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In adult acute myeloid leukemia (AML), the acquisition of driver somatic mutations may be preceded by a benign state termed clonal hematopoiesis (CH). To develop therapeutic strategies to prevent leukemia development from CH, it is important to understand the mechanisms by which CH-driving and AML-driving mutations cooperate. Here, we use mice with inducible mutant alleles common in human CH (DNMT3AR882; mouse Dnmt3aR878H) and AML (NPM1c; mouse Npm1cA). We find that Dnmt3aR878H/+ hematopoietic stem cells (HSCs), but not multipotent progenitor cell (MPP) subsets, have reduced cytokine expression and proinflammatory transcriptional signatures and a functional competitive advantage over their wild-type counterparts. Dnmt3aR878H/+ HSCs are the most potent cell type transformed by Npm1cA, generating myeloid malignancies in which few additional cooperating somatic mutation events were detected. At a molecular level, Npm1cA, in cooperation with Dnmt3aR878H, acutely increased the accessibility of a distinct set of promoters in HSCs compared with MPP cells. These promoters were enriched for cell cycling, PI3K/AKT/mTOR signaling, stem cell signatures, and targets of transcription factors, including NFAT and the chromatin binding factor HMGB1, which have been implicated in human AML. These results demonstrate cooperativity between preexisting Dnmt3aR878H and Npm1cA at the chromatin level, where specific loci altered in accessibility by Npm1cA are dependent on cell context as well as Dnmt3a mutation status. These findings have implications for biological understanding and therapeutic intervention in the transformation from CH to AML.

Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.

SummaryMicrotubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintainedin vivoremain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate thatC. elegansMAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.

DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in polyploidy but their centriole state is not known. We used a cell culture model for TGC differentiation to examine centriole and centrosome number and properties. Prior to differentiation, trophoblast stem cells (TSCs) have either two centrioles before duplication, or four centrioles after. We find that average nuclear area increases approximately 8-fold over differentiation, but most TGCs do not have more than four centrioles. However, these centrioles become disengaged, acquire centrosome proteins, and can nucleate microtubules. In addition, some TGCs undergo further duplication and disengagement of centrioles, resulting in substantially higher numbers. Live imaging revealed that disengagement and separation are centriole autonomous and can occur asynchronously. Centriole amplification, when present, occurs by the standard mechanism of one centriole generating one procentriole. PLK4 inihibition blocks centriole formation in differentiating TGCs but does not affect endocycle progression. In summary, centrioles in TGC endocycles undergo disengagement and conversion to centrosomes. This increases centrosome number, but to a limited extent compared with DNA reduplication.

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a “timely two-ness” that allows cell division to occur in absence of a SAC-dependent mitotic delay.

Clonal hematopoiesis resulting from enhanced fitness of mutant hematopoietic stem cells (HSCs) associates with both favorable and unfavorable health outcomes related to the types of mature mutant blood cells produced, but how this lineage output is regulated is unclear. Using a mouse model of a clonal hematopoiesis-associated mutation,DNMT3AR882/+(Dnmt3aR878H/+), we found that aging-induced TNFα signaling promoted the selective advantage of mutant HSCs as well as stimulated mutant B lymphoid cell production. Genetic loss of TNFα receptor TNFR1 impaired mutant HSC fitness without altering lineage output, while loss of TNFR2 reduced lymphoid cell production and favored myeloid cell production from mutant HSCs without altering overall fitness. These results support a model where clone size and mature blood lineage production can be independently controlled to harness potential beneficial aspects of clonal hematopoiesis.Statement of SignificanceThrough identification and dissection of TNFα signaling as a key driver of murineDnmt3a-mutant hematopoiesis, we report the discovery that clone size and production of specific mature blood cell types can be independently regulated.

Calcineurin, or PP2B, the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain undiscovered. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped calcineurin’s spatial distribution in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal/ciliary proteins. These include POC5, which binds centrin in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT-type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and expansion microscopy, we demonstrate that calcineurin co-localizes with POC5 at the centrosome, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centrosomal proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes primary cilia elongation without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in cilia length maintenance, a process frequently disrupted in ciliopathies.Summary statementCalcineurin phosphatase participates in centrosome and cilia regulation. Calcineurin localizes to centrosomes, where it interacts with partner POC5, and its inhibition promotes cilia elongation.

Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50 to 100 μm from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 μm/min. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium prior to other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.

Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50–100 μm from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 µm/minute. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium before other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.

Multiciliated cells (MCCs) are extremely highly-differentiated, presenting >100 cilia and basal bodies. Therefore, MCC fate is thought to be terminal and irreversible. We analyzed how MCCs are removed from the airway-like mucociliary Xenopus epidermis during developmental tissue remodeling. We found that a subset of MCCs undergoes lateral line-induced apoptosis, but that the majority coordinately trans-differentiate into Goblet secretory cells. Both processes are dependent on Notch signaling, while the cellular response to Notch is modulated by Jak/STAT, Thyroid hormone and mTOR signaling. At the cellular level, trans-differentiation is executed through loss of ciliary gene expression including foxj1 and pcm1, altered proteostasis, cilia retraction, basal body elimination as well as initiation of mucus production and secretion. Our work describes two modes for MCC loss during vertebrate development, the signaling regulation of these processes, and demonstrates that even cells with extreme differentiation features can undergo direct fate conversion.

The mechanisms of vertebrate Hedgehog signaling are linked to the biology of the primary cilium, an antenna-like organelle that projects from the surface of most vertebrate cell types. Although the advantages of restricting signal transduction to cilia are often noted, the constraints imposed are less frequently considered, and yet they are central to how Hedgehog signaling operates in developing tissues. In this Review, we synthesize current understanding of Hedgehog signal transduction, ligand secretion and transport, and cilia dynamics to explore the temporal and spatial constraints imposed by the primary cilium on Hedgehog signaling in vivo.

Introduction: B7-H3 (encoded by the CD276 gene) is suggested to act as an immune checkpoint molecule and is highly expressed in some pediatric solid tumors. Monoclonal antibodies targeting B7-H3 (8H9 and MGA271) are well-tolerated but have demonstrated limited success in clinical trials. DS-7300a is a B7-H3 targeting ADC with a payload of DXd (an exatecan derivative that inhibits DNA topoisomerase I). DS-7300a has a drug-to-antibody ratio of 4 and it has shown promising early clinical activity in adults with advanced solid cancers (Johnson, Annals of Oncology 2021; 32:S583-S585). Methods: Xenograft models for several pediatric cancer types were selected for preclinical testing of DS-7300a based on the high rates of B7-H3 expression in these cancers. Models were dosed at 10 mg/kg administered intravenously every other week for two doses (Q2wk x 2). Different experimental designs were used for efficacy testing. For rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, and other pediatric solid tumors a single mouse trial (SMT) design with 40 models was used. A cohort design (N=10) was used for efficacy testing in 6 osteosarcoma models, 3 patient-derived orthotopic xenograft glioblastoma (PDOX) models, and 3 PDOX ependymoma models. For neuroblastoma models, 10 models (2 mice per model) were tested. To evaluate treatment efficacy, objective response measures based on changes in relative tumor volume were used (Houghton, Pediatr Blood Cancer 2007;49:928-940). Results: As a single agent, DS-7300a induced a statistically significant prolongation of survival in 2 of 3 orthotopic glioblastoma and 1 of 3 ependymal tumor models. DS-7300a demonstrated high efficacy (maintained complete response, complete response, and partial response) in most non-CNS models tested: 17 of 21 rhabdomyosarcomas, 5 of 7 osteosarcomas, 7 of 10 neuroblastomas and 2 of 2 Wilms tumors. For Ewing sarcoma, most models were classified as progressive disease (9 of 15). For osteosarcoma models, the log cell kill per dose values ranged from 0.95 to 2.76 indicating high activity of the agent in these models. The activity for DS-7300a followed the general pattern of protein and RNA expression levels for B7-H3/CD276 in the models, with Ewing sarcoma models showing lower expression compared to the other models. Conclusions: DS-7300a shows tumor-regressing anticancer activity across a wide range of pediatric solid tumor models. The maintained complete remissions observed for osteosarcoma models are noteworthy, as this level of response is uncommon for these models and as osteosarcoma shows the highest B7-H3 expression among pediatric cancers. The high level of preclinical activity observed for DS-7300a combined with the promising early clinical activity observed for adult patients provide strong rationale for studying DS-7300a in children with B7-H3 expressing solid tumors. Citation Format: Richard Gorlick, E. Anders Kolb, Yifei Wang, Peter Houghton, Raushan Kurmasheva, Yael Mosse, John Maris, Matthew Tsang, David Groff, Kateryna Krytska, Xiao-Nan Li, Yuchen Du, Jun Hasegawa, Nanae Izumi, Steven Neuhauser, Anuj Srivastava, Tim Stearns, Vivek Philip, Emily L. Jocoy, Jeff Chuang, Carol J. Bult, Beverly Teicher, Malcolm Smith. Evaluation of the in vivo efficacy of the B7-H3 targeting antibody-drug conjugate (ADC) DS7300a: A report fro the Pediatric Preclinical In Vivo Resting (PIVOT) program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB061.

