Isabel Porto-Carreiro

CCS Associates, Mountain View, California, United States

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Publications (9)35.09 Total impact

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    ABSTRACT: Coagulation proteins play a critical role in numerous aspects of tumor biology. Cancer cells express tissue factor (TF), the protein that initiates blood clotting, which frequently correlates with processes related to cell aggressiveness, including primary tumor growth, invasion, and metastasis. It has been demonstrated that TF gets incorporated into tumor-derived microvesicles (MVs), a process that has been correlated with cancer-associated thrombosis. Here, we describe the exchange of TF-bearing MVs between breast cancer cell lines with different aggressiveness potential. The highly invasive and metastatic MDA-MB-231 cells displayed higher surface levels of functional TF compared with the less aggressive MCF-7 cells. MVs derived from MDA-MB-231 cells were enriched in TF and accelerated plasma coagulation, but MCF-7 cell-derived MVs expressed very low levels of TF. Incubating MCF-7 cells with MDA-MB-231 MVs significantly increased the TF activity. This phenomenon was not observed upon pretreatment of MVs with anti-TF or annexin-V, which blocks phosphatidylserine sites on the surface of MVs. Our data indicated that TF-bearing MVs can be transferred between different populations of cancer cells and may therefore contribute to the propagation of a TF-related aggressive phenotype among heterogeneous subsets of cells in a tumor.
    Thrombosis Research 08/2013; · 3.13 Impact Factor
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    ABSTRACT: The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling.
    Cellular and Molecular Life Sciences CMLS 03/2013; · 5.62 Impact Factor
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    ABSTRACT: Reservosomes are endocytic organelles from Trypanosoma cruzi epimastigotes that store proteins and lipids for future use. The lack of molecular markers for the compartments of this parasite makes it difficult to clarify all reservosome functions, as they present characteristics of pre-lysosomes, lysosomes and recycling compartments.
    Parasitology Research 10/2006; 99(4):325-7. · 2.85 Impact Factor
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    ABSTRACT: Exosomes are membrane vesicles that are released by cells upon fusion of multivesicular bodies with the plasma membrane. Their molecular composition reflects their origin in endosomes as intraluminal vesicles. In addition to a common set of membrane and cytosolic molecules, exosomes harbor unique subsets of proteins linked to cell type-associated functions. Exosome secretion participates in the eradication of obsolete proteins but several findings, essentially in the immune system, indicate that exosomes constitute a potential mode of intercellular communication. Release of exosomes by tumor cells and their implication in the propagation of unconventional pathogens such as prions suggests their participation in pathological situations. These findings open up new therapeutic and diagnostic strategies.
    Journal of Biochemistry 08/2006; 140(1):13-21. · 3.07 Impact Factor
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    ABSTRACT: Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.
    The EMBO Journal 07/2006; 25(12):2674-85. · 9.82 Impact Factor
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    ABSTRACT: Exosomes are membrane vesicles that are released by cells upon fusion of multivesicular bodies with the plasma membrane. Their molecular composition reflects their origin in endosomes as intraluminal vesicles. In addition to a common set of membrane and cytosolic molecules, exosomes harbor unique subsets of proteins linked to cell type– associated functions. Exosome secretion participates in the eradication of obsolete pro-teins but several findings, essentially in the immune system, indicate that exosomes constitute a potential mode of intercellular communication. Release of exosomes by tumor cells and their implication in the propagation of unconventional pathogens such as prions suggests their participation in pathological situations. These findings open up new therapeutic and diagnostic strategies. Multivesicular bodies (MVBs), and their intraluminal vesi-cles (ILVs), are involved in the sequestration of proteins destined for degradation in lysosomes (1). An alternative fate of MVBs is their exocytic fusion with the plasma mem-brane leading to the release of the 50–90 nm ILVs into the extracellular milieu (Fig. 1). The secreted ILVs are then called exosomes (reviewed in Refs. 2 and 3). After their initial description as vesicles of endosomal origin secreted by reticulocytes during differentiation (4), vesicles with the hallmarks of exosomes appeared to be released by other cells. Exosomes are present in the culture superna-tant of several cell types of hematopoietic origin [B cells (5), dendritic cells (6), mast cells (7), T cells (8) and platelets (9)] and of non hematopoietic origin [intestinal epithelial cells (10), tumor cells (11), Schwann cells (12) and neuronal cells (13)]. In addition there is increasing evidence for the presence of exosomes in physiological fluids such as plasma (14), malignant and pleural effusions (15, 16) and urine (17). As a consequence of proteins and lipids sorting at the limiting membrane of endosomes during the formation of the ILVs in MVBs, exosomes harbour a specific set of molecules. The sorting process and the generation of ILVs require the recognition of cargo proteins by a series of multiprotein complexes that form the ESCRT machinery [for review (18)] (Fig. 2). There is increasing evidence, how-ever, that cargo sorting and MVB generation is not solely dependent on ESCRT components. The mechanisms lead-ing to the fusion of MVBs with the plasma membrane and the consequent release of exosomes are unknown,
