demonstrate region-specific variations in the abundance and timing of circadian cilia genes' peaks, indicating an adaptation to the distinct physiological roles of each brain region. Additionally, we show that the rhythmic patterns of cilia transcripts are shifted under various physiological and pathological conditions, including modulation of the dopamine system, high-fat diet, and epileptic conditions, indicating the adaptable nature of cilia transcripts' oscillation. While limited to a few mouse brain regions, our study provides initial insights into the distinct circadian patterns of cilia transcripts and highlights the need for future research to expand the mapping across wider brain areas to fully understand the role of cilia's spatiotemporal dynamics in brain functions.

Cdon is essential for organ left-right patterning by regulating dorsal forerunner cells clustering and Kupffer's vesicle morphogenesis.

Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they have been reported to be involved in muscle and neural development at late developmental stage, their early roles in embryonic development remain unknown. Here, we discovered that in zebrafish, cdon, but not boc, is expressed in dorsal forerunner cells (DFCs) and the epithelium of Kupffer's vesicle (KV), suggesting a potential role for cdon in organ left-right (LR) patterning. Further data showed that liver and heart LR patterning were disrupted in cdon morphants and cdon mutants. Mechanistically, we found that loss of cdon function led to defect in DFCs clustering, reduced KV lumen, and defective cilia, resulting in randomized Nodal/spaw signaling and subsequent organ LR patterning defects. Additionally, predominant distribution of a cdon morpholino (MO) in DFCs caused defects in DFC clustering, KV morphogenesis, cilia number/length, Nodal/spaw signaling, and organ LR asymmetry, similar to those observed in cdon morphants and cdon -/- embryos, indicating a cell-autonomous role for cdon in regulating KV formation during LR patterning. In conclusion, our data demonstrate that during gastrulation and early somitogenesis, cdon is essential for proper DFC clustering, KV formation, and normal cilia, thereby playing a critical role in establishing organ LR asymmetry.

A prioritization tool for cilia-associated genes and their in vivo resources unveil new avenues for ciliopathy research.

Defects in ciliary signaling or mutations in proteins that localize to primary cilia lead to a class of human diseases known as ciliopathies. About 10% of mammalian genes encode cilia-associated proteins and a major gap in the cilia research field is prioritizing which genes to study and finding the in vivo vertebrate mutant alleles and reagents available for their study. Here we present a unified resource listing the cilia-associated human genes cross-referenced to available mouse and zebrafish mutant alleles, their associated phenotypes as well as expression data in kidney and functional data for vertebrate Hedgehog signaling. This resource empowers researchers to easily sort and filter genes based on their own expertise and priorities, cross-reference with newly-generated -omics datasets, and quickly find in vivo resources and phenotypes associated with a gene of interest.

Fly Fam161 is an essential centriole and cilium transition zone protein with unique and diverse cell type-specific localizations.

Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.

Multiciliated ependymal cells: an update on biology and pathology in the adult brain.

Mature multiciliated ependymal cells line the cerebral ventricles where they form a partial barrier between the cerebrospinal fluid (CSF) and brain parenchyma and regulate local CSF microcirculation through coordinated ciliary beating. Although the ependyma is a highly specialized brain interface with barrier, trophic, and perhaps even regenerative capacity, it remains a misfit in the canon of glial neurobiology. We provide an update to seminal reviews in the field by conducting a scoping review of the post-2010 mature multiciliated ependymal cell literature. We delineate how recent findings have either called into question or substantiated classical views of the ependymal cell. Beyond this synthesis, we document the basic methodologies and study characteristics used to describe multiciliated ependymal cells since 1980. Our review serves as a comprehensive resource for future investigations of mature multiciliated ependymal cells.

Two Tetrahymena kinesin-9 family members exhibit slow plus-end-directed motility in vitro.

The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family.

Constructing condylar cartilage organoid to explore primary cilia functions.