Micronuclei, whole or fragmented chromosomes spatially separated from the main nucleus, are associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to asynchronous segregation of unaligned chromosomes in vivo but do not develop spontaneous tumors. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. Micronuclei in cultured KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of nuclear envelope components and successful expansion of decondensing chromatin compared with those induced by nocodazole washout or radiation. Lagging chromosomes were also positioned closer to the main chromatin masses in KIF18A KO cells. These data suggest that not all micronuclei actively promote tumorigenesis.

CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors. This assay can also easily determine the effect of various interventions on obtaining a larger pool of Cas9-treated cells.

Mitosis is a dramatic cellular process that affects all parts of the cell. In Xenopus embryos and extracts it is driven by the activation of a bistable trigger circuit, whose various components are localized in the nucleus, centrosome, and cytoplasm. In principle, whichever cellular location has the fastest intrinsic rhythm should act as a pacemaker for the process. Here we followed tubulin polymerization and depolymerization in Xenopus egg extracts supplemented with demembranated sperm, and thereby identified locations where mitosis first occurred. We found that mitosis was commonly first initiated at sperm-derived nuclei and their accompanying centrosomes, and then spread outward in circular trigger waves. The cell cycle was ∼20% more rapid at the nucleus/centrosome-associated trigger wave sources than in the regions of the extract that appeared not to be entrained by trigger waves. Nuclei produced from phage DNA, which did not possess centrosomes, also acted as trigger wave sources, but purified centrosomes in the absence of nuclei did not. We conclude that the nucleus accelerates mitotic entry and propose that it acts as a pacemaker for cell cycle.One Sentence SummaryStudies in cycling Xenopus egg extracts show that mitosis first occurs in the nucleus and then spreads outward through the cytoplasm in circular trigger waves.

Micronuclei, whole or fragmented chromosomes which are spatially separated from the main nucleus, are strongly associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to unaligned chromosomes in vivo but do not develop spontaneous tumors, raising questions about whether all micronuclei contribute similarly to genomic instability and cancer. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. To further explore micronuclear envelope stability in KIF18A KO cells, we compared micronuclei induced via different insults in cultured cells. Micronuclei in KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of core and non-core nuclear envelope components and successful expansion of decondensing chromatin compared to those induced by microtubule drug washout or exposure to radiation. We also observed that lagging chromosomes, which lead to micronucleus formation, were positioned closer to the main chromatin masses, and further from the central spindle, in KIF18A KO cells. Our studies provide in vivo support to models suggesting that micronuclear fate depends on the sub-cellular location of late lagging chromosomes and suggest that not all micronuclei actively promote tumorigenesis.

