Stéphanie Balor

Proteolipidic composition of exosomes changes during reticulocyte maturation.

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Macrophage podosomes go 3D.

Macrophage tissue infiltration is a critical step in the immune response against microorganisms and is also associated with disease progression in chronic inflammation and cancer. Macrophages are constitutively equipped with specialized structures called podosomes dedicated to extracellular matrix (ECM) degradation. We recently reported that these structures play a critical role in trans-matrix mesenchymal migration mode, a protease-dependent mechanism. Podosome molecular components and their ECM-degrading activity have been extensively studied in two dimensions (2D), but yet very little is known about their fate in three-dimensional (3D) environments. Therefore, localization of podosome markers and proteolytic activity were carefully examined in human macrophages performing mesenchymal migration. Using our gelled collagen I 3D matrix model to obligate human macrophages to perform mesenchymal migration, classical podosome markers including talin, paxillin, vinculin, gelsolin, cortactin were found to accumulate at the tip of F-actin-rich cell protrusions together with β1 integrin and CD44 but not β2 integrin. Macrophage proteolytic activity was observed at podosome-like protrusion sites using confocal fluorescence microscopy and electron microscopy. The formation of migration tunnels by macrophages inside the matrix was accomplished by degradation, engulfment and mechanic compaction of the matrix. In addition, videomicroscopy revealed that 3D F-actin-rich protrusions of migrating macrophages were as dynamic as their 2D counterparts. Overall, the specifications of 3D podosomes resembled those of 2D podosome rosettes rather than those of individual podosomes. This observation was further supported by the aspect of 3D podosomes in fibroblasts expressing Hck, a master regulator of podosome rosettes in macrophages. In conclusion, human macrophage podosomes go 3D and take the shape of spherical podosome rosettes when the cells perform mesenchymal migration. This work sets the scene for future studies of molecular and cellular processes regulating macrophage trans-migration.

Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes

Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.

The Process of Macrophage Migration Promotes Matrix Metalloproteinase-Independent Invasion by Tumor Cells

Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck−/− macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.

Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines

Exosomes are important mediators in cell‐to‐cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma‐specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro‐migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells’ aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment.

Zeta potential of anoxygenic phototrophic bacteria and Ca adsorption at the cell surface: Possible implications for cell protection from CaCO3 precipitation in alkaline solutions

Electrophoretic mobility measurements and surface adsorption of Ca on living, inactivated, and heat-killed haloalkaliphilic Rhodovulum steppense, A-20s, and halophilic Rhodovulum sp., S-17-65 anoxygenic phototrophic bacteria (APB) cell surfaces were performed to determine the degree to which these bacteria metabolically control their surface potential equilibria. Zeta potential of both species was measured as a function of pH and ionic strength, calcium and bicarbonate concentrations. For both live APB in 0.1M NaCl, the zeta potential is close to zero at pH from 2.5 to 3 and decreases to -30 to -40 mV at pH of 5-8. In alkaline solutions, there is an unusual increase of zeta potential with a maximum value of -10 to -20 mV at a pH of 9-10.5. This increase of zeta potential in alkaline solutions is reduced by the presence of NaHCO(3) (up to 10 mM) and only slightly affected by the addition of equivalent amount of Ca. At the same time, for inactivated (exposure to NaN(3), a metabolic inhibitor) and heat-killed bacteria cells, the zeta potential was found to be stable (-30 to -60 mV, depending upon the ionic strength) between pH 5 and 11 without any increase in alkaline solutions. Adsorption of Ca ions on A-20s cells surface was more significant than that on S-17-65 cells and started at more acidic pHs, consistent with zeta potential measurements in the presence of 0.001-0.01 mol/L CaCl(2). Overall, these results indicate that APB can metabolically control their surface potential to electrostatically attract nutrients at alkaline pH, while rejecting/avoiding Ca ions to prevent CaCO(3) precipitation in the vicinity of cell surface and thus, cell incrustation.

Ligand Binding, but Undetected Functional Response of FcR after Their Capture by T Cells via Trogocytosis

Intercellular transfer of cell surface proteins by trogocytosis is common and could affect T cell responses. Yet, the role of trogocytosis in T cell function is still elusive, and it is unknown whether a molecule, once captured by T cells, harbors the same biological properties as in donor APC. In this study, we showed that FcγR as well as the associated FcRγ subunit could be detected at high levels on murine and human T cells after their intercellular transfer from FcγR-expressing APC. Capture of FcγR occurred during coculture of T cells with FcγR-expressing APC upon Ab- or Ag-mediated T cell stimulation. Once captured by T cells, FcγR were expressed in a conformation compatible with physiological function and conferred upon T cells the ability to bind immune complexes and to provision B cells with this source of Ag. However, we were unable to detect downstream signal or signaling-dependent function following the stimulation of FcγR captured by T cells, and biochemical studies suggested the improper integration of FcγR in the recipient T cell membrane. Thus, our study demonstrates that T cells capture FcγR that can efficiently exert ligand-binding activity, which, per se, could have functional consequences in T cell-B cell cooperation.

Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.