Yongli Zhang, Xuecheng Li, Zhengmao Wang, Junmin Pan
{"title":"SAXO proteins mediate tubulin turnover in axonemal microtubules of Chlamydomonas.","authors":"Yongli Zhang, Xuecheng Li, Zhengmao Wang, Junmin Pan","doi":"10.1242/jcs.264074","DOIUrl":"10.1242/jcs.264074","url":null,"abstract":"<p><p>Cilia and eukaryotic flagella are microtubule-based organelles that are crucial for cell motility and signaling. SAXO proteins (denoted for 'stabilizers of axonemal microtubules') are found exclusively in flagellated or ciliated organisms, but their physiological functions remain unclear. We investigated four SAXO proteins (SAXO1-SAXO4) in Chlamydomonas reinhardtii, identified via bioinformatics. All localize to cilia but differ in axonemal binding and spatial distribution. Single SAXO knockouts had no effect, whereas double mutants (saxo1/2, saxo1/3 and saxo2/3) showed shorter cilia. This phenotype intensified in the saxo1/2/3 triple mutant but not further in the quadruple mutant. Ciliary beating remained normal in saxo1/2/3 mutants, even under mechanical stress, indicating that SAXO1-SAXO3 are not essential for ciliary rigidity. Biochemical and proteomic analyses revealed no significant changes in the ciliary proteome or in tubulin acetylation, tyrosination and glutamylation within cilia. However, dikaryon assays with labeled tubulin showed that there was increased axonemal tubulin turnover in saxo1/2/3 mutant. Our findings underscore a crucial role of SAXO proteins in stabilizing axonemal microtubules by reducing tubulin turnover, thereby regulating ciliary length and assembly, and provide new insights into their function in cilia.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144150414","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Phosphorylation-induced SUMOylation promotes Ulk4 condensation at the ciliary tip to transduce Hedgehog signal.","authors":"Mengmeng Zhou, Yuhong Han, Jin Jiang","doi":"10.1242/jcs.263695","DOIUrl":"10.1242/jcs.263695","url":null,"abstract":"<p><p>Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the Gli family of transcription factors. In vertebrates, Hh signal transduction depends on the primary cilium, where Gli proteins are thought to be activated at the ciliary tip, but the underlying mechanism has remained poorly understood. Here, we provide evidence that two Unc-51-like kinase (Ulk) family members, Stk36 and Ulk4, regulate Gli2 ciliary tip localization and activation through phosphorylation and SUMOylation-mediated condensation in response to the Hh family protein Shh. We find that Stk36-mediated phosphorylation of Ulk4 promotes its SUMOylation in response to Shh, and the subsequent interaction between SUMO and a SUMO-interacting-motif (SIM) in the C-terminal region of Ulk4 drives Ulk4 self-assembly to form biomolecular condensates that also recruit Stk36 and Gli2. SUMOylation or SIM-deficient Ulk4 failed to accumulate at ciliary tip to activate Gli2 whereas phospho-mimetic mutation of Ulk4 sufficed to drive Ulk4, Stk36 and Gli2 condensation at ciliary tip, leading to constitutive Shh pathway activation in a manner dependent on Ulk4 SUMOylation. Taken together, our results suggest that phosphorylation-dependent SUMOylation of Ulk4 promotes kinase-substrate condensation at ciliary tip to transduce the Hh signal.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12148026/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Skeletal ciliopathy variants of the dynein-2 DYNC2LI1 subunit impair osteogenic differentiation of mesenchymal stem cells.","authors":"Yamato Ishida, Haruka Hoshi, Kenichi Kawano, Hye-Won Shin, Yohei Katoh, Kazuhisa Nakayama","doi":"10.1242/jcs.263737","DOIUrl":"10.1242/jcs.263737","url":null,"abstract":"<p><p>Skeletal ciliopathies result from defects in primary cilia, which are crucial for embryonic development because they transduce extracellular signals, including Hedgehog. Selective transport of ciliary proteins is mediated by the intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes and the kinesin-2 and dynein-2 motors. Biallelic loss-of-function variants in genes encoding dynein-2-specific subunits, including DYNC2LI1, cause skeletal ciliopathies. As mesenchymal stem cells (MSCs) differentiate into osteoblasts, we investigated the effects of pathogenic variants of DYNC2LI1 on osteogenic differentiation