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.

Sperm-contributed centrioles segregate stochastically into blastomeres of 4-cell stage Caenorhabditis elegans embryos.

Whereas both sperm and egg contribute nuclear genetic material to the zygote in metazoan organisms, the inheritance of other cellular constituents is unequal between the 2 gametes. Thus, 2 copies of the centriole are contributed solely by the sperm to the zygote in most species. Centrioles can have a stereotyped distribution in some asymmetric divisions, but whether sperm-contributed centrioles are distributed in a stereotyped manner in the resulting embryo is not known. Here, we address this question in Caenorhabditis elegans using marked mating experiments, whereby the presence of the 2 sperm-contributed centrioles is monitored in the embryo using the stable centriolar component SAS-4::GFP, as well as GFP::SAS-7. Our analysis demonstrates that the distribution of sperm-contributed centrioles is stochastic in 4-cell stage embryos. Moreover, using sperm from zyg-1 mutant males that harbor a single centriole, we show that the older sperm-contributed centriole is likewise distributed stochastically in the resulting embryo. Overall, we conclude that, in contrast to the situation during some asymmetric cell divisions, centrioles contributed by the male germ line are distributed stochastically in embryos of C. elegans.

A novel biocompatible polymer derived from D-mannitol used as a vector in the field of genetic engineering of eukaryotic cells.

The design and preparation of new vectors to transport genetic material and increase the transfection efficiency continue being an important research line. Here, a novel biocompatible sugar-based polymer derived from D-mannitol has been synthesized to be used as a gene material nanocarrier in human (gene transfection) and microalga cells (transformation process). Its low toxicity allows its use in processes with both medical and industrial applications. A multidisciplinary study about the formation of polymer/p-DNA polyplexes has been carried out using techniques such as gel electrophoresis, zeta potential, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy. The nucleic acids used were the eukaryotic expression plasmid pEGFP-C1 and the microalgal expression plasmid Phyco69, which showed different behaviors. The importance of DNA supercoiling in both transfection and transformation processes was demonstrated. Better results were obtained in microalga cells nuclear transformation than in human cells gene transfection. This was related to the plasmid's conformational changes, in particular to their superhelical structure. It is noteworthy that the same nanocarrier has been used with eukaryotic cells from both human and microalga.

TRIM37: a critical orchestrator of centrosome function.

Loss of function mutations in the E3 ubiquitin ligase TRIM37 result in MULIBREY nanism, a disease characterized by impaired organ growth and a high propensity to develop different tumor types. Additionally, increased copy number of TRIM37 is a feature of some breast cancers and neuroblastomas. The molecular role played by TRIM37 in such loss and gain of function conditions has been a focus of research in the last decade, which led notably to the identification of critical roles of TRIM37 in centrosome biology. Specifically, deletion of TRIM37 results in the formation of aberrant centrosomal proteins assemblies, including Centrobin-PLK4 assemblies, which can act as extra MTOCs, thus resulting in defective chromosome segregation. Additionally, TRIM37 overexpression targets the centrosomal protein CEP192 for degradation, thereby preventing centrosome maturation and increasing the frequency of mitotic errors. Interestingly, increased TRIM37 protein levels sensitize cells to the PLK4 inhibitor centrinone. In this review, we cover the emerging roles of TRIM37 in centrosome biology and discuss how this knowledge may lead to new therapeutic strategies to target specific cancer cells.

Multivalent Calixarene-Based Liposomes as Platforms for Gene and Drug Delivery.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

The Emerging Role of RNA Modifications in DNA Double-Strand Break Repair.

The correct repair of DNA double-strand breaks is essential for maintaining the stability of the genome, thus ensuring the survival and fitness of any living organism. Indeed, the repair of these lesions is a complicated affair, in which several pathways compete for the DNA ends in a complex balance. Thus, the fine-tuning of the DNA double-strand break repair pathway choice relies on the different regulatory layers that respond to environmental cues. Among those different tiers of regulation, RNA modifications have just emerged as a promising field.

TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin.

