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International audience; The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A-CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A-CHMP3 membrane tubes. We therefore conclude that CHMP2A-CHMP3-VPS4 act as a minimal membrane fission machinery.

Spermiogenesis is a radical process of differentiation whereby sperm cells acquire a compact and specialized morphology to cope with the constraints of sexual reproduction while preserving their main cargo, an intact copy of the paternal genome. In animals, this often involves the replacement of most histones by sperm-specific nuclear basic proteins (SNBPs). Yet, how the SNBP-structured genome achieves compaction and accommodates shaping remain largely unknown. Here, we exploited confocal, electron and super-resolution microscopy observations, coupled with polymer modeling simulations to identify the higher-order architecture of sperm chromatin in the needle-shaped nucleus of the emerging model cricket Gryllus bimaculatus. Accompanying spermatid differentiation and shaping, the SNBP-based genome was strikingly reorganized as ~25nm-thick fibers orderly coiled along the elongated nucleus axis. This chromatin spool was further found to achieve large-scale helical twisting in the final stages of spermiogenesis, favoring its ultracompaction. Through a combination of microscopy observations and polymer simulations, we revealed that these dramatic transitions may be recapitulated by a surprisingly simple biophysical principle based on a nucleated rigidification of chromatin linked to the histone-to-SNBP transition within a confined nuclear space. Our work highlights a unique, liquid crystal-like mode of higher-order genome organization in ultracompact cricket sperm completely distinct from nucleosomal chromatin, and establishes a multidisciplinary methodological framework to explore the diversity of non-canonical modes of DNA organization.SIGNIFICANCE STATEMENTAnimal sperm cells are highly compact and atypically shaped compared to other cell types. How DNA is packaged and organized in the 3D space of sperm cell nuclei to cope with these constraints is poorly understood. In this work, we identified an original and elegant solution to this problem in crickets, whereby DNA fibers orderly spool and twist to fit into ultracompact, needle-shaped sperm cells. To understand this reorganization, we modeled DNA fibers in the nucleus as polymers and found that a relatively simple mechanism through which fibers become more rigid bit by bit can largely recapitulate our observations. Our multidisciplinary work highlights a simple solution to compact DNA to extreme levels in specialized nuclei.

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo electron microscopy structures of membrane-coated CHMP2A-CHMP3 filaments of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence towards the tube interior. Inter-filament interactions are electrostatic, which facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high speed atomic force microscopy imaging corroborate that CHMP2A-CHMP3 polymers and VPS4 can constrict and cleave narrow membrane tubes, thus acting as a minimal membrane fission machinery.

International audience; Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

Significance Bacteria possess a sophisticated arsenal of defense mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, Escherichia coli and many other enterobacteria activate a proton-consuming resistance system based on the acid stress-inducible lysine decarboxylase LdcI. Here we develop generally applicable tools to uncover the spatial localization of LdcI inside the cell by superresolution fluorescence microscopy and investigate the in vitro supramolecular organization of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations., Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with K Ds in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction.

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statementBacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.

We designed a particularly simple, compact and robust microscope for phase and fluorescent imaging. The phase-contrast image is reconstructed from a single, approximately 100 µm defocused image with an algorithm based on a constrained optimization of Fresnel diffraction model. Fluorescence image is recorded in-focus. No mechanical movement of neither sample nor objective or any other part of the system is needed to change between the phase-contrast and fluorescence modality. The change of focus between phase (out-of-focus) and fluorescence (in-focus) imaging is achieved with chromatic aberration specifically enhanced by the optical design of our system. Our microscope is sufficiently compact (10x10x10 cm^3) to fit into a standard biological incubator. The simple and robust design reduces the vibration and the drift of the sample. The absence of motorized components makes the system robust and resistant to the humid conditions inside the biological incubator. These aspects greatly facilitate the long-time observation of cell cultures. We can observe a thousand of cells in parallel in a single field of view (1mm^2) with resolution down to 2 µm. We show FUCCI marked HeLa cell culture observed over three days directly in the incubator. FUCCI (fluorescence ubiquitination cell-cycle indicator), is a genetically encoded, two-colour (red and green), indicator of the progression through the cell cycle: the cells in G1 phase show red fluorescence nuclei while the cells in S, G2 and M phase display green fluorescence within the nuclei. We use phase images for segmentation and tracking of the individual cells which allows us to determine the level of fluorescence in each cell in the green and red fluorescence channel. We compare the obtained statistics with the data from flow cytometer acquired at the end of the observation. We show that we can produce a statistically relevant time-resolved measurement of a cell population while keeping access to the individual cells.

