CEP164-null cells generated by genome editing show a ciliation defect with intact DNA repair capacity.

Primary cilia are microtubule structures that extend from the distal end of the mature, mother centriole. CEP164 is a component of the distal appendages carried by the mother centriole that is required for primary cilium formation. Recent data have implicated CEP164 as a ciliopathy gene and suggest that CEP164 plays some roles in the DNA damage response (DDR). We used reverse genetics to test the role of CEP164 in the DDR. We found that conditional depletion of CEP164 in chicken DT40 cells using an auxin-inducible degron led to no increase in sensitivity to DNA damage induced by ionising or ultraviolet irradiation. Disruption of CEP164 in human retinal pigmented epithelial cells blocked primary cilium formation but did not affect cellular proliferation or cellular responses to ionising or ultraviolet irradiation. Furthermore, we observed no localisation of CEP164 to the nucleus using immunofluorescence microscopy and analysis of multiple tagged forms of CEP164. Our data suggest that CEP164 is not required in the DDR.

Ulk4 Is Essential for Ciliogenesis and CSF Flow.

UNLABELLED: Ciliopathies are an emerging class of devastating disorders with pleiotropic symptoms affecting both the central and peripheral systems and commonly associated with hydrocephalus. Even though ciliary components and three master transcriptional regulators have been identified, little is known about the signaling molecules involved. We previously identified a novel gene, Unc51-like-kinase 4 (ULK4), as a risk factor of neurodevelopmental disorders. Here we took multidisciplinary approaches and uncovered essential roles of Ulk4 in ciliogenesis. We show that Ulk4 is predominantly expressed in the ventricular system, and Ulk4(tm1a/tm1a) ependymal cells display reduced/disorganized cilia with abnormal axonemes. Ulk4(tm1a/tm1a) mice exhibit dysfunctional subcommissural organs, obstructive aqueducts, and impaired CSF flow. Mechanistically, we performed whole-genome RNA sequencing and discovered that Ulk4 regulates the Foxj1 pathway specifically and an array of other ciliogenesis molecules. This is the first evidence demonstrating that ULK4 plays a vital role in ciliogenesis and that deficiency of ULK4 can cause hydrocephalus and ciliopathy-related disorders. SIGNIFICANCE STATEMENT: Ciliopathies are an emerging class of devastating disorders with pleiotropic symptoms affecting both the central and peripheral systems. Ciliopathies are commonly associated with hydrocephalus, and Unc51-like-kinase 4 (Ulk4) has been identified as one of 12 genes causing hydrocephalus in mutants. Here we uncover an essential role of Ulk4 in ciliogenesis. Ulk4 is predominantly expressed in the ventricles, and mutant ependymal cells display reduced/disorganized/nonfunctional motile cilia with abnormal axonemes and impaired CSF flow. Ulk4 modulates expression of the master regulator of ciliogenesis, Foxj1, and other ciliogenesis molecules. This is the first report demonstrating a vital role of Ulk4 in ciliogenesis. ULK4 deficiency may be implicated in human hydrocephalus and other ciliopathy-related disorders.

Tandem Amplification of the Staphylococcal Cassette Chromosome mec Element Can Drive High-Level Methicillin Resistance in Methicillin-Resistant Staphylococcus aureus.

Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) ß-lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the mecA gene, carried on the staphylococcal cassette chromosome mec element (SCCmec), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 µg/ml to 130 µg/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in gdpP, clpX, guaA, and camS Closer inspection of the genome sequence data further revealed that reads covering SCCmec were ∼10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 µg/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.

Three-Dimensional Microgel Platform for the Production of Cell Factories Tailored for the Nucleus Pulposus.

