CEP120-mediated KIAA0753 recruitment onto centrioles is required for timely neuronal differentiation and germinal zone exit in the developing cerebellum.

Joubert syndrome (JS) is a recessive ciliopathy in which all affected individuals have congenital cerebellar vermis hypoplasia. Here, we report that CEP120, a JS-associated protein involved in centriole biogenesis and cilia assembly, regulates timely neuronal differentiation and the departure of granule neuron progenitors (GNPs) from their germinal zone during cerebellar development. Our results show that depletion of Cep120 perturbs GNP cell cycle progression, resulting in a delay of cell cycle exit in vivo. To dissect the potential mechanism, we investigated the association between CEP120 interactome and the JS database and identified KIAA0753 (a JS-associated protein) as a CEP120-interacting protein. Surprisingly, we found that CEP120 recruits KIAA0753 to centrioles, and that loss of this interaction induces accumulation of GNPs in the germinal zone and impairs neuronal differentiation. Importantly, the replenishment of wild-type CEP120 rescues the above defects, whereas expression of JS-associated CEP120 mutants, which hinder KIAA0753 recruitment, does not. Together, our data reveal a close interplay between CEP120 and KIAA0753 for the germinal zone exit and timely neuronal differentiation of GNPs during cerebellar development, and mutations in CEP120 and KIAA0753 may participate in the heterotopia and cerebellar hypoplasia observed in JS patients.

DNA Damage-Induced Nucleosome Depletion Enhances Homology Search Independently of Local Break Movement.

To determine whether double-strand break (DSB) mobility enhances the physical search for an ectopic template during homology-directed repair (HDR), we tested the effects of factors that control chromatin dynamics, including cohesin loading and kinetochore anchoring. The former but not the latter is altered in response to DSBs. Loss of the nonhistone high-mobility group protein Nhp6 reduces histone occupancy and increases chromatin movement, decompaction, and ectopic HDR. The loss of nucleosome remodeler INO80-C did the opposite. To see whether enhanced HDR depends on DSB mobility or the global chromatin response, we tested the ubiquitin ligase mutant uls1Δ, which selectively impairs local but not global movement in response to a DSB. Strand invasion occurs in uls1Δ cells with wild-type kinetics, arguing that global histone depletion rather than DSB movement is rate limiting for HDR. Impaired break movement in uls1Δ correlates with elevated MRX and cohesin loading, despite normal resection and checkpoint activation.

The CEP170B-KIF2A complex destabilizes microtubule minus ends to generate polarized microtubule network.

Microtubule (MT) minus ends are stabilized by CAMSAP family proteins at noncentrosomal MT-organizing centers. Despite progress in identifying diverse positive regulators, knowledge on the negative regulation of the MT minus-end distribution is lacking. Here, we identify CEP170B as a MT minus-end-binding protein that colocalizes with the microtubule-stabilizing complex at the cortical patches. CEP170B depends on the scaffold protein liprin-α1 for its cortical targeting and requires liprin-α1-bound PP2A phosphatase for its MT localization. CEP170B excludes CAMSAPs-stabilized MT minus ends from the cell periphery in HeLa cells and the basal cortex in human epithelial cells and is required for directional vesicle trafficking and cyst formation in 3D culture. Reconstitution experiments demonstrate that CEP170B autonomously tracks growing MT minus ends and blocks minus-end growth. Furthermore, CEP170B in a complex with the kinesin KIF2A acts as a potent MT minus-end depolymerase capable of antagonizing the stabilizing effect of CAMSAPs. Our study uncovers an antagonistic mechanism for controlling the spatial distribution of MT minus ends, which contributes to the establishment of polarized MT network and cell polarity.

Cep57 regulates human centrosomes through multivalent interactions.

Human Cep57 is a coiled-coil scaffold at the pericentriolar matrix (PCM), controlling centriole duplication and centrosome maturation for faithful cell division. Genetic truncation mutations of Cep57 are associated with the mosaic-variegated aneuploidy (MVA) syndrome. During interphase, Cep57 forms a complex with Cep63 and Cep152, serving as regulators for centrosome maturation. However, the molecular interplay of Cep57 with these essential scaffolding proteins remains unclear. Here, we demonstrate that Cep57 undergoes liquid-liquid phase separation (LLPS) driven by three critical domains (NTD, CTD, and polybasic LMN). In vitro Cep57 condensates catalyze microtubule nucleation via the LMN motif-mediated tubulin concentration. In cells, the LMN motif is required for centrosomal microtubule aster formation. Moreover, Cep63 restricts Cep57 assembly, expansion, and microtubule polymerization activity. Overexpression of competitive constructs for multivalent interactions, including an MVA mutation, leads to excessive centrosome duplication. In Cep57-depleted cells, self-assembly mutants failed to rescue centriole disengagement and PCM disorganization. Thus, Cep57's multivalent interactions are pivotal for maintaining the accurate structural and functional integrity of human centrosomes.

