Phage production is blocked in the adherent-invasive Escherichia coli LF82 upon macrophage infection.

Adherent-invasive Escherichia coli (AIEC) strains are frequently recovered from stools of patients with dysbiotic microbiota. They have remarkable properties of adherence to the intestinal epithelium, and survive better than other E. coli in macrophages. The best studied of these AIEC is probably strain LF82, which was isolated from a Crohn's disease patient. This strain contains five complete prophages, which have not been studied until now. We undertook their analysis, both in vitro and inside macrophages, and show that all of them form virions. The Gally prophage is by far the most active, generating spontaneously over 108 viral particles per mL of culture supernatants in vitro, more than 100-fold higher than the other phages. Gally is also over-induced after a genotoxic stress generated by ciprofloxacin and trimethoprim. However, upon macrophage infection, a genotoxic environment, this over-induction is not observed. Analysis of the transcriptome and key steps of its lytic cycle in macrophages suggests that the excision of the Gally prophage continues to be repressed in macrophages. We conclude that strain LF82 has evolved an efficient way to block the lytic cycle of its most active prophage upon macrophage infection, which may participate to its good survival in macrophages.

A bacterial virulence factor interacts with the splicing factor RBM5 and stimulates formation of nuclear RBM5 granules.

L. monocytogenes causes listeriosis, a foodborne disease that is particularly dangerous for immunocompromised individuals and fetuses. Several virulence factors of this bacterial pathogen belong to a family of leucine-rich repeat (LRR)-containing proteins called internalins. Among these, InlP is known for its role in placental infection. We report here a function of InlP in mammalian cell nucleus organization. We demonstrate that bacteria do not produce InlP under in vitro culture conditions. When ectopically expressed in human cells, InlP translocates into the nucleus and changes the morphology of nuclear speckles, which are membrane-less organelles storing splicing factors. Using yeast two-hybrid screen, immunoprecipitation and pull-down experiments, we identify the tumor suppressor and splicing factor RBM5 as a major nuclear target of InlP. InlP inhibits RBM5-induced cell death and stimulate the formation of RBM5-induced nuclear granules, where the SC35 speckle protein redistributes. Taken together, these results suggest that InlP acts as a nucleomodulin controlling compartmentalization and function of RBM5 in the nucleus and that L. monocytogenes has developed a mechanism to target the host cell splicing machinery.

The unforeseen intracellular lifestyle of Enterococcus faecalis in hepatocytes.

Enterococcus faecalis is a bacterial species present at a subdominant level in the human gut microbiota. This commensal turns into an opportunistic pathogen under specific conditions involving dysbiosis and host immune deficiency. E. faecalis is one of the rare pathobionts identified to date as contributing to liver damage in alcoholic liver disease. We have previously observed that E. faecalis is internalized in hepatocytes. Here, the survival and fate of E. faecalis was examined in hepatocytes, the main epithelial cell type in the liver. Although referred to as an extracellular pathogen, we demonstrate that E. faecalis is able to survive and divide in hepatocytes, and form intracellular clusters in two distinct hepatocyte cell lines, in primary mouse hepatocytes, as well as in vivo. This novel process extends to kidney cells. Unraveling the intracellular lifestyle of E. faecalis, our findings contribute to the understanding of pathobiont-driven diseases.

BAHD1 haploinsufficiency results in anxiety-like phenotypes in male mice.

BAHD1 is a heterochomatinization factor recently described as a component of a multiprotein complex associated with histone deacetylases HDAC1/2. The physiological and patho-physiological functions of BAHD1 are not yet well characterized. Here, we examined the consequences of BAHD1 deficiency in the brains of male mice. While Bahd1 knockout mice had no detectable defects in brain anatomy, RNA sequencing profiling revealed about 2500 deregulated genes in Bahd1-/- brains compared to Bahd1+/+ brains. A majority of these genes were involved in nervous system development and function, behavior, metabolism and immunity. Exploration of the Allen Brain Atlas and Dropviz databases, assessing gene expression in the brain, revealed that expression of the Bahd1 gene was limited to a few territories and cell subtypes, particularly in the hippocampal formation, the isocortex and the olfactory regions. The effect of partial BAHD1 deficiency on behavior was then evaluated on Bahd1 heterozygous male mice, which have no lethal or metabolic phenotypes. Bahd1+/- mice showed anxiety-like behavior and reduced prepulse inhibition (PPI) of the startle response. Altogether, these results suggest that BAHD1 plays a role in chromatin-dependent gene regulation in a subset of brain cells and support recent evidence linking genetic alteration of BAHD1 to psychiatric disorders in a human patient.

Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.

GBF1 and Arf1 interact with Miro and regulate mitochondrial positioning within cells.

The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a two-fold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein- and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.

Overexpression of the Heterochromatinization Factor BAHD1 in HEK293 Cells Differentially Reshapes the DNA Methylome on Autosomes and X Chromosome.

