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International audience; Myosin Vb (Myo5b) is an unconventional myosin involved in the actin-dependent transport and tethering of intracellular organelles. In the epidermis, granular keratinocytes accumulate cytoplasmic lamellar bodies (LBs), secretory vesicles released at the junction with the stratum corneum that participate actively in the maintenance of the epidermal barrier. We have previously demonstrated that LB biogenesis is controlled by the Rab11a guanosine triphosphate hydrolase, known for its ability to recruit the Myo5b motor. In order to better characterize the molecular pathway that controls LB trafficking, we analyzed the role of F-actin and Myo5b in the epidermis. We demonstrated that LB distribution in granular keratinocytes was dependent on a dynamic F-actin cytoskeleton. Myo5b was shown to be highly expressed in granular keratinocytes and associated with corneodesmosin-loaded LB. In reconstructed human epidermis, Myo5b silencing led to epidermal barrier defects associated with structural alterations of the stratum corneum and a reduced pool of LB showing signs of disordered maturation. Myo5b depletion also disturbed the expression and distribution of both LB cargoes and junctional components, such as claudin-1, which demonstrates its action on both LB trafficking and junctional complex composition. Together, our data reveal the essential role of Myo5b in maintaining the epidermal barrier integrity.

Summary Faithful copying of the genome is essential for life. In eukaryotes, a single archaeo-eukaryotic primase (AEP), DNA primase, is required for the initiation and progression of DNA replication. Here we have identified additional eukaryotic AEP-like proteins with DNA-dependent primase and/or polymerase activity. Uniquely, the genomes of trypanosomatids, a group of kinetoplastid protozoa of significant medical importance, encode two PrimPol-like (PPL) proteins. In the African trypanosome, PPL2 is a nuclear enzyme present in G2 phase cells. Following PPL2 knockdown, a cell-cycle arrest occurs after the bulk of DNA synthesis, the DNA damage response is activated, and cells fail to recover. Consistent with this phenotype, PPL2 replicates damaged DNA templates in vitro, including templates containing the UV-induced pyrimidine-pyrimidone (6-4) photoproduct. Furthermore, PPL2 accumulates at sites of nuclear DNA damage. Taken together, our results indicate an essential role for PPL2 in postreplication tolerance of endogenous DNA damage, thus allowing completion of genome duplication., Graphical Abstract, Highlights • Trypanosomatids contain two archaeo-eukaryotic primase-polymerase-like proteins • PPL2 is essential in the pathogenic bloodstream form African trypanosome • PPL2 suppresses DNA damage and allows completion of chromosomal replication • PPL2 mediates translesion DNA synthesis

International audience; Type III secretion systems (T3SS) are macromolecular complexes that translocate a wide number of effector proteins into eukaryotic host cells. Once within the cytoplasm, many T3SS effectors mimic the structure and/or function of eukaryotic proteins in order to manipulate signaling cascades, and thus play pivotal roles in colonization, invasion, survival and virulence. Structural biology techniques have played key roles in the unraveling of bacterial strategies employed for mimicry and targeting. This review provides an overall view of our current understanding of structure and function of T3SS effectors, as well as of the different classes of eukaryotic proteins that are targeted and the consequences for the infected cell.

Graphical abstract Highlights ► Histone H2A phosphorylated at Thr130 is identified as trypanosomal γH2A. ► γH2A nuclear foci mark the putative sites of replication fork stalling. ► γH2A nuclear foci form in response to meganuclease or chemical-induced DNA damage. ► Trypanosomal γH2A and RAD51 positive damage and repair foci are most often seen in S-phase and G2. ► The findings implicate γH2A in mitosis-entry checkpoint signaling., DNA damage and repair in trypanosomatids impacts virulence, drug resistance and antigenic variation but, currently, little is known about DNA damage responses or cell cycle checkpoints in these divergent protozoa. One of the earliest markers of DNA damage in eukaryotes is γH2A(X), a serine phosphorylated histone H2A (variant). Here, we report the identification and initial characterization of γH2A in Trypanosoma brucei. We identified Thr130 within the replication-dependent histone H2A as a candidate phosphorylation site and found that the abundance of this trypanosomal γH2A increased in vivo in response to DNA damage. Nuclear γH2A foci mark the sites of putative natural replication fork stalling, sites of meganuclease-induced DNA double strand breaks and sites of methyl methanesulphonate-induced DNA damage. Naturally occurring and meganuclease-induced γH2A and RAD51 double-positive repair foci are typically found in S-phase or G2 nuclei. The results link trypanosomal γH2A, with an unusual histone modification motif, to DNA damage sensing and mitotic checkpoint signaling.

