scholarly journals Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mathilde Mathieu ◽  
Nathalie Névo ◽  
Mabel Jouve ◽  
José Ignacio Valenzuela ◽  
Mathieu Maurin ◽  
...  
Keyword(s):  
Intracellular Trafficking ◽  
Surface Proteins ◽  
Cell Type ◽  
Comparative Proteomic Analysis ◽  
Late Endosomes ◽  
Functional Discrimination ◽  
Different Populations ◽  
Endosomal Ph ◽  
Endocytic Compartments ◽  
Intercellular Communications

AbstractDespite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

scholarly journals Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking and synchronized extracellular vesicle release of CD9 and CD63

2020 ◽  
Author(s):  
Mathilde Mathieu ◽  
Nathalie Névo ◽  
Mabel Jouve ◽  
José Ignacio Valenzuela ◽  
Mathieu Maurin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Extracellular Vesicle ◽  
Multiple Roles ◽  
Surface Proteins ◽  
Intracellular Location ◽  
Functional Discrimination ◽  
Different Populations ◽  
Endocytic Compartments ◽  
Intercellular Communications

ABSTRACTDespite their important and multiple roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized yet. In particular, how and to what extent EVs form either as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (ectosomes) remains unclear. We followed in HeLa cells the intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment and identified transient co-localization both at the plasma membrane (PM) and in endosomes, before they finally segregate. CD9 was more abundantly released in EVs than CD63. However, when forcing expression of CD63 at the PM, by mutating its lysosome-addressing motive, its secretion in EVs was increased. Thus, in HeLa cells, small ectosomes are more prominently released than exosomes. By comparative proteomic analysis, we identified a few surface proteins likely specific of either exosomes (e.g. LAMP1) or ectosomes (e.g. BSG, SLC3A2), based on their known intracellular location in lysosomes or the PM, and on the different effects on their release of Bafilomycin A1, a drug that neutralizes endosomal pH. Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

scholarly journals Adenovirus Serotype 7 Retention in a Late Endosomal Compartment prior to Cytosol Escape Is Modulated by Fiber Protein

2001 ◽  
Vol 75 (3) ◽  
pp. 1387-1400 ◽  
Author(s):  
Naoki Miyazawa ◽  
Ronald G. Crystal ◽  
Philip L. Leopold
Keyword(s):  
Intracellular Trafficking ◽  
A549 Cells ◽  
Ph Optimum ◽  
Trafficking Pathway ◽  
Marker Proteins ◽  
Late Endosomes ◽  
Fiber Protein ◽  
Membrane Lysis ◽  
Endocytic Compartments ◽  
Acidic Compartments

ABSTRACT The intracellular trafficking of adenovirus (Ad) subgroup B (e.g., Ad7) differs from that of subgroup C (e.g., Ad5) in that Ad5 rapidly escapes from endocytic compartments following infection whereas Ad7 accumulates in organelles. To assess the hypothesis that Ad7 is targeted to the lysosomal pathway, Ad7 and Ad5 were conjugated with fluorophores and their trafficking in A549 epithelial cells was analyzed by fluorescence microscopy. Within 1 h after infection, Ad7, but not Ad5, accumulated in the cytoplasm of A549 cells. The pH in the environment of Ad5 was nearly neutral (pH 7), while Ad7 occupied acidic compartments (pH 5) over the first 2 h with a gradual shift toward neutrality by 8 h. Ad7 partially colocalized with α2-macroglobulin and late endosomal and lysosomal marker proteins, including Rab7, mannose-6-phosphate receptor, and LAMP-1. The pH optimum for membrane lysis by Ad7, as well as a chimeric Ad5 capsid that expressed the Ad7 fiber (Ad5fiber7), was pH 5.5, while that for lysis by Ad5 was pH 6.0. Thus, the native trafficking pathway for Ad7 involves residence in late endosomes and lysosomes, with information encoded in the Ad7 fiber acting as a pH-dependent trigger for membrane lysis and escape to the cytosol.

scholarly journals Evidence for Recycling of Invariant Surface Transmembrane Domain Proteins in African Trypanosomes

2012 ◽  
Vol 12 (2) ◽  
pp. 330-342 ◽  
Author(s):  
V. Lila Koumandou ◽  
Cordula Boehm ◽  
Katy A. Horder ◽  
Mark C. Field
Keyword(s):  
Intracellular Trafficking ◽  
Intracellular Transport ◽  
Transmembrane Domain ◽  
Life Stage ◽  
Surface Proteins ◽  
Invariant Surface ◽  
Content Type ◽  
African Trypanosomes ◽  
Surface Proteome ◽  
Endocytic Compartments

ABSTRACT Intracellular trafficking is a vital component of both virulence mechanisms and drug interactions in Trypanosoma brucei , the causative agent of human African trypanosomiasis and n'agana of cattle. Both maintaining the surface proteome composition within a life stage and remodeling the composition when progressing between life stages are important features of immune evasion and development for trypanosomes. Our recent work implicates the abundant transmembrane invariant surface glycoproteins (ISGs) in the uptake of first-line therapeutic suramin, suggesting a potential therapeutic route into the cell. RME-8 is a mediator of recycling pathways in higher eukaryotes and is one of a small cohort of intracellular transport gene products upregulated in mammal-infective trypanosomes, suggesting a role in controlling the copy number of surface proteins in trypanosomes. Here we investigate RME-8 function and its contribution to intracellular trafficking and stability of ISGs. RME-8 is a highly conserved protein and is broadly distributed across multiple endocytic compartments. By knockdown we find that RME-8 is essential and mediates delivery of endocytic probes to late endosomal compartments. Further, we find ISG accumulation within endosomes, but that RME-8 knockdown also increases ISG turnover; combined with previous data, this suggests that it is most probable that ISGs are recycled, and that RME-8 is required to support recycling.