BAH domain-containing protein 1 (BAHD1) is involved in heterochromatin formation and gene repression in human cells. BAHD1 also localizes to the inactive X chromosome (Xi), but the functional significance of this targeting is unknown. So far, research on this protein has been hampered by its low endogenous abundance and its role in epigenetic regulation remains poorly explored. In this work, we used whole-genome bisulfite sequencing (BS-seq) to compare the DNA methylation profile of HEK293 cells expressing low levels of BAHD1 (HEK-CT) to that of isogenic cells stably overexpressing BAHD1 (HEK-BAHD1). We show that increasing BAHD1 levels induces de novo DNA methylation on autosomes and a marked hypomethylation on the X chromosome (chrX). We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed "BAHD1-DMRs"), of which 83,850 mapped on autosomes and 7508 on the X chromosome (chrX). Autosomal BAHD1-DMRs were predominantly hypermethylated and located to satellites, interspersed repeats, and intergenic regions. In contrast, BAHD1-DMRs on chrX were mainly hypomethylated and located to gene bodies and enhancers. We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains. Half of these "BAHD1-Associated differentially methylated Domains" (BADs) overlapped with lamina-associated domains (LADs). Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.

JMY is involved in anterograde vesicle trafficking from the trans-Golgi network.

Junction-mediating and regulatory protein (JMY) was originally identified as a transcriptional co-factor in the p53-response to DNA damage. Aside from this nuclear function, recent years have uncovered an additional function of JMY, namely in cytoskeleton remodelling and actin assembly. The C-terminus of JMY comprises a canonical VCA-module, the sequence signature of Arp2/3 complex activators. Furthermore, tandem repeats of 3 WH2 (V, or more recently also W) domains render JMY capable of Arp2/3 independent actin assembly. The motility promoting cytoplasmic function of JMY is abrogated upon DNA-damage and nuclear translocation of JMY. To address the precise cellular function of JMY in cellular actin rearrangements, we have searched for potential new interaction partners by mass spectrometry. We identified several candidates and correlated their localization with the subcellular dynamics of JMY. JMY is localized to dynamic vesiculo-tubular structures throughout the cytoplasm, which are decorated with actin and Arp2/3 complex. Moreover, JMY partially colocalizes and interacts with VAP-A, which is involved in vesicle-based transport processes. Finally, overexpression of JMY results in Golgi dispersal by loss from the trans-site and affects VSV-G transport. These analyses, together with biochemical experiments, indicate that JMY drives vesicular trafficking in the trans-Golgi region and at ER-membrane contact sites (MCS), distinct from other Arp2/3 activators involved in vesicle transport processes such as the related WHAMM or WASH.

A role for Ral GTPase-activating protein subunit ß in mitotic regulation.

Ral proteins are small GTPases that play critical roles in normal physiology and in oncogenesis. There is little information on the GTPase-activating proteins (GAPs) that downregulate their activity. Here, we provide evidence that the noncatalytic ß subunit of RalGAPα1/2 ß complexes is involved in mitotic control. RalGAPß localizes to the Golgi and nucleus during interphase, and relocalizes to the mitotic spindle and cytokinetic intercellular bridge during mitosis. Depletion of RalGAPß causes chromosome misalignment and decreases the amount of mitotic cyclin B1, disturbing the metaphase-to-anaphase transition. Overexpression of RalGAPß interferes with cell division, leading to binucleation and multinucleation, and cell death. We propose that RalGAPß plays an essential role in the sequential progression of mitosis by controlling the spatial and temporal activation of Ral GTPases in the spindle assembly checkpoint (SAC) and cytokinesis. Deregulation of RalGAPß might cause genomic instability, leading to human carcinogenesis.

Inhibitory signalling to the Arp2/3 complex steers cell migration.

Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.

Endosomal recruitment of the WASH complex: active sequences and mutations impairing interaction with the retromer.

BACKGROUND INFORMATION: The Wiskott-Aldrich syndrome protein and scar homolog (WASH) complex is the major Arp2/3 activator at the surface of endosomes. The branched actin network, that the WASH complex induces, contributes to cargo sorting and scission of transport intermediates destined for most endosomal routes. A major challenge is to understand how the WASH molecular machine is recruited to the surface of endosomes. The retromer endosomal machinery has been proposed by us and others to play a role in this process. RESULTS: In this work, we used an unbiased approach to identify the endosomal receptor of the WASH complex. We have delineated a short fragment of the FAM21 subunit that is able to displace the endogenous WASH complex from endosomes. Using a proteomic approach, we have identified the retromer cargo selective complex (CSC) as a partner of the active FAM21 sequence displacing the endogenous WASH complex. A point mutation in FAM21 that abolishes CSC interaction also impairs WASH complex displacement activity. The CSC is composed of three subunits, VPS35, VPS29 and VPS26. FAM21 directly binds the VPS35 subunit of the retromer CSC. Additionally, we show that a point mutant of VPS35 that blocks binding to VPS29 also prevents association with FAM21 and the WASH complex revealing a novel role for the VPS35-VPS29 interaction in regulating retromer association with the WASH complex. CONCLUSIONS: This novel approach of endogenous WASH displacement confirms previous suggestions that the retromer is the receptor of the WASH complex at the surface of endosomes and identify key residues that mediate this interaction. The interaction between these two endosomal machineries, the WASH complex and the retromer, is likely to play a critical role in forming platforms at the surface of endosomes for efficient sorting of cargoes.

A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response.

Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon (IFN)-stimulated genes (ISGs). IFN-λ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1(+/-) mice or when lntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-λ-mediated immune response to control bacterial colonization of the host.