The Y-box binding protein 1 (YB1) is an established metastatic marker: high expression and nuclear localization of YB1 correlate with tumor aggressiveness, drug resistance, and poor patient survival in various tumors. In the nucleus, YB1 interacts with and regulates the activities of several nuclear proteins, including the DNA glycosylase, human endonuclease III (hNTH1). In the present study, we used Förster resonance energy transfer (FRET) and AlphaLISA technologies to further characterize this interaction and define the minimal regions of hNTH1 and YB1 required for complex formation. This work led us to design an original and cost-effective FRET-based biosensor for the rapid

We propose a simple and compact microscope combining phase imaging with multi-color fluorescence using a standard bright-field objective. The phase image of the sample is reconstructed from a single, approximately 100 μm out-of-focus image taken under semi-coherent illumination, while fluorescence is recorded in-focus in epi-fluorescence geometry. The reproducible changes of the focus are achieved with specifically introduced chromatic aberration in the imaging system. This allows us to move the focal plane simply by changing the imaging wavelength. No mechanical movement of neither sample nor objective or any other part of the setup is therefore required to alternate between the imaging modality. Due to its small size and the absence of motorized components the microscope can easily be used inside a standard biological incubator and allows long-term imaging of cell culture in physiological conditions. A field-of-view of 1.2 mm2 allows simultaneous observation of thousands of cells with micro-meter spatial resolution in phase and multi-channel fluorescence mode. In this manuscript we characterize the system and show a time-lapse of cell culture in phase and multi-channel fluorescence recorded inside an incubator. We believe that the small dimensions, easy usage and low cost of the system make it a useful tool for biological research.

Measles virus proteins form liquid droplets where they can encapsidate their genomic material., Many viruses are known to form cellular compartments, also called viral factories. Paramyxoviruses, including measles virus, colocalize their proteomic and genomic material in puncta in infected cells. We demonstrate that purified nucleoproteins (N) and phosphoproteins (P) of measles virus form liquid-like membraneless organelles upon mixing in vitro. We identify weak interactions involving intrinsically disordered domains of N and P that are implicated in this process, one of which is essential for phase separation. Fluorescence allows us to follow the modulation of the dynamics of N and P upon droplet formation, while NMR is used to investigate the thermodynamics of this process. RNA colocalizes to droplets, where it triggers assembly of N protomers into nucleocapsid-like particles that encapsidate the RNA. The rate of encapsidation within droplets is enhanced compared to the dilute phase, revealing one of the roles of liquid-liquid phase separation in measles virus replication.

Our knowledge of bacterial nucleoids originates mostly from studies of rod- or crescent-shaped bacteria. Here we reveal that Deinococcus radiodurans, a relatively large spherical bacterium with a multipartite genome, constitutes a valuable system for the study of the nucleoid in cocci. Using advanced microscopy, we show that D. radiodurans undergoes coordinated morphological changes at both the cellular and nucleoid level as it progresses through its cell cycle. The nucleoid is highly condensed, but also surprisingly dynamic, adopting multiple configurations and presenting an unusual arrangement in which oriC loci are radially distributed around clustered ter sites maintained at the cell centre. Single-particle tracking and fluorescence recovery after photobleaching studies of the histone-like HU protein suggest that its loose binding to DNA may contribute to this remarkable plasticity. These findings demonstrate that nucleoid organization is complex and tightly coupled to cell cycle progression in this organism., Deinococcus radiodurans is a relatively large spherical bacterium with a multipartite genome. Here, the authors study the coordinated morphological changes at the cellular and nucleoid level as the bacteria progress through the cell cycle, showing complex nucleoid organization and dynamics.