An organoid culture system better recapitulates the cellular structure, function, and interaction between cells and the extracellular matrix (ECM) than a two-dimensional (2D) culture system. We here constructed a condylar cartilage organoid to explore the regulatory role of primary cilia. Similar to the natural condylar cartilage, the condylar cartilage organoid exhibited abundant ECM and comprised superficial, proliferative, and hypertrophic zones. Primary cilia in the condylar cartilage organoid were shorter on average than those in the 2D culture chondrocytes, but their average length was equivalent to those in the natural condylar cartilage. Notably, primary cilia in each zone of the condylar cartilage organoid had an average length similar to that of primary cilia in the natural condylar cartilage. According to transcriptomic and biochemical analyses, the expression of cilia-related genes and cilia-related Hedgehog (HH) signaling differed between the condylar cartilage organoid and 2D culture systems. IFT88 knockdown promoted the protein levels of COL-Ⅹ, TRPV4, and HH signaling molecules in the condylar cartilage organoid, but decreased them in the 2D culture system. Notably, the protein levels of COL-Ⅹ, TRPV4, and HH signaling molecules increased in the superficial zone of the si IFT88 condylar cartilage organoid compared with the condylar cartilage organoid. However, the protein levels of aforementioned molecules were not significantly different in proliferative and hypertrophic zones. Collectively, we successfully constructed the condylar cartilage organoid with a better tissue structure and abundant ECM. Moreover, the condylar cartilage organoid is more suitable for exploring primary cilia functions.

Identifying the roles of miR-17 in ciliogenesis and cell cycle.

Emerging evidence suggests a significant contribution of primary cilia to cell division and proliferation. MicroRNAs, especially miR-17, contribute to cell cycle regulation and proliferation. Recent investigations have highlighted the dysregulated expression of miR-17 in various malignancies, underlining its potential role in cancer. However, the correlation between primary cilia and miR-17 has yet to be fully elucidated. The present study examines the presence of miR-17 in primary cilia. The miR-17 expression is studied in selected ciliary protein knockdown cells. Using in situ hybridization (ISH), we identified the subcellular localization of miR-17 in both cilium and cell body. We confirmed the importance of miR-17, progesterone receptor membrane component-2 (PGRMC2), and monosialodihexosylganglioside (GM3S) in cilia formation, as shown by the significant reduction in cilia and cilia length in knockdown cells compared to control. We also demonstrated the involvement of PGRMC2, GM3S, polycystin-2 (PKD2), and miR-17 in cellular proliferation and cell growth. Our studies revealed a hyperproliferative effect in the knockdown cells compared to control cells, suggesting the regulatory roles of PGRMC2/GM3S/PKD2/miR-17 in promoting cell proliferation. Overall, our studies conclude that ciliary proteins are involved in cell division and proliferation. We further hypothesize that primary cilia can serve as compartments to store and control genetic materials, further implicating their complex involvement in cellular processes.

HYDIN variants cause primary ciliary dyskinesia in the Finnish population.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by chronic respiratory tract infections and in some cases laterality defects and infertility. The symptoms of PCD are caused by malfunction of motile cilia, hair-like organelles protruding out of the cell. Thus far, disease causing variants in over 50 genes have been identified and these variants explain around 70% of all known cases. Population specific genetics underlying PCD has been reported highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics and management. METHODS: Whole exome sequencing was used to identify disease causing variants in Finnish PCD cohort. The effect of the identified HYDIN variants on cilia structure and function was confirmed by high-speed video analysis, immunofluorescence and electron tomography. RESULTS: In this study, we identified three Finnish PCD patients carrying homozygous loss-of-function variants and one patient with compound heterozygous variants within HYDIN. The functional studies showed defects in the axonemal central pair complex. All patients had clinical PCD symptoms including chronic wet cough and recurrent airway infections, associated with mostly static airway cilia. CONCLUSION: Our results are consistent with the previously identified important role of HYDIN in the axonemal central pair complex and improve specific diagnostics of PCD in different national populations.

Haploinsufficiency of intraflagellar transport protein 172 causes autism-like behavioral phenotypes in mice through BDNF.