Intradiscal injection of growth factors or cells has been shown to attenuate symptoms of intervertebral disc degeneration. However, different approaches are needed to overcome limitations such as short-term efficacy and leakage of the injected solutions. The current study aims at creating a platform for the realization of functional cell factories by using in parallel cell delivery and gene therapy approaches. Superfect, a transfecting agent, was used as nonviral gene vector because of its ability to form complexes with plasmid DNA (polyplexes). Polyplexes were loaded into collagen hollow microsphere reservoirs, and their ability to transfect cells was ascertained in vitro. Adipose-derived stem cells were then embedded in three-dimensional (3D) microgels composed of type II collagen/hyaluronan, which mimics the environmental cues typical of the healthy nucleus pulposus. These were functionalized with polyplex-loaded collagen hollow spheres and the secretion of the target protein was assessed quantitatively. Delivery of polyplexes from a reservoir system lowered their toxicity significantly while maintaining high levels of transfection in a monolayer culture. In 3D microgels, lower levels of transfection were observed, however; increasing levels of luciferase were secreted from the microgels over 7 days of culture. These results indicate that 3D microgels, functionalized with polyplex-loaded reservoirs offer a reliable platform for the production of cell factories that are able to manufacture targeted therapeutic proteins for regenerative therapies that have applications in nucleus pulposus repair.

Centrobin controls primary ciliogenesis in vertebrates.

The BRCA2 interactor, centrobin, is a centrosomal protein that has been implicated in centriole duplication and microtubule stability. We used genome editing to ablate CNTROB in hTERT-RPE1 cells and observed an increased frequency of monocentriolar and acentriolar cells. Using a novel monoclonal antibody, we found that centrobin primarily localizes to daughter centrioles but also associates with mother centrioles upon serum starvation. Strikingly, centrobin loss abrogated primary ciliation upon serum starvation. Ultrastructural analysis of centrobin nulls revealed defective axonemal extension after mother centriole docking. Ciliogenesis required a C-terminal portion of centrobin that interacts with CP110 and tubulin. We also depleted centrobin in zebrafish embryos to explore its roles in an entire organism. Centrobin-depleted embryos showed microcephaly, with curved and shorter bodies, along with marked defects in laterality control, morphological features that indicate ciliary dysfunction. Our data identify new roles for centrobin as a positive regulator of vertebrate ciliogenesis.

Phytochrome A and B Regulate Primary Metabolism in Arabidopsis Leaves in Response to Light.

Primary metabolism is closely linked to plant productivity and quality. Thus, a better understanding of the regulation of primary metabolism by photoreceptors has profound implications for agricultural practices and management. This study aims at identifying the role of light signaling in the regulation of primary metabolism, with an emphasis on starch. We first screened seven cryptochromes and phytochromes mutants for starch phenotype. The phyAB mutant showed impairment in starch accumulation while its biomass, chlorophyll fluorescence parameters, and leaf anatomy were unaffected, this deficiency being present over the whole vegetative growth period. Mutation of plastidial nucleoside diphosphate kinase-2 (NDPK2), acting downstream of phytochromes, also caused a deficit in starch accumulation. Besides, the glucose-1-phosphate adenylyltransferase small subunit (APS1) was down-regulated in phyAB. Those results suggest that PHYAB affect starch accumulation through NDPK2 and APS1. Then, we determined changes in starch and primary metabolites in single phyA, single phyB, double phyAB grown in light conditions differing in light intensity and/or light spectral content. PHYA is involved in starch accumulation in all the examined light conditions, whereas PHYB only exhibits a role under low light intensity (44 ± 1 µmol m-2 s-1) or low R:FR (11.8 ± 0.6). PCA analysis of the metabolic profiles in the mutants and wild type (WT) suggested that PHYB acts as a major regulator of the leaf metabolic status in response to light intensity. Overall, we propose that PHYA and PHYB signaling play essential roles in the control of primary metabolism in Arabidopsis leaves in response to light.

Screening of exosomal microRNAs from colorectal cancer cells.