Our knowledge of bacterial nucleoids originates mostly from studies of rod- or crescent-shaped bacteria. Here, we reveal that Deinococcus radiodurans, a relatively large, spherical bacterium, possessing a multipartite genome, and well-known for its radioresistance, constitutes a valuable system for the study of nucleoids in cocci. Using advanced microscopy, we show that as D. radiodurans progresses through its cell cycle, it undergoes coordinated morphological changes at both the cellular and nucleoid level. D. radiodurans nucleoids were found to be highly condensed, but also surprisingly dynamic, adopting multiple distinct configurations and presenting a novel chromosomal arrangement in which oriC loci are radially distributed around clustered ter sites maintained at the centre of cells. Single-molecule and ensemble studies of the abundant histone-like HU protein suggest that its loose binding to DNA may contribute to this remarkable plasticity. These findings clearly demonstrate that nucleoid organization is complex and tightly coupled to cell cycle progression.

We propose a simple and compact microscope combining phase imaging with fluorescence. This compact setup can be easily inserted in a standard biological incubator and allows observation of cellular cultures over several days. Phase image of the sample is reconstructed from a single, slightly (~50 μm) defocused image taken under semi-coherent illumination. Fluorescence in-focus image is recorded in epi-fluorescence geometry. The phase and fluorescence images are taken sequentially using a single CMOS camera. No mechanical movement of neither sample nor objective is required to change the imaging modality. The only change is the wavelength of illumination and excitation light for phase and fluorescence imaging, respectively. The slight defocus needed for phase imaging is achieved due to specifically introduced chromatic aberration in the imaging system. We present dual modality time-lapse movies of cellular cultures observed over several days in physiological conditions inside an incubator. A field-of-view of 3 mm2 allows observation up to thousands of cells with micro-meter spatial resolution in quasi-simultaneous phase and fluorescence mode. We believe that the simplicity, small dimensions, ease-of-use and low cost of the system make it a useful tool for biological research

International audience; Calreticulin (CRT) is a well-known "eat-me" signal harbored by dying cells participating in their recognition by phagocytes. CRT is also recognized to deeply impact the immune response to altered self-cells. In this study, we focus on the role of the newly exposed CRT following cell death induction. We show that if CRT increases at the outer face of the plasma membrane and is well recognized by C1q even when phosphatidylserine is not yet detected, CRT is also released in the surrounding milieu and is able to interact with phagocytes. We observed that exogenous CRT is endocytosed by THP1 macrophages through macropinocytosis and that internalization is associated with a particular phenotype characterized by an increase of cell spreading and migration, an upregulation of CD14, an increase of interleukin-8 release, and a decrease of early apoptotic cell uptake. Importantly, CRT-induced pro-inflammatory phenotype was confirmed on human monocytes-derived macrophages by the overexpression of CD40 and CD274, and we found that monocyte-derived macrophages exposed to CRT display a peculiar polarization notably associated with a downregulation of the histocompatibility complex of class II molecules hampering its description through the classical M1/M2 dichotomy. Altogether our results highlight the role of soluble CRT with strong possible consequences on the macrophage-mediated immune response to dying cell.

Unusual intramolecular cross-links present in adhesins from Gram-positive bacteria have been used to develop a generic process amenable to biotechnology applications. Based on the crystal structure of RrgA, the Streptococcus pneumoniae pilus adhesin, we provide evidence that two engineered protein fragments retain their ability to associate covalently with high specificity, in vivo and in vitro, once isolated from the parent protein. We determined the optimal conditions for the assembly of the complex and we solved its crystal structure at 2 Å. Furthermore, we demonstrate biotechnological applications related to antibody production, nanoassembly and cell-surface labeling based on this process we named Bio Molecular Welding.