of the MSC-like line C3H10T1/2. Dync2li1-knockout cells expressing disease-causing DYNC2LI1 variants demonstrated defects in the retrograde ciliary protein trafficking, including Hedgehog pathway GPCRs, Smoothened and GPR161. Furthermore, Dync2li1-knockout cells expressing the pathogenic variants demonstrated impaired Hedgehog signaling, in particular, a reduced ratio of the GLI3 repressor form to total GLI3, resulting in impaired osteogenic differentiation of MSCs. By contrast, osteogenic differentiation via BMP signaling was derepressed in Dync2li1-knockout cells. This suggests that skeletal ciliopathies caused by DYNC2LI1 variants could be attributable in part to impaired osteogenic differentiation due to defects in Hedgehog signaling, resulting from defects in retrograde ciliary protein trafficking.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144110669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"β-Arrestin mediates the export of ciliary GPR161 but not Smoothened together with the BBSome and intraflagellar transport machinery.","authors":"Taiju Fujii, Norihito Murai, Shinya Aso, Hiroyuki Takatsu, Hye-Won Shin, Yohei Katoh, Kazuhisa Nakayama","doi":"10.1242/jcs.263793","DOIUrl":"10.1242/jcs.263793","url":null,"abstract":"<p><p>Specific G-protein-coupled receptors (GPCRs) exist on the ciliary membrane. Hedgehog signaling activation triggers the import of Smoothened into and export of GPR161 from cilia. The BBSome, which comprises eight Bardet-Biedl syndrome (BBS) proteins, mediates GPCR export, together with the intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes. The absence of any BBSome subunit or IFT27 (also known as BBS19) (an IFT-B subunit) impairs ciliary GPCR export, including that of GPR161. Plasma membrane GPCRs undergo phosphorylation by GPCR kinases (GRKs) and subsequent binding of β-arrestins [β-arrestin1 (ARRB1) and β-arrestin2 (ARRB2)], which is crucial for clathrin-mediated endocytosis. We here confirmed that GPR161 and β-arrestin are accumulated within cilia in the absence of IFT27 or the BBSome, and that ARRB1 and ARRB2 double-knockout impairs GPR161 export. Notably, we found that activation-mimetic β-arrestin mutants can interact with both the BBSome and ciliary GPCRs, and cause constitutive export of GPR161. Moreover, we demonstrated that GRK2 plays a crucial role in GPR161 export. We here propose that phosphorylated GPR161 recruits β-arrestins, converting them into their activated conformation. Activated β-arrestins then interact with the BBSome, which connects them to the IFT machinery to facilitate GPR161 export.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144093707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Primary cilia shape postnatal astrocyte development through Sonic Hedgehog signaling.","authors":"Rachel Bear, Steven A Sloan, Tamara Caspary","doi":"10.1242/jcs.263965","DOIUrl":"10.1242/jcs.263965","url":null,"abstract":"<p><p>Primary cilia function as specialized signaling centers that regulate many cellular processes including neuron and glia development. Astrocytes possess cilia, but the function of cilia in astrocyte development remains largely unexplored. Crucially, dysfunction of either astrocytes or cilia contributes to the molecular changes observed in neurodevelopmental disorders. Here, we show that a subpopulation of developing astrocytes in the prefrontal cortex are ciliated. This population corresponds to proliferating astrocytes and largely expresses the ciliary protein ARL13B. Genetic ablation of astrocyte cilia in vivo at two distinct stages of astrocyte development results in changes to Sonic Hedgehog (Shh) transcriptional targets. We show that Shh activity is decreased in immature and mature astrocytes upon loss of cilia. Furthermore, loss of cilia in immature astrocytes results in decreased astrocyte proliferation and loss of cilia in mature astrocytes causes enlarged astrocyte morphology. Together, these results indicate that astrocytes require cilia for Shh signaling throughout development and uncover functions for astrocyte cilia in regulating astrocyte proliferation and maturation. This expands our fundamental knowledge of astrocyte development and cilia function to advance our understanding of neurodevelopmental disorders.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12148023/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Daniel Abbühl, Martina Pružincová, Luděk Štěpánek, Eleonore Bouscasse, Rita Azevedo, Mariette Matondo, Vladimir Varga, Serge Bonnefoy, Philippe Bastin