BACKGROUND: Cells release extracellular membrane vesicles including microvesicles known as exosomes. Exosomes contain microRNAs (miRNAs) however the full range within colorectal cancer cell secreted exosomes is unknown. OBJECTIVE: To identify the full range of exosome encapsulated miRNAs secreted from 2 colorectal cancer cell lines and to investigate engineering of exosomes over-expressing miRNAs. METHODS: Exosomes were isolated from HCT-116 and HT-29 cell lines. RNA was extracted from exosomes and microRNA array performed. Cells were engineered to express miR-379 (HCT-116-379) or a non-targeting control (HCT-116-NTC) and functional effects were determined. Exosomes secreted by engineered cells were transferred to recipient cells and the impact examined. RESULTS: Microvesicles 40-100 nm in size secreted by cell lines were visualised and confirmed to express exosomal protein CD63. HT-29 exosomes contained 409 miRNAs, HCT-116 exosomes contained 393, and 338 were common to exosomes from both cell lines. Selected targets were validated. HCT-116-379 cells showed decreased proliferation (12-15% decrease, p < 0.001) and decreased migration (32-86% decrease, p < 0.001) compared to controls. HCT-116-379 exosomes were enriched for miR-379. Confocal microscopy visualised transfer of HCT-116-379 exosomes to recipient cells. CONCLUSIONS: Colorectal cancer cells secrete a large number of miRNAs within exosomes. miR-379 decreases cell proliferation and migration, and miR-379 enriched exosomes can be engineered.

A chondromimetic microsphere for in situ spatially controlled chondrogenic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have been identified as a viable cell source for cartilage tissue engineering. However, to undergo chondrogenic differentiation hMSCs require growth factors, in particular members of the transforming growth factor beta (TGF-ß) family. While in vitro differentiation is feasible through continuous supplementation of TGF-ß3, mechanisms to control and drive hMSCs down the chondrogenic lineage in their native microenvironment remain a significant challenge. The release of TGF-ß3 from an injectable microsphere composed of the cartilage-associated extracellular matrix molecule hyaluronan represents a readily translatable approach for in situ differentiation of hMSCs for cartilage repair. In this study, chondromimetic hyaluronan microspheres were used as a growth factor delivery source for hMSC chondrogenesis. Cellular compatibility of the microspheres (1.2 and 14.1 µm) with hMSCs was shown and release of TGF-ß3 from the most promising 14.1 µm microspheres to control differentiation of hMSCs was evaluated. Enhanced accumulation of cartilage-associated glycosaminoglycans by hMSCs incubated with TGF-ß3-loaded microspheres was seen and positive staining for collagen type II and proteoglycan confirmed successful in vitro chondrogenesis. Gene expression analysis showed significantly increased expression of the chondrocyte-associated genes, collagen type II and aggrecan. This delivery platform resulted in significantly less collagen type X expression, suggesting the generation of a more stable cartilage phenotype. When evaluated in an ex vivo osteoarthritic cartilage model, implanted hMSCs with TGF-ß3-loaded HA microspheres were detected within cartilage fibrillations and increased proteoglycan staining was seen in the tissue. In summary, data presented here demonstrate that TGF-ß3-bound hyaluronan microspheres provide a suitable delivery system for induction of hMSC chondrogenesis and their use may represent a clinically feasible tissue engineering approach for the treatment of articular cartilage defects.

Ultrastructure of the ciliated cells of the free-swimming larva, and sessile stages, of the marine sponge Haliclona indistincta (Demospongiae: Haplosclerida).

We provide a detailed, comparative study of the ciliated cells of the marine haplosclerid sponge Haliclona indistincta, in order to make data available for future phylogenetic comparisons at the ultrastructural level. Our study focuses on the description and analysis of the larval epithelial cells, and choanocytes of the metamorphosed juvenile sponge. The ultrastructure of the two cell types is sufficiently different to prevent our ability to conclusively determine the origin of the choanocytes from the larval ciliated cells. However, ciliated, epithelial cells were observed in a migratory position within the inner cell mass of the larval stages. Some cilia were observed within the cell's cytoplasm, which is indicative of the ciliated epithelial cell undergoing transdifferentiation into a choanocyte; while traces of other ciliated epithelial cells were contained within phagosomes, suggesting they are phagocytosed. We compared our data with other species described in the literature. However, any phylogenetic inference must wait until further detailed comparisons can be made with species whose phylogenetic position has been determined by other means, such as phylogenomics, in order to more closely link genomic, and morphological information.