The centriole duplication cycle normally ensures that centriole number is maintained at two centrioles per G1 cell. However, some circumstances can result in an aberrant increase in centriole number-a phenotype that is particularly prevalent in several types of cancer. Following an artificial increase in centriole number without tetraploidization due to transient overexpression of the kinase PLK4, human cells return to a normal centriole number during the proliferation of the population. We examine the mechanisms responsible for this return to normal centriole number at the population level in human retinal pigment epithelial cells. We find that the return to normal centriole number in the population of induced cells cannot be explained by limited duplication of centrioles, instability of extra centrioles, or by grossly asymmetric segregation of extra centrioles in mitosis. However, cells with extra centrioles display heterogenous phenotypes including extended cell cycle arrest, longer interphase durations, and death, which overall results in a proliferative disadvantage relative to normal cells in the population. Although about half of cells with extra centrioles in a population were able to divide, the extent of the disadvantages conferred by other fates is sufficient to account for the observed rate of return to normal centriole number. These results suggest that only under conditions of positive selection for cells with extra centrioles, continuous generation of such centrioles, or alleviation of the disadvantageous growth phenotypes would they be maintained in a population.

Background The complement cascade is increasingly implicated in development of a variety of diseases with strong immune contributions such as Alzheimer’s disease and Systemic Lupus Erythematosus. Mouse models have been used to determine function of central components of the complement cascade such as C1q and C3. However, species differences in their gene structures mean that mice do not adequately replicate human complement regulators, including CR1 and CR2. Genetic variation in CR1 and CR2 have been implicated in modifying disease states but the mechanisms are not known. Results To decipher the roles of human CR1 and CR2 in health and disease, we engineered C57BL/6J (B6) mice to replace endogenous murine Cr2 with human complement receptors, CR1 and CR2 (B6.CR2CR1). CR1 has an array of allotypes in human populations and using traditional recombination methods (Flp-frt and Cre-loxP) two of the most common alleles (referred to here as CR1long and CR1short) can be replicated within this mouse model, along with a CR1 knockout allele (CR1KO). Transcriptional profiling of spleens and brains identified genes and pathways differentially expressed between mice homozygous for either CR1long, CR1short or CR1KO. Gene set enrichment analysis predicts hematopoietic cell number and cell infiltration are modulated by CR1long, but not CR1short or CR1KO. Conclusion The B6.CR2CR1 mouse model provides a novel tool for determining the relationship between human-relevant CR1 alleles and disease.

Loss of the cilium is important for cell cycle progression and certain developmental transitions. Chytrid fungi are a group of basal fungi that have retained centrioles and cilia, and they can disassemble their cilia via axoneme internalization as part of the transition from free-swimming spores to sessile sporangia. While this type of cilium disassembly has been observed in many single-celled eukaryotes, it has not been well characterized because it is not observed in common model organisms. To better characterize cilium disassembly via axoneme internalization, we focused on chytrids Rhizoclosmatium globosum and Spizellomyces punctatus to represent two lineages of chytrids with different motility characteristics. Our results show that each chytrid species can reel in its axoneme into the cell body along its cortex on the order of minutes, while S. punctatus has additional faster ciliary compartment loss and lash-around mechanisms. S. punctatus retraction can also occur away from the cell cortex and is partially actin dependent. Post-internalization, the tubulin of the axoneme is degraded in both chytrids over the course of about 2 hr. Axoneme disassembly and axonemal tubulin degradation are both partially proteasome dependent. Overall, chytrid cilium disassembly is a fast process that separates axoneme internalization and degradation.

Hematopoietic stem cells (HSCs) are responsible for lifelong maintenance and regeneration of the blood system. With aging, loss of HSC function is a major contributor to decline in overall hematopoietic function, leading to increased rate of infection, poor vaccination response, clonal hematopoiesis, and increased risk of hematologic malignancies. While cellular and molecular hallmarks of HSC aging have been defined1–3, the lack of understanding of the nature and timing of the initiating events that cause HSC aging is a barrier to achieving the goal of extending healthy hematopoietic function into older age. Here we discover that hallmarks of HSC aging and myeloid-biased hematopoiesis accumulate by middle age in mice, and that the bone marrow (BM) microenvironment at middle age induces and is indispensable for hematopoietic aging phenotypes. Using unbiased transcriptome-based approaches, we identify decreased production of IGF1 by cells in the middle-aged BM microenvironment as a factor causing hematopoietic stem and progenitor cell aging and show that direct stimulation with IGF1 rescues hallmarks of hematopoietic aging. Declining IGF1 in the BM microenvironment at middle age represents a compelling target for intervention using prophylactic therapies to effectively extend healthspan and to prevent functional decline during aging.