International audience; Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function.

International audience; BACKGROUND: We aimed to accurately estimate the frequency of a hexanucleotide repeat expansion in C9orf72 that has been associated with a large proportion of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). METHODS: We screened 4448 patients diagnosed with ALS (El Escorial criteria) and 1425 patients with FTD (Lund-Manchester criteria) from 17 regions worldwide for the GGGGCC hexanucleotide expansion using a repeat-primed PCR assay. We assessed familial disease status on the basis of self-reported family history of similar neurodegenerative diseases at the time of sample collection. We compared haplotype data for 262 patients carrying the expansion with the known Finnish founder risk haplotype across the chromosomal locus. We calculated age-related penetrance using the Kaplan-Meier method with data for 603 individuals with the expansion. FINDINGS: In patients with sporadic ALS, we identified the repeat expansion in 236 (7*0%) of 3377 white individuals from the USA, Europe, and Australia, two (4*1%) of 49 black individuals from the USA, and six (8*3%) of 72 Hispanic individuals from the USA. The mutation was present in 217 (39*3%) of 552 white individuals with familial ALS from Europe and the USA. 59 (6*0%) of 981 white Europeans with sporadic FTD had the mutation, as did 99 (24*8%) of 400 white Europeans with familial FTD. Data for other ethnic groups were sparse, but we identified one Asian patient with familial ALS (from 20 assessed) and two with familial FTD (from three assessed) who carried the mutation. The mutation was not carried by the three Native Americans or 360 patients from Asia or the Pacific Islands with sporadic ALS who were tested, or by 41 Asian patients with sporadic FTD. All patients with the repeat expansion had (partly or fully) the founder haplotype, suggesting a one-off expansion occurring about 1500 years ago. The pathogenic expansion was non-penetrant in individuals younger than 35 years, 50% penetrant by 58 years, and almost fully penetrant by 80 years. INTERPRETATION: A common Mendelian genetic lesion in C9orf72 is implicated in many cases of sporadic and familial ALS and FTD. Testing for this pathogenic expansion should be considered in the management and genetic counselling of patients with these fatal neurodegenerative diseases. FUNDING: Full funding sources listed at end of paper (see Acknowledgments).

International audience; Secretion and translocation of bacterial pathogen effectors into host cells via dedicated secretion machineries like type III secretion systems (T3SSs) or type IV secretion systems (T4SSs) is a key feature employed by pathogens to attack host cells. Innovative fluorescence and imaging approaches have blossomed during recent years, and became instrumental in revealing the dynamics of effector secretion and function in interfering with host cellular processes, particularly signaling events, gene expression regulation, membrane trafficking, and autophagy. Furthermore, imaging-based screening approaches have demonstrated the mode of action of several bacterial effectors upon host cellular translocation. The rapid technological advancement of imaging technologies indicates that these techniques will continue to be at the center of numerous future breakthroughs delineating the dynamic processes of bacterial effector actions.

International audience; We recently characterized a novel heme biogenesis pathway required for heme c(i)' covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme c(i)' binding.

Magali Irla, Murielle Saade, Geneviève Chazal, and Catherine Nguyen contributed equally to this work; International audience; We identified the Spatial (Stromal Protein Associated with Thymii and Lymph-node) gene from an adult thymus mouse library of cDNA clones. By RT-PCR, we reported that Spatial was highly expressed in restricted areas of the central nervous system. Here, we characterize the precise cellular localization of Spatial during mouse brain development in the cerebellum, hippocampus and cortex. Five different transcript isoforms have been described for Spatial and among those, only Spatial-epsilon and -beta present a tightly controlled expression. In the cerebellum, Spatial expression is detected in the external precursor granular layer and persists as these cells migrate and differentiate to form the internal granular layer. It is also expressed in differentiating Purkinje cells with a specific somatodendritic distribution. Spatial expression in the hippocampus is spatially and temporally regulated: it is first expressed in the CA3 field, then in CA1 and later in the dentate gyrus. Interestingly, Spatial-beta expression tightly overlaps with the beginning of neuronal differentiation in both structures. Using cultured hippocampal neurons, we show that Spatial also exhibits a somatodendritic distribution and it is concentrated in some synaptic regions. Moreover, the vesicle-like cellular distribution of Spatial protein in dendrites is similar to that described for the kinesin motor protein KIF17. Immunofluorescence analyses show that Spatial colocalizes with KIF17 in dendrites of hippocampal neurons in primary culture. Additionally, coimmunoprecipitation experiments of endogenous proteins from hippocampus confirmed that Spatial and KIF17 physically interact. These findings suggest that Spatial may play a role in neuronal morphogenesis and synaptic plasticity through its interaction with the kinesin motor KIF17 in dendrites.

