scholarly journals Development of Fluorescently Labeled SSEA-3, SSEA-4, and Globo-H Glycosphingolipids for Elucidating Molecular Interactions in the Cell Membrane

2019 ◽  
Vol 20 (24) ◽  
pp. 6187 ◽  
Author(s):  
Sachi Asano ◽  
Rita Pal ◽  
Hide-Nori Tanaka ◽  
Akihiro Imamura ◽  
Hideharu Ishida ◽  
...  
Keyword(s):  
Single Molecule ◽  
Pluripotent Stem Cells ◽  
Membrane Vesicles ◽  
Biological Processes ◽  
Plasma Membrane Vesicles ◽  
Liquid Ordered ◽  
Embryonic Antigen ◽  
Globo Series ◽  
Detergent Resistant Membrane ◽  
Stage Specific Embryonic Antigen

Glycosphingolipids (GSLs), such as the globo-series GSLs stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, and Globo-H, are specifically expressed on pluripotent stem cells and cancer cells, and are known to be associated with various biological processes such as cell recognition, cell adhesion, and signal transduction. However, the behavior and biological roles of these GSLs are still unclear. In our previous study, we observed the interactions between the lipid raft and GSLs in real-time using single-molecule imaging, where we successfully synthesized various fluorescent analogs of GSLs (e.g., GM1 and GM3). Here, we have developed fluorescent analogs of SSEA-3, SSEA-4, and Globo-H using chemical synthesis. The biophysical properties of these analogs as raft markers were examined by partitioning giant plasma membrane vesicles from RBL-2H3 cells into detergent-resistant membrane fractions and liquid-ordered/liquid-disordered phases. The results indicated that the analogs were equivalent to native-type GSLs. The analogs could be used to observe the behavior of globo-series GSLs for detailing the structure and biological roles of lipid rafts and GSL-enriched nanodomains during cell differentiation and cell malignancy.

scholarly journals Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles

2009 ◽  
Vol 424 (2) ◽  
pp. 163-167 ◽  
Author(s):  
Ilya Levental ◽  
Fitzroy J. Byfield ◽  
Pramit Chowdhury ◽  
Feng Gai ◽  
Tobias Baumgart ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Membrane Vesicles ◽  
Correlation Spectroscopy ◽  
Model Systems ◽  
Live Cells ◽  
Lipid Phase ◽  
Plasma Membrane Vesicles ◽  
Liquid Ordered ◽  
Lipid Phase Separation

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

Differentiation of bone marrow-derived stage-specific embryonic antigen 1 positive pluripotent stem cells into male germ cells

2016 ◽  
Vol 80 (4) ◽  
pp. 430-440 ◽  
Author(s):  
Reza Shirazi ◽  
Amir Hassan Zarnani ◽  
Masoud Soleimani ◽  
Karim Nayernia ◽  
Iraj Ragerdi Kashani
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Male Germ Cells ◽  
Embryonic Antigen ◽  
Stage Specific Embryonic Antigen

scholarly journals Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains

2021 ◽  
Vol 118 (9) ◽  
pp. e2025343118
Author(s):  
Jongyun Myeong ◽  
Cheon-Gyu Park ◽  
Byung-Chang Suh ◽  
Bertil Hille
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Membrane Vesicles ◽  
Membrane Lipid ◽  
Phosphoinositide Metabolism ◽  
Protein Markers ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Liquid Ordered

Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

scholarly journals Molecular Dynamics and Concentration in Raft and Boundary Domains in Actin-Depleted Plasma Membrane Vesicles as Revealed by Single-Molecule Imaging

2013 ◽  
Vol 104 (2) ◽  
pp. 191a
Author(s):  
Rahul Chadda ◽  
Takahiro K. Fujiwara ◽  
Ziya Kalay ◽  
Shusaku Shibutani ◽  
Kenichi G.N. Suzuki ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Plasma Membrane ◽  
Single Molecule ◽  
Membrane Vesicles ◽  
Single Molecule Imaging ◽  
Plasma Membrane Vesicles

scholarly journals Burkholderia cenocepacia transcriptome during the early contacts with giant plasma membrane vesicles derived from live bronchial epithelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andreia I. Pimenta ◽  
Nuno Bernardes ◽  
Marta M. Alves ◽  
Dalila Mil-Homens ◽  
Arsenio M. Fialho
Keyword(s):  
Membrane Vesicles ◽  
Bronchial Epithelial Cell ◽  
Host Cells ◽  
Burkholderia Cenocepacia ◽  
Transport Systems ◽  
Epithelial Cell Line ◽  
Plasma Membrane Vesicles ◽  
Bronchial Epithelial ◽  
Cellular Processes ◽  
Associated Functions

AbstractBurkholderia cenocepacia is known for its capacity of adherence and interaction with the host, causing severe opportunistic lung infections in cystic fibrosis patients. In this work we produced Giant Plasma Membrane Vesicles (GPMVs) from a bronchial epithelial cell line and validated their use as a cell-like alternative to investigate the steps involved in the adhesion process of B. cenocepacia. RNA-sequencing was performed and the analysis of the B. cenocepacia K56-2 transcriptome after the first contacts with the surface of host cells allowed the recognition of genes implicated in bacterial adaptation and virulence-associated functions. The sensing of host membranes led to a transcriptional shift that caused a cascade of metabolic and physiological adaptations to the host specific environment. Many of the differentially expressed genes encode proteins related with central metabolic pathways, transport systems, cellular processes, and virulence traits. The understanding of the changes in gene expression that occur in the early steps of infection can uncover new proteins implicated in B. cenocepacia-host cell adhesion, against which new blocking agents could be designed to control the progression of the infectious process.

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