Significance Primary cilia are antenna-like sensory organelles critical for many signal transduction pathways. One such pathway, called Hedgehog signaling, is essential for normal embryonic development and linked to tissue homeostasis and tumorigenesis. While the tightly regulated spatial and temporal localization of proteins in cilia controls activity, the mechanistic details are not fully understood. To characterize the movements of the Hedgehog receptor Patched1 in the cilium, we used single-molecule fluorescence microscopy, a tool that separates the behavior of individual proteins from the ensemble average, unveiling the underlying hidden physical behaviors. Our study reveals Hedgehog-induced changes in the motion of individual Patched1 molecules, which precede the exodus of Patched1 from cilia. These changes constitute one of the earliest measurable steps of Hedgehog-signal transduction., The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

The cilium, once considered a vestigial structure, is a conserved, microtubule-based organelle critical for transducing extracellular chemical and mechanical signals that control cell polarity, differentiation, and proliferation. The cilium undergoes cycles of assembly and disassembly that are controlled by complex inter-relationships with the cytoskeleton. Microtubules form the core of the cilium, the axoneme, and are regulated by post-translational modifications, associated proteins, and microtubule dynamics. Although actin and septin cytoskeletons are not major components of the axoneme, they also regulate cilium organization and assembly state. Here, we discuss recent advances on how these different cytoskeletal systems­ affect cilium function, structure, and organization.

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2cub/cub) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2cub/cub and Rhbdf2+/+ mice at 0h, 15 min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15 min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds.

SummaryShort linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins – structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

SummaryThe regulation of proliferation is one of the primary functions of Hedgehog (Hh) signaling in development. Transduction of Hh signaling requires the primary cilium, a microtubule-based organelle that is necessary for several steps in the pathway (Corbit et al., 2005; Huangfu and Anderson, 2005; Huangfu et al., 2003; Liu et al., 2005; Rohatgi et al., 2007). Many cells only build a primary cilium upon cell cycle arrest in G0. In those proliferating cells that do make a cilium, it is a transient organelle, being assembled in G1 and disassembled sometime after, although exactly when is not well-characterized (Ford et al., 2018; Pugacheva et al., 2007; Wang and Dynlacht, 2018). Thus the requirement for primary cilia presents a conundrum: how are proliferative signals conveyed through an organelle that is present for only part of the cell cycle? Here we investigate this question in a mouse medulloblastoma cell line, SMB55, that requires cilium-mediated Hh pathway activity for proliferation (Zhao et al., 2015). We show that SMB55 cells are often ciliated beyond G1 into S phase, and the presence of the cilium determines the periods of Hh pathway activity. Using live imaging over multiple cell cycles, we define two windows of opportunity for Hh pathway activity, either of which is sufficient to effect cell cycle entry. The first is in the ciliated phase of the previous cell cycle, and the second is in G1 of the cell cycle in which the decision is made. We propose that the ability of cells to integrate Hh pathway activity from more than one cell cycle imparts robustness on Hh pathway control of proliferation and may have implications for other Hh-mediated events in development.

Olfaction in most animals is mediated by sensory neurons bearing receptors on cilia that are accessible to the environment. Within chordates, olfactory sensory neurons (OSNs) usually have multiple cilia, each with a centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how mature cells with multiple centrioles are generated during this process is not yet understood. OSNs in the mouse olfactory epithelium have about 15 cilia each, and we show that centrioles are amplified in precursor cells via formation of centriole rosette structures both during embryonic development and during turnover of the olfactory epithelium in adults. We also found free centrioles present in rosette-bearing cells, suggesting that more than one pathway contributes to total centriole number. Cells with amplified centrioles can go on to divide, with clustered centrioles at each pole. Additionally, we found that centrioles are amplified in early immediate neuronal precursors, coincident with elevation of mRNA for Plk4 and Stil, two key regulators of centriole duplication. Our findings highlight the importance of accounting for centriole amplification in the development of olfactory epithelium-derived neuron regeneration therapies.