{"title":"A novel approach to tagging tubulin reveals microtubule assembly dynamics of the axoneme in Trypanosoma brucei.","authors":"Daniel Abbühl, Martina Pružincová, Luděk Štěpánek, Eleonore Bouscasse, Rita Azevedo, Mariette Matondo, Vladimir Varga, Serge Bonnefoy, Philippe Bastin","doi":"10.1242/jcs.264145","DOIUrl":"10.1242/jcs.264145","url":null,"abstract":"<p><p>The protozoan parasite Trypanosoma brucei assembles a new flagellum while maintaining the existing one in the same cell. Our group has previously proposed a model where the mature flagellum is locked after construction to full length. To test this hypothesis directly, we monitored flagellum assembly dynamics through inducible expression of tubulin marked with an intragenic tag. We found that addition of new tubulin occurs at the distal flagellum tip at a linear rate and is indeed restricted to the new flagellum in bi-flagellated cells. Depleting the locking protein CEP164C prior to induction resulted in simultaneous integration of new tubulin in both flagella. This is direct evidence that trypanosomes avoid competition between the two flagella by allowing tubulin incorporation only in the new organelle. However, by tracing flagella over several cell cycles we also found that flagella do not remain locked forever. An orthogonal approach with HaloTag-tagged radial spoke protein 4/6 (GeneID Tb927.11.4480) supported these findings. Given that flagellum length in trypanosomes is stable, this indicates regular events of transient disassembly, followed by assembly, at the distal tip.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144173944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The mammalian protein MTCH1 can function as an insertase.","authors":"Anna Roza Dimogkioka, Anni Elias, Doron Rapaport","doi":"10.1242/jcs.263736","DOIUrl":"https://doi.org/10.1242/jcs.263736","url":null,"abstract":"<p><p>The outer mitochondrial membrane (OMM) hosts a variety of proteins such as import machineries, enzymes, fission/fusion factors, and pore proteins. In Saccharomyces cerevisiae, the MIM complex, consisting of Mim1 and Mim2, mediates the insertion of α-helical proteins into the OMM. Until recently, it was unclear which proteins serve this function in higher eukaryotes. Recent studies identified MTCH2 as the insertase of α-helical proteins into the OMM in mammals. MTCH1 is a paralogue of MTCH2 but its general function and contribution to the biogenesis process are not clear. To better characterize MTCH1, we explored whether MTCH1 or MTCH2 could functionally replace Mim1/Mim2 in yeast. Expression of MTCH1 and MTCH2 in yeast cells lacking Mim1, Mim2, or both revealed that MTCH1, but not MTCH2, could compensate the growth defects upon deleting the MIM complex. Furthermore, MTCH1 could restore the biogenesis of MIM substrates, TOM complex stability, and morphology of mitochondria. These findings indicate that MTCH1 by itself has insertase activity and is a functional homologue of the MIM complex, despite the absence of any evolutionary relation between the mammalian and yeast insertases.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144698631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Din Baruch, Ioannis Tsirkas, Ehud Sass, Benjamin Dubreuil, Yeynit Asraf, Amir Aharoni, Maya Schuldiner, Ofir Klein
{"title":"Creation and validation of a proteome-wide yeast library for protein detection and analysis.","authors":"Din Baruch, Ioannis Tsirkas, Ehud Sass, Benjamin Dubreuil, Yeynit Asraf, Amir Aharoni, Maya Schuldiner, Ofir Klein","doi":"10.1242/jcs.263848","DOIUrl":"https://doi.org/10.1242/jcs.263848","url":null,"abstract":"<p><p>A significant challenge in cell biology is to uncover the function of uncharacterized proteins. Surprisingly a quarter of the proteome is still poorly understood even in the most well studied model organisms. Systematic methodologies, including the use of tagged protein collections, have emerged as a powerful approach to address this gap. Despite the availability of proteome-wide collections featuring various fused proteins, the impact of different tags on protein function highlights the need for diversifying the tags used for functional genomic studies. To rise to this challenge, we created a proteome-wide collection of yeast strains in which proteins are N-terminally tagged with the broadly utilized and compact Hemagglutinin (HA) epitope. We showcase the potential uses of our library for systematically evaluating protein size, abundance and localization using an in vivo labeling approach. Our characterization underscores the potential utility of a proteome-wide HA-tagged library in revealing novel aspects of cell biology, providing an additional powerful tool for functional genomics.