Chemokines stimulate signals in cells by binding to G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. These chemoattractant cytokines also interact with heparan sulfate (HS), which provides positional information within tissues in the form of haptotactic gradients along which cells can migrate directionally. To investigate the mechanism by which HS modulates chemokine functions, we used the CXC chemokine CXCL12, which exists in different isoforms that all signal through CXCR4 but have distinct HS-binding domains. In experiments with both cell-associated and solubilized CXCR4, we found that although CXCL12γ bound to CXCR4 with a higher affinity than did CXCL12α, CXCL12γ displayed reduced signaling and chemotactic activities. These properties were caused by the specific carboxyl-terminal region of CXCL12γ, which, by interacting with CXCR4 sulfotyrosines, mediated high-affinity, but nonproductive, binding to CXCR4. HS prevented CXCL12γ from interacting with the CXCR4 sulfotyrosines, thereby functionally presenting the chemokine to its receptor such that its activity was similar to that of CXCL12α. HS had no effects on the binding of CXCL12α to CXCR4 or its biological activity, suggesting that this polysaccharide controls CXCL12 in an isoform-specific manner. These data suggest that the HS-dependent regulation of chemokine functions extends beyond the simple process of immobilization and directly modulates receptor ligation and activation.

International audience; Acoustofluidics is acknowledged as a powerful tool offering a contactless and label-free manipulation of fluids, micro-beads, and living cells. To date, most techniques rely on the use of propagating acoustic waves and take advantage of the associated acoustic radiation force in standing or progressive fields. Here, we present a new approach based on the generation of an evanescent acoustic field above a substrate. This field is obtained by means of subsonic interfacial waves giving rise to a well-defined standing wave pattern. By both imaging and probing the evanescent acoustic field, we show that these interfacial waves are guided waves known as quasi-Scholte acoustic waves. Scholte waves present very interesting features for applications in acoustofluidics. Namely, they confine the acoustic energy to the vicinity of the surface, they are nearly lossless and thus can propagate over long distances along the substrate, and finally they do not require any particular material for the substrate. With a very simple and low-cost device we show several examples of applications including patterning lines or arrays of cells, triggering spinning of living cells, and separating plasma from RBC in a whole blood microdroplet.

Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. IMPORTANCE A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response.

International audience; Rationale: An alternative approach for the improvement of radiotherapy consists in increasing differentially the radiation dose between tumors and healthy tissues using nanoparticles (NPs) that have been beforehand internalized into the tumor. These high-Z NPs can be photo-activated by monochromatic synchrotron X-rays, leading to a local dose enhancement delivered to the neighboring tumor cells[1]. This enhancement is due to secondary and Auger electrons expelled from the NPs by the radiations. In order to carry the NPs into the tumor center, macrophages are currently under study for their phagocytosis and diapedesis abilities[2] (cf. Figure adapted from [3] and [4]). In this study we characterized J774A.1 macrophages’ internalization kinetics and subcellular distribution of iron NPs and compared them to the internalization abilities of the F98 glioblastoma cell line.Materials and Methods: Three aspects of internalization were examined: first, the location of internalized NPs in J774A.1 macrophages and F98 glioblastoma cells following a 24h incubation with iron NPs (0.3 mg/mL in the cell culture medium) was determined by optical microscopy after cellslicing. Subsequently, the iron intake after a 24h incubation with NPs (0.3 mg/mL and 0.06 mg/mL in the cell culture medium) was characterized for the two types of cells using ICP-MS. Finally, the internalization dynamics were studied by live phase-contrast microscopy imagining for 11 hours and by absorbance measurements for 24 hours using a plate reader.Results: F98 tumor cells and J774A.1 macrophages are both able to endocytose NPs: we measured ~61±10 pg of internalized iron per macrophage compared with ~33±5 pg per F98 cell (initial iron concentration: 0.3 mg/mL in culture medium). F98 internalizing NPs for 10 hours showed stress signs during the first minutes after the NPs injection, but behaved like F98 control cells during the rest of the experiment. Finally, we determined that the internalization kinetics for J774A.1 had a typical saturation time of one hour.Conclusion: Macrophages seem to be promising vectors for NPs, being able to endocytose and retain in their cytoplasm larger quantities of NPs than tumor cells. Our following studies will attempt to shed light on their other potential abilities as “Trojan Horses”.

