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Coxiella burnetii, the causative agent of Q fever, is a highly infectious pathogen capable of invading diverse cell types, from alveolar macrophages to trophoblasts. Within host cells, it establishes a replicative niche named Coxiella-containing vacuole (CCV). This is driven by effector proteins secreted by the bacterium into the host cell cytoplasm via a Type 4b Secretion System (T4SS). Advances in axenic culture and mutagenesis allowed the characterization of Coxiella effector proteins, revealing their host targets and strategies of cellular subversion. This review highlights recent insights into Coxiella effector proteins and their manipulation of host processes, from vesicular trafficking to innate immunity.

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.

Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCFFbw7 ubiquitin ligase. In human cancers bearing FBXW7-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in Xenopus eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.

Key Points Polo-like kinases (PLKs) are a family of Ser/Thr kinases that have a pivotal role in cell cycle progression, the centrosome cycle, mitosis and cellular responses to DNA damage, which makes them attractive targets for treatments against several diseases. PLK1 is the most ancestral and best-conserved member of the family; it is found in most eukaryotic organisms, except for higher land plants. PLK4 is the most divergent member of the family. PLK2, PLK3 and PLK5 have evolved very recently, probably from a PLK1 gene duplication in vertebrates. PLK1 and PLK4 have distinct structural organizations and are phosphorylated at different residues, which correlate with different mode of actions. The amino-terminal kinase domain and carboxy-terminal polo box domains that characterize PLKs are crucial for regulation of their kinase catalytic activity in time and space, and for controlling subcellular PLK localization. Recent studies show non-canonical functions for PLKs in asymmetric cell division and cilia disassembly. PLKs function in centriole and centrosome biogenesis; PLK1 integrates various external stimuli with cell cycle inputs to coordinate mitotic progression and the centrosome cycle, whereas PLK4 drives centriole assembly. PLK2 and PLK3 have roles in DNA replication and in the DNA damage response and are also expressed in non-proliferative tissues, in which they have a role in cell differentiation and homeostasis (for example, PLK2 and PLK5 regulate neuronal activity). Members of the polo-like kinase (PLK) family are crucial regulators of cell cycle progression, centriole duplication, mitosis, cytokinesis and the DNA damage response. Recent structural and molecular studies have revealed how such processes depend on the tight regulation of PLK abundance, activity, localization and interactions with other proteins, and how dysregulation may be associated with disease. Members of the polo-like kinase (PLK) family are crucial regulators of cell cycle progression, centriole duplication, mitosis, cytokinesis and the DNA damage response. PLKs undergo major changes in abundance, activity, localization and structure at different stages of the cell cycle. They interact with other proteins in a tightly controlled spatiotemporal manner as part of a network that coordinates key cell cycle events. Their essential roles are highlighted by the fact that alterations in PLK function are associated with cancers and other diseases. Recent knowledge gained from PLK crystal structures, evolution and interacting molecules offers important insights into the mechanisms that underlie their regulation and activity, and suggests novel functions unrelated to cell cycle control for this family of kinases.

In this paper, a speech enhancement method based on correlation canceling approach associated with the Log- minimum mean-square-error estimator is presented. Unlike the conventional statistical-model methods based on the nonlinear estimation of the enhanced speech signal, such as Maximum-Likelihood estimator (ML), Maximum A Posteriori (MAP) estimator, Minimum Mean Square Error (MMSE) estimator and log MMSE estimator, in the proposed hybrid method (CC/Log-MMSE), the nonlinear estimation is transformed into a linear estimation by exploiting the orthogonal projection of clean signal into the noisy signal. Thus, the enhanced signal represents the best “copy,” or estimate, of clean signal that can be made on the basis of the noisy signal vector. This is also seen as a canceling of the component of the noisy vector residing in the noise subspace, which therefore leads to improve the intelligibility of the enhanced signal. Extensive simulations are carried out using speech files corrupted by different noises available in the NOIZEUS corpus, show that the proposed hybrid method CC/Log-MMSE consistently outperforms the baseline methods of speech enhancement at different levels of SNR in terms of objective and subjective measures, spectrogram analysis and the overall SNR improvement.

