The placenta experiences a low-oxygen stage during early pregnancy. Aspirin is an effective preventative treatment for preeclampsia if applied early in pregnancy. Elevation of fibronectin (FN) level has been reported to be associated with preeclampsia; however, the role of FN in the physiological hypoxic phase and whether aspirin exerts its effect on FN at this hypoxic stage remain unknown. We determined pregnancy outcomes by injecting saline or recombinant FN protein into C57BL/6 pregnant mice and one group of FN-injected mice was fed aspirin. The effects of FN, the underlying pathways on trophoblast biology, and cilia formation under hypoxia were investigated in FN-pretreated or FN-knockdown HTR-8/SVneo cells in a hypoxic chamber (0.1 % O2). Preeclampsia-like phenotypes, including blood pressure elevation and proteinuria, developed in FN-injected pregnant mice. The fetal weight of FN-injected mice was significantly lower than that of non-FN-injected mice (p < 0.005). Trophoblast FN expression was upregulated under hypoxia, which could be suppressed by aspirin treatment. FN inhibited trophoblast invasion and migration under hypoxia, and this inhibitory effect occurred through downregulating ZEB1/2, MMP 9 and the Akt and MAPK signaling pathways. Ciliogenesis of trophoblasts was stimulated under hypoxia but was inhibited by FN treatment. Aspirin was shown to reverse the FN-mediated inhibitory effect on trophoblast invasion/migration and ciliogenesis. In conclusion, FN overexpression induces preeclampsia-like symptoms and impairs fetal growth in mice. Aspirin may exert its suppressive effect on FN upregulation and FN-mediated cell function in the hypoxic stage of pregnancy and therefore provides a preventative effect on preeclampsia development.

BACKGROUND: Primary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear.
METHODS: TonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis.
RESULTS: We identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation.
CONCLUSIONS: This study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP.

The primary cilium is a microtubule-based sensory organelle that plays a critical role in signaling pathways and cell cycle progression. Defects in the structure and/or function of the primary cilium result in developmental diseases collectively known as ciliopathies. However, the constituents and regulatory mechanisms of the primary cilium are not fully understood. In recent years, the activity of the epigenetic modifier SMYD3 has been shown to play a key role in the regulation of cell cycle progression. However, whether SMYD3, a histone/lysine methyltransferase, contributes to the regulation of ciliogenesis remains unknown. Here, we report that SMYD3 drives ciliogenesis via the direct and indirect regulation of cilia-associated components. We show that SMYD3 is a novel component of the distal appendage and is required for centriolar appendage assembly. The loss of SMYD3 decreased the percentage of ciliated cells and resulted in the formation of stumpy cilia. We demonstrated that SMYD3 modulated the recruitment of centrosome proteins (Cep164, Fbf1, Ninein, Ttbk2 and Cp110) and the trafficking of intraflagellar transport proteins (Ift54 and Ift140) important for cilia formation and maintenance, respectively. In addition, we showed that SMYD3 regulated the transcription of cilia genes and bound to the promoter regions of C2cd3, Cep164, Ttbk2, Dync2h1 and Cp110. This study provides insights into the role of SMYD3 in cilia biology and suggests that SMYD3-mediated cilia formation/function may be relevant for cilia-dependent signaling in ciliopathies.

The unicellular green alga, Chlamydomonas reinhardtii, has played a central role in discovering much of what is currently known about the composition, assembly, and function of cilia and flagella. Chlamydomonas combines excellent genetics, such as the ability to grow cells as haploids or diploids and to perform tetrad analysis, with an unparalleled ability to detach and isolate flagella in a single step without cell lysis. The combination of genetics and biochemistry that is possible in Chlamydomonas has allowed many of the key components of the cilium to be identified by looking for proteins that are missing in a defined mutant. Few if any other model organisms allow such a seamless combination of genetic and biochemical approaches. Other major advantages of Chlamydomonas compared to other systems include the ability to induce flagella to regenerate in a highly synchronous manner, allowing the kinetics of flagellar growth to be measured, and the ability of Chlamydomonas flagella to adhere to glass coverslips allowing Intraflagellar Transport to be easily imaged inside the flagella of living cells, with quantitative precision and single-molecule resolution. These advantages continue to work in favor of Chlamydomonas as a model system going forward, and are now augmented by extensive genomic resources, a knockout strain collection, and efficient CRISPR gene editing. While Chlamydomonas has obvious limitations for studying ciliary functions related to animal development or organ physiology, when it comes to studying the fundamental biology of cilia and flagella, Chlamydomonas is simply unmatched in terms of speed, efficiency, cost, and the variety of approaches that can be brought to bear on a question.