scholarly journals SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates

2020 ◽  
Vol 36 (11) ◽  
pp. 3447-3456 ◽  
Author(s):  
Matthew Waas ◽  
Shana T Snarrenberg ◽  
Jack Littrell ◽  
Rachel A Jones Lipinski ◽  
Polly A Hansen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Specific Surface ◽  
Web Application ◽  
Rank Order ◽  
Surface Proteins ◽  
Supplementary Information ◽  
Live Cells ◽  
Specific Marker ◽  
Cell Type ◽  
Cell Type Specific

Abstract Motivation Cell-type-specific surface proteins can be exploited as valuable markers for a range of applications including immunophenotyping live cells, targeted drug delivery and in vivo imaging. Despite their utility and relevance, the unique combination of molecules present at the cell surface are not yet described for most cell types. A significant challenge in analyzing ‘omic’ discovery datasets is the selection of candidate markers that are most applicable for downstream applications. Results Here, we developed GenieScore, a prioritization metric that integrates a consensus-based prediction of cell surface localization with user-input data to rank-order candidate cell-type-specific surface markers. In this report, we demonstrate the utility of GenieScore for analyzing human and rodent data from proteomic and transcriptomic experiments in the areas of cancer, stem cell and islet biology. We also demonstrate that permutations of GenieScore, termed IsoGenieScore and OmniGenieScore, can efficiently prioritize co-expressed and intracellular cell-type-specific markers, respectively. Availability and implementation Calculation of GenieScores and lookup of SPC scores is made freely accessible via the SurfaceGenie web application: www.cellsurfer.net/surfacegenie. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

scholarly journals Internalization and endosomal degradation of receptor-bound antigens regulate the efficiency of cross presentation by human dendritic cells

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2011-2020 ◽  
Author(s):  
Bithi Chatterjee ◽  
Anna Smed-Sörensen ◽  
Lillian Cohn ◽  
Cécile Chalouni ◽  
Richard Vandlen ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Vaccine Development ◽  
Mannose Receptor ◽  
Antigen Delivery ◽  
Cross Presentation ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Antibody Conjugates ◽  
Human Dendritic Cells ◽  
Endocytic Compartments

Abstract Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.

scholarly journals Rab9 GTPase Regulates Late Endosome Size and Requires Effector Interaction for Its Stability

2004 ◽  
Vol 15 (12) ◽  
pp. 5420-5430 ◽  
Author(s):  
Ian G. Ganley ◽  
Kate Carroll ◽  
Lenka Bittova ◽  
Suzanne Pfeffer
Keyword(s):  
Membrane Protein ◽  
Cultured Cells ◽  
Late Endosome ◽  
Interacting Protein ◽  
Late Endosomes ◽  
Specific Effector ◽  
Mannose 6 Phosphate ◽  
Endocytic Compartments ◽  
Gdp Dissociation Inhibitor ◽  
Multivesicular Endosomes

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule–containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.

scholarly journals Recent Progress in Cardiovascular Research Involving Single-Cell Omics Approaches

2021 ◽  
Vol 8 ◽  
Author(s):  
Zhehao Dai ◽  
Seitaro Nomura
Keyword(s):  
Single Cell ◽  
Recent Progress ◽  
Disease Gene ◽  
Cell Type ◽  
Cardiovascular Research ◽  
Omics Technologies ◽  
Technological Advances ◽  
Cell Type Specific ◽  
Intercellular Communications ◽  
Research Resources

Cardiovascular diseases are among the leading causes of morbidity and mortality worldwide. Although the spectrum of the heart from development to disease has long been studied, it remains largely enigmatic. The emergence of single-cell omics technologies has provided a powerful toolbox for defining cell heterogeneity, unraveling previously unknown pathways, and revealing intercellular communications, thereby boosting biomedical research and obtaining numerous novel findings over the last 7 years. Not only cell atlases of normal and developing hearts that provided substantial research resources, but also some important findings regarding cell-type-specific disease gene program, could never have been established without single-cell omics technologies. Herein, we briefly describe the latest technological advances in single-cell omics and summarize the major findings achieved by such approaches, with a focus on development and homeostasis of the heart, myocardial infarction, and heart failure.

scholarly journals Comprehensive multi-omics single-cell data integration reveals greater heterogeneity in the human immune system

2021 ◽  
Author(s):  
Congmin Xu ◽  
Junkai Yang ◽  
Astrid Kosters ◽  
Benjamin R Babcock ◽  
Peng Qiu ◽  
...  
Keyword(s):  
Immune System ◽  
Single Cell ◽  
Immune Cell ◽  
Cell Types ◽  
Cell Receptor ◽  
Surface Proteins ◽  
Cell Type ◽  
Human Immune System ◽  
Receptor Repertoire ◽  
Human Immune

Single-cell transcriptomics enables the definition of diverse human immune cell types across multiple tissue and disease contexts. Still, deeper biological understanding requires comprehensive integration of multiple single-cell omics (transcriptomic, proteomic, and cell receptor repertoire). To improve the identification of diverse cell types and the accuracy of cell-type classification in our multi-omics single-cell datasets, we developed SuPERR-seq, a novel analysis workflow to increase the resolution and accuracy of clustering and allow for the discovery and characterization of previously hidden cell subsets. We show that by incorporating information from cell-surface proteins and immunoglobulin transcript counts, we accurately remove cell doublets and prevent widespread cell-type misclassification. This approach uniquely improves the identification of heterogeneous cell types in the human immune system, including a novel subset of antibody-secreting cells in the bone marrow.

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