The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity.

Highlights • We determine the mode of interaction between ELMO1 and Hck with direct methods. • The SH3 domain of Hck interacts specifically with the polyproline motif of ELMO1. • ELMO1 polyproline affinity for the SH3 of Hck is modulated by Tyr-phosphorylation., Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling.

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in α-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

International audience; C1q has been shown to recognize apoptotic cells, to enhance their uptake and to modulate cytokine release by phagocytes and thus promote immune tolerance. Surface-exposed calreticulin (CRT), known as a C1q receptor, is also considered to be an early eat-me signal that enhances the phagocytosis of apoptotic cells and is capable of eliciting an immunogenic response. However, the molecular mechanisms that trigger these functions are not clear. We hypothesized that CRT and C1q might act together in these processes. We first showed, by means of fluorescence resonance energy transfer (FRET), that CRT interacts with the C1q globular region at the surface of early apoptotic cells. Next, we pointed out that knockdown of CRT on early apoptotic HeLa cells impairs the enhancement effect of C1q on their uptake by THP-1 monocyte-derived macrophages. Furthermore, a deficiency of CRT induces contrasting effects on cytokine release by THP-1 macrophages, increasing interleukin (IL)-6 and monocyte chemotactic protein 1/CCL2 and decreasing IL-8. Remarkably, these effects were greatly reduced when apoptotic cells were opsonized by C1q, which counterbalanced the effect of the CRT deficiency. These results demonstrate that CRT-C1q interaction is involved in the C1q bridging function and they highlight the particular ability of C1q to control the phagocyte inflammatory status, i.e. by integrating the molecular changes that could occur at the surface of dying cells.

Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie-shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein-protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its alpha-helical extensions.

Collagens V and XI are thought to form a core around which the major interstitial collagens, I and II, respectively, are organized during fibrillogenesis. We previously reported the presence of a heterotypic form of collagens V and XI, [alpha 1(XI)]2 alpha 2(V), in cultures of A204 rhabdomyosarcoma cells [Kleman, J.-P., Hartmann, D. J., Ramirez, F., & van der Rest, M. (1992) Eur. J. Biochem. 210, 329-335]. This collagen forms a matrix which remains highly insoluble, even when cells were cultured in the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase and thereby of "classical" collagen cross-linking. When the cells were cultured in the presence of putrescine, a competitive inhibitor of transglutaminase-catalyzed protein cross-linking, a drastic increase in collagen solubility was observed. This result indicates that a transglutaminase contributes to the covalent stabilization of the collagen matrix of these cells. A204 rhabdomyosarcoma cells express tissue transglutaminase as revealed by specific antibodies, and enzyme activity was detected in the cell layer during culture and in cell extracts. Both collagens V and XI are specific glutaminyl substrates for tissue transglutaminase in vitro, as shown by incorporation of [3H]putrescine. The highly homologous alpha 1 chains of collagens V and XI were the major targets for the cross-linking. Trypsin cleaved the [3H] label from the alpha 1 chain of collagen V, demonstrating that the cross-linking occurs in the non triple helical propeptide domains.