Ciliary defects cause several ciliopathies, some of which have late onset, suggesting cilia are actively maintained. Still, we have a poor understanding of the mechanisms underlying their maintenance. Here, we show Drosophila melanogaste r IFT88 ( Dm IFT88/nompB) continues to move along fully formed sensory cilia. We further identify Inactive, a TRPV channel subunit involved in Drosophila hearing and negative-gravitaxis behaviour, and a yet uncharacterised Drosophila Guanylyl Cyclase 2d ( Dm Gucy2d/CG34357) as Dm IFT88 cargoes. We also show Dm IFT88 binding to the cyclase´s intracellular part, which is evolutionarily conserved and mutated in several degenerative retinal diseases, is important for the ciliary localisation of Dm Gucy2d. Finally, acute knockdown of both Dm IFT88 and Dm Gucy2d in ciliated neurons of adult flies caused defects in the maintenance of cilium function, impairing hearing and negative-gravitaxis behaviour, but did not significantly affect ciliary ultrastructure. We conclude that the sensory ciliary function underlying hearing in the adult fly requires an active maintenance program which involves Dm IFT88 and at least two of its signalling transmembrane cargoes, Dm Gucy2d and Inactive.

Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCFFbw7 ubiquitin ligase. In human cancers bearing FBXW7-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in Xenopus eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state

Abstract Cilia are involved in a plethora of motility and sensory-related functions. Ciliary defects cause several ciliopathies, some of which with late-onset, suggesting cilia are actively maintained. While much is known about cilia assembly, little is understood about the mechanisms of their maintenance. Given that intraflagellar transport (IFT) is essential for cilium assembly, we investigated the role of one of its main players, IFT88, in ciliary maintenance. We show that DmIFT88, the Drosophila melanogaster orthologue of IFT88, continues to move along fully formed sensory cilia, and that its acute knockdown in the ciliated neurons of the adult affects sensory behaviour. We further identify DmGucy2d, the Drosophila guanylyl cyclase 2d, as a DmIFT88 cargo, whose loss also leads to defects in sensory behaviour maintenance. DmIFT88 binds to the intracellular part of DmGucy2d, which is evolutionarily conserved and mutated in several degenerative retina diseases, taking the cyclase into the cilia. Our results offer a novel mechanism for the maintenance of sensory cilia function and its potential role in human diseases. Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵7 Shared lead authors.

The centriole duplication cycle must be tightly controlled and coordinated with the chromosome cycle. Aberrations in centriole biogenesis can lead to cancer, developmental disorders and ciliopathies. Here, we show that McIdas -previously implicated in cell cycle control and centriole amplification in multiciliated cells- is critical to maintain centriole numbers. Using expansion microscopy, we demonstrate that McIdas is present at the middle part of centrioles, where it exhibits a differential localization during the cell cycle. McIdas loss perturbs daughter centriole biogenesis and centrosomal SAS6 recruitment, whereas its overexpression induces centriole overduplication. Consistently, McIdas depletion reduces PLK4-induced centriole amplification. McIdas interacts with and is phosphorylated by PLK4 in multiple sites identified by mass spectrometry. Mutational analysis shows that McIdas phosphorylation is important for centriole number control. Overall, our results identify a novel, direct role of McIdas on centriole duplication that can link its previously characterized roles in the chromosome cycle and multiciliogenesis. Competing Interest Statement The authors have declared no competing interest.

DNA replication initiates with pre-replication complex (pre-RC) formation at replication origins in G1 (replication origin licensing), followed by activation of a pre-RC subset in the S phase. It has been suggested that a checkpoint prevents S phase entry when too few origins are licensed. Yet, we found that in normal cells, complete DNA synthesis inhibition by overexpression of a non-degradable geminin variant, or by CDT1 silencing prevents DNA replication without inducing any checkpoint. Cells continue cycling and enter mitosis, despite the absence of replicated DNA. Most of these unlicensed cells exit mitosis without dividing and enter senescence; however, about 25% of them successfully divide without previous DNA replication, producing daughter cells with half the normal diploid complement of chromosomes (1C). This suggests a potentially attractive strategy to derive haploid cells from any somatic cell type and unveil undescribed aspects of the coordination between DNA replication and cell division in mammals. Competing Interest Statement The authors have declared no competing interest.