Neuronal IL-17 controls Caenorhabditis elegans developmental diapause through CEP-1/p53.

During metazoan development, how cell division and metabolic programs are coordinated with nutrient availability remains unclear. Here, we show that nutrient availability signaled by the neuronal cytokine, ILC-17.1, switches Caenorhabditis elegans development between reproductive growth and dormancy by controlling the activity of the tumor suppressor p53 ortholog, CEP-1. Specifically, upon food availability, ILC-17.1 signaling by amphid neurons promotes glucose utilization and suppresses CEP-1/p53 to allow growth. In the absence of ILC-17.1, CEP-1/p53 is activated, up-regulates cell-cycle inhibitors, decreases phosphofructokinase and cytochrome C expression, and causes larvae to arrest as stress-resistant, quiescent dauers. We propose a model whereby ILC-17.1 signaling links nutrient availability and energy metabolism to cell cycle progression through CEP-1/p53. These studies describe ancestral functions of IL-17 s and the p53 family of proteins and are relevant to our understanding of neuroimmune mechanisms in cancer. They also reveal a DNA damage-independent function of CEP-1/p53 in invertebrate development and support the existence of a previously undescribed C. elegans dauer pathway.

CEP41, a ciliopathy-linked centrosomal protein, regulates microtubule assembly and cell proliferation.

Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies. Understanding the multifaceted roles of centrosomal proteins is vital to comprehend their involvement in disease development. Here, we report novel cellular functions of CEP41, a centrosomal and ciliary protein implicated in Joubert syndrome. We show that CEP41 is an essential microtubule-associated protein with microtubule-stabilizing activity. Purified CEP41 binds to preformed microtubules, promotes microtubule nucleation and suppresses microtubule disassembly. When overexpressed in cultured cells, CEP41 localizes to microtubules and promotes microtubule bundling. Conversely, shRNA-mediated knockdown of CEP41 disrupts the interphase microtubule network and delays microtubule reassembly, emphasizing its role in microtubule organization. Further, we demonstrate that the association of CEP41 with microtubules relies on its conserved rhodanese homology domain (RHOD) and the N-terminal region. Interestingly, a disease-causing mutation in the RHOD domain impairs CEP41-microtubule interaction. Moreover, depletion of CEP41 inhibits cell proliferation and disrupts cell cycle progression, suggesting its potential involvement in cell cycle regulation. These insights into the cellular functions of CEP41 hold promise for unraveling the impact of its mutations in ciliopathies.

Roles for CEP170 in cilia function and dynein-2 assembly.

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.

Architecture of RabL2-associated complexes at the ciliary base: A structural modeling perspective: Deciphering the structural organization of ciliary RabL2 complexes.

Cilia are slender, micrometer-long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so-called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N-terminal domain of CEP43 forms a homo-dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N-terminal domain of CEP43 from the C-terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2-CEP19 associates with the IFT-B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT-B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled-coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.

Separation-of-function MCPH-associated mutations in CPAP affect centriole number and length.

Centrioles are microtubule-based cylindrical ultrastructures characterized by their definite size and robustness. The molecular capping protein, CPAP (also known as CENPJ) engages its N-terminal region with the centriole microtubules to regulate their length. Nevertheless, the conserved C-terminal glycine-rich G-box of CPAP, which interacts with the centriole inner cartwheel protein STIL, is frequently mutated in primary microcephaly (MCPH) patients. Here, we show that two different MCPH-associated variants, E1235V and D1196N in the CPAP G-box, affect distinct functions at centrioles. The E1235V mutation reduces CPAP centriole recruitment and causes overly long centrioles. The D1196N mutation increases centriole numbers without affecting centriole localization. Both mutations prevent binding to STIL, which controls centriole duplication. Our work highlights the involvement of an alternative CEP152-dependent route for CPAP centriole localization. Molecular dynamics simulations suggest that E1235V leads to an increase in G-box flexibility, which could have implications on its molecular interactions. Collectively, we demonstrate that a CPAP region outside the microtubule-interacting domains influences centriole number and length, which translates to spindle defects and reduced cell viability. Our work provides new insights into the molecular causes of primary microcephaly.