INTRODUCTION: Primary cilia are hair-like solitary organelles growing on most mammalian cells that play fundamental roles in embryonic patterning and organogenesis. Defective cilia often cause a suite of inherited diseases called ciliopathies with multifaceted manifestations. Intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium, actively facilitates the formation and absorption of primary cilia. IFT172 is the largest component of the IFT-B complex, and its roles in Bardet-Biedl Syndrome (BBS) have been appreciated with unclear mechanisms. OBJECTIVES: We performed a battery of behavioral tests with Ift172 haploinsufficiency (Ift172+/-) and WT littermates. We use RNA sequencing to identify the genes and signaling pathways that are differentially expressed and enriched in the hippocampus of Ift172+/- mice. Using AAV-mediated sparse labeling, electron microscopic examination, patch clamp and local field potential recording, western blot, luciferase reporter assay, chromatin immunoprecipitation, and neuropharmacological approach, we investigated the underlying mechanisms for the aberrant phenotypes presented by Ift172+/- mice. RESULTS: Ift172+/- mice displayed excessive self-grooming, elevated anxiety, and impaired cognition. RNA sequencing revealed enrichment of differentially expressed genes in pathways relevant to axonogenesis and synaptic plasticity, which were further confirmed by less spine density and synaptic number. Ift172+/- mice demonstrated fewer parvalbumin-expressing neurons, decreased inhibitory synaptic transmission, augmented theta oscillation, and sharp-wave ripples in the CA1 region. Moreover, Ift172 haploinsufficiency caused less BDNF production and less activated BDNF-TrkB signaling pathway through transcription factor Gli3. Application of 7,8-Dihydroxyflavone, a potent small molecular TrkB agonist, fully restored BDNF-TrkB signaling activity and abnormal behavioral phenotypes presented by Ift172+/- mice. With luciferase and chip assays, we provided further evidence that Gli3 may physically interact with BDNF promoter I and regulate BDNF expression. CONCLUSIONS: Our data suggest that Ift172 per se drives neurotrophic effects and, when defective, could cause neurodevelopmental disorders reminiscent of autism-like disorders.

Centriole Translational Planar Polarity in Monociliated Epithelia.

Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left-right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process.

Multicellularity and increasing Reynolds number impact on the evolutionary shift in flash-induced ciliary response in Volvocales.

BACKGROUND: Volvocales in green algae have evolved by multicellularity of Chlamydomonas-like unicellular ancestor. Those with various cell numbers exist, such as unicellular Chlamydomonas, four-celled Tetrabaena, and Volvox species with different cell numbers (~1,000, ~5,000, and ~10,000). Each cell of these organisms shares two cilia and an eyespot, which are used for swimming and photosensing. They are all freshwater microalgae but inhabit different fluid environments: unicellular species live in low Reynolds-number (Re) environments where viscous forces dominate, whereas multicellular species live in relatively higher Re where inertial forces become non-negligible. Despite significant changes in the physical environment, during the evolution of multicellularity, they maintained photobehaviors (i.e., photoshock and phototactic responses), which allows them to survive under changing light conditions. RESULTS: In this study, we utilized high-speed imaging to observe flash-induced changes in the ciliary beating manner of 27 Volvocales strains. We classified flash-induced ciliary responses in Volvocales into four patterns: "1: temporal waveform conversion", "2: no obvious response", "3: pause in ciliary beating", and "4: temporal changes in ciliary beating directions". We found that which species exhibit which pattern depends on Re, which is associated with the individual size of each species rather than phylogenetic relationships. CONCLUSIONS: These results suggest that only organisms that acquired different patterns of ciliary responses survived the evolutionary transition to multicellularity with a greater number of cells while maintaining photobehaviors. This study highlights the significance of the Re as a selection pressure in evolution and offers insights for designing propulsion systems in biomimetic micromachines.

mTOR potentiates senescent phenotypes and primary cilia formation after cisplatin-induced G2 arrest in retinal pigment epithelial cells.

Cisplatin, a platinum-based anticancer drug, is used to treat several types of cancer. Despite its effectiveness, cisplatin-induced side effects have often been reported. Although cisplatin-induced toxicities, such as apoptosis and/or necrosis, have been well studied, the fate of cells after exposure to sublethal doses of cisplatin needs further elucidation. Treatment with a sublethal dose of cisplatin induced cell cycle arrest at the G2 phase in retinal pigment epithelial cells. Following cisplatin withdrawal, the cells irreversibly exited the cell cycle and became senescent. Notably, the progression from the G2 to the G1 phase occurred without mitotic entry, a phenomenon referred to as mitotic bypass, resulting in the accumulation of cells containing 4N DNA content. Cisplatin-exposed cells exhibited morphological changes associated with senescence, including an enlarged size of cell and nucleus and increased granularity. In addition, the senescent cells possessed primary cilia and persistent DNA lesions. Senescence induced by transient exposure to cisplatin involves mTOR activation. Although transient co-exposure with an mTORC1 inhibitor rapamycin did not prevent mitotic bypass and entry into senescence, it delayed the progression of senescence and attenuated senescent phenotypes, resulting in shorter primary cilia formation. Conclusively, cisplatin induces senescence in retinal pigment epithelial cells by promoting mTOR activation.