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease with impaired organ growth and increased tumor formation. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterize these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We find that an atypical de novo assembly pathway can generate Cenpas that act as microtubule-organizing centers (MTOCs), including in Mulibrey patient cells. Correlative light electron microscopy reveals that Cenpas are centriole-related or electron-dense structures with stripes. TRIM37 regulates the stability and solubility of Centrobin, which accumulates in elongated entities resembling the striped electron dense structures upon TRIM37 depletion. Furthermore, Cenpas formation upon TRIM37 depletion requires PLK4, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents Cenpas formation, which would otherwise threaten genome integrity.

Methylation of the central transcriptional regulator KLF4 by PRMT5 is required for DNA end resection and recombination.

Cell fitness and survival upon exposure to DNA damage depends on the repair of DNA lesions. Interestingly, cellular identity does affect and finetunes such response, although the molecular basis of such differences between tissues and cell types is not well understood. Thus, a possibility is that DNA repair itself is controlled by the mechanisms that govern cell identity. Here we show that the KLF4, involved in cellular homeostasis, proliferation, cell reprogramming and cancer development, directly regulates resection and homologous recombination proficiency. Indeed, resection efficiency follows KLF4 protein levels, i.e. decreases upon KLF4 downregulation and increases when is overexpressed. Moreover, KLF4 role in resection requires its methylation by the methyl-transferase PRMT5. Thus, PRMT5 depletion not only mimics KLF4 downregulation, but also showed an epistatic genetic relationship. Our data support a model in which the methylation of KLF4 by PRMT5 is a priming event required to license DNA resection and homologous recombination.

Metallo-Liposomes of Ruthenium Used as Promising Vectors of Genetic Material.

Gene therapy is a therapeutic process consisting of the transport of genetic material into cells. The design and preparation of novel carriers to transport DNA is an important research line in the medical field. Hybrid compounds such as metallo-liposomes, containing a mixture of lipids, were prepared and characterized. Cationic metal lipids derived from the [Ru(bpy)3]2+ complex, RuC11C11 or RuC19C19, both with different hydrophobic/lipophilic ratios, were mixed with the phospholipid DOPE. A relation between the size and the molar fraction α was found and a multidisciplinary study about the interaction between the metallo-liposomes and DNA was performed. The metallo-liposomes/DNA association was quantified and a relationship between Kapp and α was obtained. Techniques such as AFM, SEM, zeta potential, dynamic light scattering and agarose gel electrophoresis demonstrated the formation of lipoplexes and showed the structure of the liposomes. L/D values corresponding to the polynucleotide's condensation were estimated. In vitro assays proved the low cell toxicity of the metallo-liposomes, lower for normal cells than for cancer cell lines, and a good internalization into cells. The latter as well as the transfection measurements carried out with plasmid DNA pEGFP-C1 have demonstrated a good availability of the Ru(II)-based liposomes for being used as non-toxic nanovectors in gene therapy.

The dual role of the centrosome in organizing the microtubule network in interphase.

Here, we address the regulation of microtubule nucleation during interphase by genetically ablating one, or two, of three major mammalian γ-TuRC-binding factors namely pericentrin, CDK5Rap2, and AKAP450. Unexpectedly, we find that while all of them participate in microtubule nucleation at the Golgi apparatus, they only modestly contribute at the centrosome where CEP192 has a more predominant function. We also show that inhibiting microtubule nucleation at the Golgi does not affect centrosomal activity, whereas manipulating the number of centrosomes with centrinone modifies microtubule nucleation activity of the Golgi apparatus. In centrosome-free cells, inhibition of Golgi-based microtubule nucleation triggers pericentrin-dependent formation of cytoplasmic-nucleating structures. Further depletion of pericentrin under these conditions leads to the generation of individual microtubules in a γ-tubulin-dependent manner. In all cases, a conspicuous MT network forms. Strikingly, centrosome loss increases microtubule number independently of where they were growing from. Our results lead to an unexpected view of the interphase centrosome that would control microtubule network organization not only by nucleating microtubules, but also by modulating the activity of alternative microtubule-organizing centers.