Loss of ATG4B and ATG4A results in two-stage cell cycle defects in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) exhibits elevated levels of autophagy, which promote tumor progression and treatment resistance. ATG4B is an autophagy-related cysteine protease under consideration as a potential therapeutic target, but it is largely unexplored in PDAC. Here, we investigated the clinical and functional relevance of ATG4B expression in PDAC. Using two PDAC patient cohorts, we found that low ATG4B mRNA or protein expression is associated with worse patient survival outcomes, poorly differentiated PDAC tumors and a lack of survival benefit from adjuvant chemotherapy. In PDAC cell lines, ATG4B knockout reduced proliferation, abolished processing of LC3B (also known as MAP1LC3B), and reduced GABARAP and GABARAPL1 levels, but increased ATG4A levels. ATG4B and ATG4A double knockout lines displayed a further reduction in proliferation, characterized by delays in G1-S phase transition and mitosis. Pro-LC3B accumulated aberrantly at the centrosome with a concomitant increase in centrosomal proteins PCM1 and CEP131, which was rescued by exogenous ATG4B. The two-stage cell cycle defects following ATG4B and ATG4A loss have important therapeutic implications for PDAC.

Centriolar subdistal appendages promote double-strand break repair through homologous recombination.

The centrosome is a cytoplasmic organelle with roles in microtubule organization that has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA damage response. However, whether the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosome presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection. Cells lacking centrosomes or depleted for CEP170 are, consequently, hypersensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but could also be targeted to improve current oncological treatments.

Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

The APC/C targets the Cep152-Cep63 complex at the centrosome to regulate mitotic spindle assembly.

The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase-promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions, including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and an APC/C substrate. Previous studies have shown that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitylation of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.

Centriole distal-end proteins CP110 and Cep97 influence centriole cartwheel growth at the proximal end.

Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. We previously showed that in Drosophila embryos an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. Here, in the same model, we show that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, in a manner that appears to be entrained by the core cyclin-dependent kinase (Cdk)-Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110 and Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, we find that changing the levels of CP110 and Cep97 appears to alter the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected potential crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which might help to coordinate centriole growth. This article has an associated First Person interview with the first authors of the paper.

CEPs suppress auxin signaling but promote cytokinin signaling to inhibit root growth in Arabidopsis.

C-terminally encoded peptides (CEPs) are peptide hormones that function as mobile signals coordinating crucial developmental programs in plants. Previous studies have revealed that CEPs exert negative regulation on root development through interaction with CEP receptors (CEPRs), CEP DOWNSTREAMs (CEPDs), the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE (AHKs) and the transcriptional repressor Auxin/Indole-3-Acetic Acid (AUX/IAA). However, the precise molecular mechanisms underlying CEPs-mediated regulation of root development via auxin and cytokinin signaling pathways still necessitate further detailed investigation. In this study, we examined prior research and elucidated the underlying molecular mechanisms. The results showed that both synthetic AtCEPs and overexpression of AtCEP5 markedly supressed primary root elongation and lateral root (LR) formation in Arabidopsis. Molecular biology and genetics elucidated how CEPs inhibit root growth by suppressing auxin signaling while promoting cytokinin signaling. In summary, this study elucidated the inhibitory effects of AtCEPs on Arabidopsis root growth and provided insights into their potential molecular mechanisms, thus enhancing our comprehension of CEP-mediated regulation of plant growth and development.

Insights into degradation and targeting of the photoreceptor channelrhodopsin-1.

In Chlamydomonas, the directly light-gated, plasma membrane-localized cation channels channelrhodopsins ChR1 and ChR2 are the primary photoreceptors for phototaxis. Their targeting and abundance is essential for optimal movement responses. However, our knowledge how Chlamydomonas achieves this is still at its infancy. Here we show that ChR1 internalization occurs via light-stimulated endocytosis. Prior or during endocytosis ChR1 is modified and forms high molecular mass complexes. These are the solely detectable ChR1 forms in extracellular vesicles and their abundance therein dynamically changes upon illumination. The ChR1-containing extracellular vesicles are secreted via the plasma membrane and/or the ciliary base. In line with this, ciliogenesis mutants exhibit increased ChR1 degradation rates. Further, we establish involvement of the cysteine protease CEP1, a member of the papain-type C1A subfamily. ΔCEP1-knockout strains lack light-induced ChR1 degradation, whereas ChR2 degradation was unaffected. Low light stimulates CEP1 expression, which is regulated via phototropin, a SPA1 E3 ubiquitin ligase and cyclic AMP. Further, mutant and inhibitor analyses revealed involvement of the small GTPase ARL11 and SUMOylation in ChR1 targeting to the eyespot and cilia. Our study thus defines the degradation pathway of this central photoreceptor of Chlamydomonas and identifies novel elements involved in its homoeostasis and targeting.