International audience; Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

International audience; Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell packing the DNA into the condensed spermatid nucleus. H1T2 is a histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis that specifically localizes to a chromatin domain at the apical pole under the acrosome. We explored the mechanisms determining polar localization of H1T2 in spermatids. In acrosome-deficient round spermatids of hrb -/- and gopc -/- mice, H1T2 localization is not altered, indicating that proper acrosome development is not required for specifying nuclear polarity. In contrast, in late round spermatids from trf2 -/- or hmgb2 -/- mice, a bipolar H1T2 localization was observed revealing that polarity is modified by loss of proteins specifying chromatin architecture. Our results show that intranuclear chromatin organization is critical for correct polar localization of H1T2 and that H1T2 can be a useful molecular marker revealing chromatin disorganization in spermatids.

SummaryCell cycle regulators such as E2F1 and retinoblastoma (RB) play crucial roles in the control of adipogenesis, mostly by controlling the transition between preadipocyte proliferation and adipocyte differentiation. The serine-threonine kinase cyclin-dependent kinase 4 (cdk4) works in a complex with D-type cyclins to phosphorylate RB, mediating the entry of cells into the cell cycle in response to external stimuli. Because cdk4 is an upstream regulator of the E2F-RB pathway, we tested whether cdk4 was a target for new factors that regulate adipogenesis. Here we find that cdk4 inhibition impairs adipocyte differentiation and function. Disruption of cdk4 or activating mutations in cdk4 in primary mouse embryonic fibroblasts results in reduced and increased adipogenic potential, respectively, of these cells. We show that the effects of cdk4 are not limited to the control of differentiation; cdk4 also participates in adipocyte function through activation of PPARγ.

International audience; Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides.

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.

We have expressed the neuroendocrine VGF protein in FRT rat thyroid cells to study the molecular mechanisms of its sorting to the regulated and polarized pathways of secretion. By immunoelectron microscopy, we have demonstrated that VGF localizes in dense-core granules. Rapid secretion of VGF is induced by PMA stimulation. Moreover, human chromogranin B, a protein of the regulated pathway, co-localizes in the same granules with VGF. In confluent, FRT monolayers on filters protein secretion occur from the apical cell domain. VGF deletion mutants have been generated. By confocal microscopy, we have found that in transient transfection, all mutant proteins are sorted into granules and co-localize with the full-length VGF. They all retain the apical polarity of secretion. We also found that intracellular VGF and its deletion mutants are largely in an aggregated form. We conclude that FRT thyroid cells correctly decode the sorting information of VGF. The signals present on the protein to enter the granules and to be secreted apically cannot be separated from each other and are not in just one discrete portion of the protein. We propose that selective aggregation might represent the signal for sorting VGF to the regulated, apical route.

International audience; Interferon-gamma (IFN-gamma) elicits a variety of activities following binding to its cell-surface-specific receptor (IFN-gammaR). This complex formation leads to the activation of the Jak-STAT pathway. Several hypotheses have been proposed to explain the role and location of the receptor and its ligand in the signalling pathway. In vivo as well as in vitro, the present study shows that IFN-gamma and its receptor were internalized in different cellular compartments including cytoplasmic matrix, mitochondria and nucleus. In order to analyse the internalization pathway of IFN-gamma and its receptor, we have study in vivo and in vitro their colocalization with clathrin and caveolin by using double immunogold-labelling experiments using electron microscopy. We demonstrate that IFN-gamma and IFN-gammaR were colocalized in the caveolin-containing structures and the clathrin-coated pits suggesting that both internalization pathways may be used. This indicates that IFN-gamma and IFN-gammaR were internalized by these two different pathways, suggesting two different intracellular routes probably for different target cell-compartments.