The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses.

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (⌬⌿m) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms. INTRODUCTIONProgrammed cell death (PCD) is a genetically regulated physiological process of cell suicide that is central to the development and homeostasis of multicellular organisms (Raff, 1992;Steller, 1995;Jacobson et al., 1997;Vaux and Korsmeyer, 1999). The basic machinery that controls the onset of PCD in roundworms (Caenorhabditis elegans), insects (Drosophila melanogaster), and vertebrates (mammals) appears to be present in all cells, at all times. Crucial aspects of PCD appear to be conserved, including both the genes encoding the basic cell death machinery, and the morphological and biochemical features of apoptosis, the most frequent phenotype of PCD (Jacobson et al., 1997;Horvitz, 1999;Song and Steller, 1999;Vaux and Korsmeyer, 1999).Mitochondria play a pivotal role in PCD in mammalian cells, in particular through the permeabilization/disruption of their outer membrane, with (or followed by) the loss of mitochondrial transmembrane potential (⌬⌿m) (Kroemer et al., 1995;Green and Reed, 1998;Petit et al., 1998;Goldstein et al., 2000;Martinou et al., 2000), leading to the release of cytochrome c (Liu et al., 1996) and apoptosis-inducing factor # Corresponding author. E-mail address: pxpetit@zeus.cochin.inserm.fr. Abbreviations used: AIF, apoptosis-inducing factor; BSA, bovine serum albumin; DIF-1, differentiation-inducing factor-1; PCR, polymerase chain reaction; ⌬⌿m, mitochondrial transmembrane potential; PCD, programmed cell death; PPIX, protoporphyrin IX; PBS, phosphate-buffered saline. ©...

Characterization of nanoscale extracellular vesicles by Raman tweezers microspectroscopy is described in detail. Intra-sample biomolecular heterogeneity is revealed for individual exosomes from human urine and rat hepatocytes.

The influence of excitation wavelength, pH, oxygen and solvents, upon fluorescence quantum yields, were measured for i n L k Indole in neutral aqueous solution exhibits the same wavelength dependence as tryptophan, which indicates that COO-absorption is not responsible for the effect. Parameters such as pH and oxygen influence only the absolute fluorescence quantum yields but not their relative variation with wavelength, indicating competition with fluorescence emission for S, deactivation. without any influence upon deactivation of higher excited states. In contrast, solvents exhibit ii specific influence upon the wavelength dependence; for indole. the decrease of fluorescence yield excited around 215 nm, compared with the yield in the first absorption band is about 40% in water. 10% in acetonitrile, 70% in n-hexane and cyclohexane, whereas no appreciable decrease occurs in methanol, ethanol or n-butanol. These observations, together with the Stokes shifts of the emission spectra, may be well correlated with Kosower's Z-values, expressing microscopic solvent-solute interactions.

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Nanomagnetism associated with cell biology enables the design of therapeutic vectors. Internalization and release of superparamagnetic nanoparticles (see image) produces submicrometric magnetic biovesicles endowed with tracer qualities for magnetic resonance imaging, targeting by magnetic guidance, and therapeutics.

A major pterin was isolated by reverse-phase high-performance liquid chromatography from cellular extract of vegetative cells of Dictyostelium discoideum after perchloric deproteinization and oxidation with acidic iodine. This compound was characterized by its chromatographic behavior, its absorption and fluorescence properties, by its oxidation product with alkaline permanganate, by secondary ion mass spectrometry and by circular dichroism. The final identification was obtained by comparison with authentic materials. It is concluded that the major pterin of D. discoideum is the compound 6-(~-threo-l,2-dihydroxypropyl)-pterin. The name dictyopterin is proposed for this new natural isomer of L-biopterin.A pteridine pathway has been demonstrated during growth and development of Dictyostelium discoideum cells [I -31. The end-products of this pathway, lumazine (2,4-dihydroxypteridine) [2-41, isoxantholumazine (2,4,7-trihydroxypteridine) [2], and a lumazine-like compound [4] were observed in the extracellular medium [2 -41. The release of extracellular pteridines was correlated with the ability of the cells to aggregate [3 -51. Whether these compounds, their precursors or related compounds (Fig