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormone signaling intersect to promote shallow lateral root angles.

Root system architecture (RSA) influences the acquisition of heterogeneously dispersed soil nutrients. Cytokinin and C-TERMINALLY ENCODED PEPTIDE (CEP) hormones affect RSA, in part by controlling the angle of lateral root (LR) growth. Both hormone pathways converge on CEP DOWNSTREAM 1 (CEPD1) and CEPD2 to control primary root growth; however, a role for CEPDs in controlling the growth angle of LRs is unknown. Using phenotyping combined with genetic and grafting approaches, we show that CEP hormone-mediated shallower LR growth requires cytokinin biosynthesis and perception in roots via ARABIDOPSIS HISTIDINE KINASE 2 (AHK2) and AHK3. Consistently, cytokinin biosynthesis and ahk2,3 mutants phenocopied the steeper root phenotype of cep receptor 1 (cepr1) mutants on agar plates, and CEPR1 was required for trans-Zeatin (tZ)-type cytokinin-mediated shallower LR growth. In addition, the cepd1,2 mutant was less sensitive to CEP and tZ, and showed basally steeper LRs on agar plates. Cytokinin and CEP pathway mutants were grown in rhizoboxes to define the role of these pathways in controlling RSA. Only cytokinin receptor mutants and cepd1,2 partially phenocopied the steeper-rooted phenotype of cepr1 mutants. These results show that CEP and cytokinin signaling intersect to promote shallower LR growth, but additional components contribute to the cepr1 phenotype in soil.

The Compact Root Architecture 2 systemic pathway is required for the repression of cytokinins and miR399 accumulation in Medicago truncatula N-limited plants.

Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N deficit or N satiety, plants were grown in a split-root experimental design in which either high or low N was provided to half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-zeatin accumulated in plants at N satiety. Cytokinin application by petiole feeding led to inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic phosphate (Pi) acquisition, miR399, accumulates in plants grown under N satiety. These two accumulation patterns are dependent on Compact Root Architecture 2 (CRA2), a receptor required for C-terminally Encoded Peptide (CEP) signaling. Constitutive ectopic expression of miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N availability affects the development of the whole legume plant root system.

Root architecture and rhizosphere-microbe interactions.

Plant roots fulfil crucial tasks during a plant's life. As roots encounter very diverse conditions while exploring the soil for resources, their growth and development must be responsive to changes in the rhizosphere, resulting in root architectures that are tailor-made for all prevailing circumstances. Using multi-disciplinary approaches, we are gaining more intricate insights into the regulatory mechanisms directing root system architecture. This Special Issue provides insights into our advancement of knowledge on different aspects of root development and identifies opportunities for future research.

CEP hormones at the nexus of nutrient acquisition and allocation, root development, and plant-microbe interactions.

A growing understanding is emerging of the roles of peptide hormones in local and long-distance signalling that coordinates plant growth and development as well as responses to the environment. C-TERMINALLY ENCODED PEPTIDE (CEP) signalling triggered by its interaction with CEP RECEPTOR 1 (CEPR1) is known to play roles in systemic nitrogen (N) demand signalling, legume nodulation, and root system architecture. Recent research provides further insight into how CEP signalling operates, which involves diverse downstream targets and interactions with other hormone pathways. Additionally, there is emerging evidence of CEP signalling playing roles in N allocation, root responses to carbon levels, the uptake of other soil nutrients such as phosphorus and sulfur, root responses to arbuscular mycorrhizal fungi, plant immunity, and reproductive development. These findings suggest that CEP signalling more broadly coordinates growth across the whole plant in response to diverse environmental cues. Moreover, CEP signalling and function appear to be conserved in angiosperms. We review recent advances in CEP biology with a focus on soil nutrient uptake, root system architecture and organogenesis, and roles in plant-microbe interactions. Furthermore, we address knowledge gaps and future directions in this research field.