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15616196; International audience; The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

F-STOP is a microtubule-associated protein that stabilizes microtubules in a calmodulin (CaM)-dependent manner. All members of the stable tubule only polypeptide (STOP) family have a central domain that contains nearly identical multiple repeats, and a CaM binding motif is present in multiple copies within this domain. We present here an analysis of this CaM binding interaction and find that it is highly unusual in nature. For this work, we synthesized two model peptides of a single STOP central repeat motif and analyzed their binding to CaM by fluorescence, circular dichroism, infrared and NMR spectroscopy. Both peptides bind to CaM with an affinity of 4 microM, similar to that of the native protein. Results indicate that the peptides bind CaM in an atypical manner. Binding is highly dependent on the concentration of cations, indicating that it is to some extent electrostatic. Further, IR and CD analysis shows that, in contrast to typical CaM binding reactions, CaM does not change in helical structure on binding. NMR mapping confirms that CaM remains in extended conformation on binding a single STOP peptide. Binding of a single peptide to CaM occurs principally in the CaM C-terminal region, and the C-terminal domain of CaM effectively competes for STOP binding. Our results establish that CaM binds STOP in an unusual manner, involving mainly the C-terminus of CaM, thus leaving CaM potentially accessible for another binding partner at the N-terminus. This intriguing possibility could be of physiological importance in F-STOP mediated CaM regulation of microtubule dynamics or stability, specifically during mitosis where CaM and STOP colocalize.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

De nombreuses proteines comportent des motifs homologues, appeles modules. Les macromolecules secretees, malgre leur grande diversite, sont construites a partir d'un nombre relativement restreint de modules differents. La modularite des proteines de la matrice extracellulaire est importante, tant par les assemblages supramoleculaires que par son role physiologique. Les modules peuvent, en effet, etre impliques a la fois dans la structure de ces proteines et dans les interactions qu'elles menagent entre elles ou avec des recepteurs cellulaires qui leur sont propres, ce qui contribue a la coherence des matrices extracellulaires. Les mutations affectant la structure d'un module et/ou la fonction qui peut lui etre associee sont a l'origine de plusieurs maladies hereditaires des matrices extracellulaires

International audience; Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.

International audience; The fibrillar collagens are the most abundant proteins of extracellular matrices. Among them, collagens V and XI are quantitatively minor components which participate in the formation of the fibrillar collagen network. Since these collagens were discovered, studies have demonstrated that they may play a fundamental role in the control of fibrillogenesis, probably by forming a core within the fibrils. Another characteristic of these collagens is the partial retention of their N-propeptide extensions in tissue forms, an unusual observation in comparison to the other known fibrillar collagens. The tissue locations of collagens V and XI are different, but their structural and biological properties seem to be closely related. It has been shown that their primary structures are highly conserved at both the gene and protein levels, and that these conserved features are the bases of their similar biological properties. In particular, they are both resistant to mammalian collagenases, and surprisingly sensitive to trypsin treatment. Collagens V and XI are usually buried within the major collagen fibrils, although they have both cell adhesion and heparin binding sites which could be of crucial importance in physiological processes such as development and wound healing. It has became evident that several molecules are in fact heterotypic associations of chains from both collagens V and XI, demonstrating that these two collagens are not distinct types but a single type which can be called collagen V/XI.The fibrillar collagens are the most abundant proteins of extracellular matrices. Among them, collagens V and XI are quantitatively minor components which participate in the formation of the fibrillar collagen network. Since these collagens were discovered, studies have demonstrated that they may play a fundamental role in the control of fibrillogenesis, probably by forming a core within the fibrils. Another characteristic of these collagens is the partial retention of their N-propeptide extensions in tissue forms, an unusual observation in comparison to the other known fibrillar collagens. The tissue locations of collagens V and XI are different, but their structural and biological properties seem to be closely related. It has been shown that their primary structures are highly conserved at both the gene and protein levels, and that these conserved features are the bases of their similar biological properties. In particular, they are both resistant to mammalian collagenases, and surprisingly sensitive to trypsin treatment. Collagens V and XI are usually buried within the major collagen fibrils, although they have both cell adhesion and heparin binding sites which could be of crucial importance in physiological processes such as development and wound healing. It has became evident that several molecules are in fact heterotypic associations of chains from both collagens V and XI, demonstrating that these two collagens are not distinct types but a single type which can be called collagen V/XI.