    Journal of Biochemistry 01/2006; 140:13-21. · 3.07 Impact Factor
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    ABSTRACT: Exosomes are membrane vesicles released into the extracellular environment upon exocytic fusion of multivesicular endosomes with the cell surface. Exosome secretion can be used by cells to eject molecules targeted to intraluminal vesicles of multivesicular bodies, but particular cell types may exploit exosomes as intercellular communication devices for transfer of proteins and lipids among cells. The glycosylphosphatyidylinositol-linked prion protein (PrP) in both its normal (PrPc) and scrappie (PrPsc) conformation is associated with exosomes. Targeting of exosomes containing the normal cellular PrP could confer susceptibility of cells that do not express PrP to prion multiplication. Furthermore, exosomes bearing proteinase-K resistant PrPsc are infectious, suggesting a model in which exosomes secreted by infected cells could serve as vehicles for propagation of prions. Thus, cells may exploit the nature of endosome-derived exosomes to communicate with each other in normal and pathological situations, providing for a novel route of cell-to-cell communication and therefore of pathogen transmission. These findings open the possibility that methods to interfere with trafficking of such unconventional pathogens could be envisioned from insights on the mechanisms involved in exosome formation, secretion and targeting.
    Blood Cells Molecules and Diseases 01/2005; 35(2):143-8. · 2.26 Impact Factor
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    ABSTRACT: Reservosomes are acidic compartments present at the posterior region of epimastigote forms of Trypanosoma cruzi that store proteins and lipids. During metacyclogenesis, they consume their contents and disappear. Reservosomes are rich in cruzipain, the main proteolytic enzyme of this parasite. By centrifugation in a sucrose gradient, we have obtained a highly purified subcellular fraction containing reservosomes from 5-day-old Y strain epimastigotes. Transmission electron microscopy showed that the fraction contained well-preserved organelles. The protein profile of the organelle analyzed by SDS-PAGE depicted a wide range of protein bands, predominating those corresponding to a triplet of 60-51 kDa and a doublet of 25-23 kDa. Protease activity in substrate-containing gels, in the presence or absence of protease inhibitors, showed that cysteine proteinase is enriched and very active in the purified fraction. Enzymatic assays demonstrated the absence of pyrophosphatase, an acidocalcisome marker, and succinate cytochrome c reductase, a mitochondrial marker, although these enzymes were active in other regions of the purification sucrose gradient. Thin layer chromatographic neutral lipid analysis of purified reservosomes demonstrated that the organelle stores large amounts of ergosterol and esterified cholesterol. Phospholipid analysis indicated phosphatidylcholine and phosphatidylethanolamine as the major constituents of reservosome membranes.
    FEMS Microbiology Letters 09/2002; 214(1):7-12. · 2.05 Impact Factor
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    ABSTRACT: SummaryIt has been known for many years that trypanosomatids require exogenous essential growth factors in order to divide. Two surface domains are involved in starting nutrient endocytosis: the flagellar pocket and the cytostome. Although the flagellar pocket plays a fundamental role in the endocytic process occurring in several trypanosomatids, we have shown the cytostome as the main structure involved in this process in epimastigote forms of T. cruzi. After one minute of endocytosis, cargo is still found at the cytostome entry as well as along the cytopharynx. After two, five and fifteen minutes of endocytosis, cargo was seen inside vesicles and tubules, prior to fusing with reservosomes. Three-dimensional reconstruction of these tubules and vesicles showed they are interconnected, forming an intricate and branched network, distributed from the perinuclear region to the posterior end of the cell. Whole unfixed parasites that had taken up gold-protein conjugates for fifteen minutes were washed and dried on electron microscope grids. Observation with an energy-filtering transmission electron microscope revealed long gold-filled tubules at the posterior end of the cell. Parasites treated with ammonium chloride had their intracellular traffic slowed down, which allowed us to observe many events of vesicle fusion. The acidic nature of this network was evidenced using acridine orange. Based on pH and protein uptake kinetics we propose that the vesicular-tubular network is the early endosome of Trypanosoma cruzi epimastigotes.
    European Journal of Cell Biology 12/2000; · 3.21 Impact Factor

Publication Stats

460 Citations
35.09 Total Impact Points

Institutions

  • 2006
    • CCS Associates
      Mountain View, California, United States
  • 2005
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2000
    • Federal University of Rio de Janeiro
      • Instituto de Biofísica Carlos Chagas Filho (IBCCF)
      Rio de Janeiro, Rio de Janeiro, Brazil