ZYG-1 promotes limited centriole amplification in the C. elegans seam lineage.

Genome stability relies notably on the integrity of centrosomes and on the mitotic spindle they organize. Structural and numerical centrosome aberrations are frequently observed in human cancer, and there is increasing evidence that centrosome amplification can promote tumorigenesis. Here, we use C. elegans seam cells as a model system to analyze centrosome homeostasis in the context of a stereotyped stem like lineage. We found that overexpression of the Plk4-related kinase ZYG-1 leads to the formation of one supernumerary centriolar focus per parental centriole during the cell cycle that leads to the sole symmetric division in the seam lineage. In the following cell cycle, such supernumerary foci function as microtubule organizing centers, but do not cluster during mitosis, resulting in the formation of a multipolar spindle and then aneuploid daughter cells. Intriguingly, we found also that supernumerary centriolar foci do not assemble in the asymmetric cell divisions that precedes or that follows the symmetric seam cell division, despite the similar presence of GFP::ZYG-1. Furthermore, we established that supernumerary centrioles form earlier during development in animals depleted of the heterochronic gene lin-14, in which the symmetric division is precocious. Conversely, supernumerary centrioles are essentially not observed in animals depleted of lin-28, in which the symmetric division is lacking. These findings lead us to conclude that ZYG-1 promotes limited centriole amplification solely during the symmetric division in the C. elegans seam lineage.

SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans.

Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome.

Multiciliogenesis: multicilin directs transcriptional activation of centriole formation.

During differentiation of multiciliated cells, numerous centrioles are generated in each cell to act as templates for the formation of a corresponding number of cilia. A new study reveals that multicilin, a protein required for multiciliogenesis, is a key component of a regulatory complex that activates the transcription of genes required for centriole formation.

Discovering regulators of centriole biogenesis through siRNA-based functional genomics in human cells.

Centrioles are essential for forming cilia, flagella, and centrosomes and are thus critical for a range of fundamental cellular processes. Despite their importance, the mechanisms governing centriole biogenesis remain incompletely understood. We performed a high-content genome-wide small-interfering-RNA-based screen to identify genes regulating centriole formation in human cells. We designed an algorithm to automatically detect GFP-Centrin foci that, combined with subsequent manual analysis, allowed us to identify 44 genes required for centriole formation and 32 genes needed for restricting centriole number. Detailed follow-up characterization uncovered that the C2 domain protein C2CD3 is required for distal centriole formation and suggests that it functions in the basal body to template primary cilia. Moreover, we found that the E3 ubiquitin ligase TRIM37 prevents centriole reduplication events. We developed a dynamic web interface containing all images and numerical features as a powerful resource to investigate facets of centrosome biology.

Spindle positioning in human cells relies on proper centriole formation and on the microcephaly proteins CPAP and STIL.

Patients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes centrosome protein 4.1 associated protein (CPAP; also known as centromere protein J, CENPJ). CPAP is a large coiled-coil protein enriched at the centrosome, a structure that comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains crucial for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects correlate with a randomization of spindle position. Moreover, we demonstrate that the MCPH protein SCL/TAL1 interrupting locus (STIL) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH because of aberrant spindle positioning in progenitor cells during brain development.

A G2-phase microtubule-damage response in fission yeast.

Microtubules assume a variety of structures throughout the different stages of the cell cycle. Ensuring the correct assembly of such structures is essential to guarantee cell division. During mitosis, it is well established that the spindle assembly checkpoint monitors the correct attachment of sister chromatids to the mitotic spindle. However, the role that microtubule cytoskeleton integrity plays for cell-cycle progression during interphase is uncertain. Here we describe the existence of a mechanism, independent of the mitotic checkpoint, that delays entry into mitosis in response to G(2)-phase microtubule damage. Disassembly of the G(2)-phase microtubule array leads to the stabilization of the universal mitotic inhibitor Wee1, thus actively delaying entry into mitosis via inhibitory Cdc2 Tyr15 phosphorylation.