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144698629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abigail R Moye, Michael A Robichaux, Melina A Agosto, Alexandre P Moulin, Alexandra Graff-Meyer, Carlo Rivolta, Theodore G Wensel
{"title":"Sub-ciliary localization of CEP290 and effects of its loss in mouse photoreceptors during development.","authors":"Abigail R Moye, Michael A Robichaux, Melina A Agosto, Alexandre P Moulin, Alexandra Graff-Meyer, Carlo Rivolta, Theodore G Wensel","doi":"10.1242/jcs.263869","DOIUrl":"https://doi.org/10.1242/jcs.263869","url":null,"abstract":"<p><p>The most common genetic cause of the childhood blinding disease Leber congenital amaurosis is mutation of the ciliopathy gene CEP290. Despite extensive study, the photoreceptor-specific roles of CEP290 remain unclear. Using advanced microscopy techniques, we investigated the sub-ciliary localization of CEP290 and its role in mouse photoreceptors during development. CEP290 was found throughout the connecting cilium between the microtubules and membrane, with nine-fold symmetry. In the absence of CEP290 ciliogenesis occurs, but the connecting cilium membrane is aberrant, and sub-structures, such as the ciliary necklace and Y-links, are confined to the proximal connecting cilium. Transition zone proteins AHI1 and NPHP1 were abnormally restricted to the proximal connecting cilium in the absence of CEP290, while others like NPHP8 and CEP89 were unaffected. Although outer segment disc formation is inhibited in Cep290 mutant retina, we observed large numbers of extracellular vesicles. These results suggest roles for CEP290 in ciliary membrane structure, outer segment disc formation and photoreceptor-specific spatial distribution of a subset of transition zone proteins, which collectively lead to failure of outer segment formation and photoreceptor degeneration.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144698630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"APEX2-based quantitative proteomics of LAT and CD3σ interactomes in living human Jurkat T cells unveils new interactors.","authors":"Juan-José Saez, Michaël Richard, Vivien Caillens, Stéphanie Dogniaux, Federico Marconi, Florent Dingli, Damarys Loew, Hermine Ferran, Loredana Saveanu, Claire Hivroz, Laurence Bataille","doi":"10.1242/jcs.263981","DOIUrl":"https://doi.org/10.1242/jcs.263981","url":null,"abstract":"<p><p>TCR stimulation induces a signaling cascade starting by the phosphorylation of immunoreceptor tyrosine-based activation motif (ITAMs) present in the TCR-CD3 complex. This is followed by the phosphorylation of proteins including LAT, which once phosphorylated interacts with multiple proteins allowing signal diversification and amplification. We take advantage of APEX2-based peroxidase- catalyzed proximity labeling combined with quantitative mass spectrometry to track the formation of CD3σ and LAT interactome dynamics in TCR activated Jurkat cells. We find more than 1 000 high confidence proteins for each bait and provide a quantitative molecular map of proteins enriched or reduced from the vicinity of CD3σ and LAT, after stimulation. We detail and compare the recruitment kinetics of signaling proteins to CD3σ and LAT and identify uncharacterized mediators of T cell activation. We show that the kinase MARK2, which is in the proximity of LAT and CD3σ at resting state and lost upon activation, is a negative regulator of cytokine production by T cells. This study provides a resource for uncovering the complex signaling networks that regulate TCR activation and highlights new players of this signaling cascade.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144690403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}