Promoter hijack reveals pericentrin functions in mitosis and the DNA damage response.

Centrosomes, the principal microtubule-organizing centers of animal somatic cells, consist of two centrioles embedded in the pericentriolar material (PCM). Pericentrin is a large PCM protein that is required for normal PCM assembly. Mutations in PCNT cause primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. To test how pericentrin is involved in cell cycle control after genotoxic stress, we disrupted the Pcnt locus in chicken DT40 cells. Pericentrin-deficient cells proceeded through mitosis more slowly, with a high level of monopolar spindles, and were more sensitive to spindle poisons than controls. Centriole structures appeared normal by light and electron microscopy, but the PCM did not recruit γ-tubulin efficiently. Cell cycle delays after ionizing radiation (IR) treatment were normal in pericentrin-deficient cells. However, pericentrin disruption in Mcph1-/- cells abrogated centrosome hyperamplification after IR. We conclude that pericentrin controls genomic stability by both ensuring appropriate mitotic spindle activity and centrosome regulation.

Abnormal centrosomal structure and duplication in Cep135-deficient vertebrate cells.

Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase-arrested cells.

Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

Loss of plakophilin 2 disrupts heart development in zebrafish.

The desmosomal armadillo protein plakophilin 2 is the only plakophilin expressed in the heart, and mutations in the human plakophilin 2 gene result in arrhythmogenic right ventricular cardiomyopathy. To investigate loss of function, we knocked down plakophilin 2 by morpholino microinjection in zebrafish. This resulted in decreased heart rate, cardiac oedema, blood pooling, a failure of the heart to pattern correctly and a twisted tail. Co-injection of plakophilin 2 mRNA rescued the morphant phenotype, indicating the specificity of the knockdown. Desmosome numbers were decreased in morphant hearts and the plaque and midline structures of the desmosomes in the intercalated discs were disrupted when examined by electron microscopy. cmlc2 and vmhc expression at 48 hours post-fertilization (hpf) showed incomplete looping of the heart in morphant embryos by whole mount in situ hybridization, and bmp4 expression was expanded into the ventricle. The domain of expression of the heart marker nkx2.5 at 24 hpf was expanded. At the 18 somite stage, expression of the cardiogenic gene lefty2 was abolished in the left cardiac field, with concomitant increases in bmp4, spaw and lefty1 expression, likely resulting in the looping defects. These results indicate that plakophilin 2 has both structural and signalling roles in zebrafish heart development.

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.

Defective nucleotide excision repair with normal centrosome structures and functions in the absence of all vertebrate centrins.

The principal microtubule-organizing center in animal cells, the centrosome, contains centrin, a small, conserved calcium-binding protein unique to eukaryotes. Several centrin isoforms exist and have been implicated in various cellular processes including nuclear export and deoxyribonucleic acid (DNA) repair. Although centrins are required for centriole/basal body duplication in lower eukaryotes, centrin functions in vertebrate centrosome duplication are less clear. To define these roles, we used gene targeting in the hyperrecombinogenic chicken DT40 cell line to delete all three centrin genes in individual clones. Unexpectedly, centrin-deficient cells underwent normal cellular division with no detectable cell cycle defects. Light and electron microscopy analyses revealed no significant difference in centrosome composition or ultrastructure. However, centrin deficiency made DT40 cells highly sensitive to ultraviolet (UV) irradiation, with Cetn3 deficiency exacerbating the sensitivity of Cetn4/Cetn2 double mutants. DNA damage checkpoints were intact, but repair of UV-induced DNA damage was delayed in centrin nulls. These data demonstrate a role for vertebrate centrin in nucleotide excision repair.