Intraflagellar transport (IFT) is required for ciliary assembly. The IFT machinery comprises the IFT motors kinesin-2 and IFT dynein plus IFT-A and IFT-B complexes, which assemble into IFT trains in cilia. To gain mechanistic understanding of IFT and ciliary assembly, here, we performed an absolute quantification of IFT machinery in Chlamydomonas reinhardtii cilium. There are ∼756, ∼532, ∼276 and ∼350 molecules of IFT-B, IFT-A, IFT dynein and kinesin-2, respectively, per cilium. The amount of IFT-B is sufficient to sustain rapid ciliary growth in terms of tubulin delivery. The stoichiometric ratio of IFT-B:IFT-A:dynein is ∼3:2:1 whereas the IFT-B:IFT-A ratio in an IFT dynein mutant is 2:1, suggesting that there is a plastic interaction between IFT-A and IFT-B that can be influenced by IFT dynein. Considering diffusion of kinesin-2 during retrograde IFT, it is estimated that one kinesin-2 molecule drives eight molecules of IFT-B during anterograde IFT. These data provide new insights into the assembly of IFT trains and ciliary assembly.

Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments (OS), which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lack OSs and undergo apoptosis. Interestingly, zebrafish mak mutants fail to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies are specified. These data suggest that MAK contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of MAK is critical in ciliary axoneme development and photoreceptor survival. Thus, MAK is required for ciliogenesis and OS formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.

Cilia and Wnt signaling have a complex relationship, wherein Wnt regulates cilia and, conversely, cilia may affect Wnt signaling. Recently, it was shown that Wnt receptors are present in flagella, primary cilia, and multicilia, where they transmit an intraciliary signal that is independent of β-catenin. Intraciliary Wnt signaling promotes ciliogenesis, affecting male fertility, adipogenesis, and mucociliary clearance. Wnt also stimulates the beating of motile cilia, highlighting that these nanomotors, too, are chemosensory. Intraciliary Wnt signaling employs a Wnt-protein phosphatase 1 (PP1) signaling axis, involving the canonical Wnt pathway\'s inhibition of glycogen synthase kinase 3 (GSK3) to repress PP1 activity. Collectively, these findings support that cilia are Wnt signaling organelles, with implications for ciliopathies and cancer.

Per- and polyfluoroalkyl substances (PFAS) are extensively utilized in varieties of products and tend to accumulate in the human body including umbilical cord blood and embryos/fetuses. In this study, we conducted an assessment and comparison of the potential early developmental toxicity of perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid, perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate, and perfluorobutyric acid at noncytotoxic concentrations relevant to human exposure using models based on human embryonic stem cells in both three-dimensional embryoid body (EB) and monolayer differentiation configurations. All six compounds influenced the determination of cell fate by disrupting the expression of associated markers in both models and, in some instances, even led to alterations in the formation of cystic EBs. The expression of cilia-related gene IFT122 was significantly inhibited. Additionally, PFOS and PFOA inhibited ciliogenesis, while PFOA specifically reduced the cilia length. Transcriptome analysis revealed that PFOS altered 1054 genes and disrupted crucial signaling pathways such as WNT and TGF-β, which play integral roles in cilia transduction and are critical for early embryonic development. These results provide precise and comprehensive insights into the potential adverse health effects of these six PFAS compounds directly concerning early human embryonic development.

The heterodimeric kinesin-2 motor (KIF3A/KIF3B with accessory protein KAP3) drives intraflagellar transport, essential for ciliogenesis and ciliary function. Three point mutations in the KIF3B subunit have recently been linked to disease in humans (E250Q and L523P) and Bengal cats (A334T) (Cogné et al., Am. J. Hum. Genet., 2020, 106, 893-904). Patients display retinal atrophy and, in some cases, other ciliopathy phenotypes. However, the molecular mechanism leading to disease is currently unknown. Here, we used Kif3a -/- ;Kif3b -/- (knockout) 3T3 cells, which cannot make cilia, to characterize these mutations. While reexpression of KIF3B(E250Q) and KIF3B(L523P) did not rescue ciliogenesis, reexpression of wildtype or KIF3B(A334T) restored ciliogenesis to wildtype levels. Fluorescent tagging revealed that the E250Q mutant decorated microtubules and thus is a rigor mutation. The L523P mutation, in the alpha-helical stalk domain, surprisingly did not affect formation of the KIF3A/KIF3B/KAP3 complex but instead impaired motility along microtubules. Lastly, expression of the A334T motor was reduced in comparison to all other motors, and this motor displayed an impaired ability to disperse the Golgi complex when artificially linked to this high-load cargo. In summary, this work uses cell-based assays to elucidate the molecular effects of disease-causing mutations in the KIF3B subunit on the kinesin-2 holoenzyme.

DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and to the centrosome, spindle midzone and bridging fiber area, and midbodies during mitosis. Loss of DPF3 causes kinetochore fiber instability, unstable kinetochore-microtubule attachment and defects in chromosome alignment, resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes to centriolar satellites at the base of primary cilia and is required for ciliogenesis by regulating axoneme extension. Taken together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis.