Intracellular trafficking of transferrin-conjugated liposome/ DNA complexes by confocal microscopy

2005 ◽  
Vol 28 (1) ◽  
pp. 93-99 ◽  
Author(s):  
Sang Mi Lee ◽  
Jin-Seok Kim
Keyword(s):  
Confocal Microscopy ◽  
Intracellular Trafficking ◽  
Dna Complexes

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

scholarly journals SNAPSwitch: A Molecular Sensor to Quantify the Localization of Proteins, DNA and Nanoparticles in Cells

2019 ◽  
Author(s):  
Laura I FitzGerald ◽  
Luigi Aurelio ◽  
Moore Chen ◽  
Daniel Yuen ◽  
Bim Graham ◽  
...  
Keyword(s):  
Immune Responses ◽  
Intracellular Trafficking ◽  
Live Cells ◽  
Quantitative Detection ◽  
Dna Complexes ◽  
Receptor Signalling ◽  
Fluorescent Markers ◽  
Molecular Sensor ◽  
Protein Nucleic Acid ◽  
In Cells

Intracellular trafficking governs receptor signalling, pathogenesis, immune responses and the cellular fate of nanomedicines. These processes are typically tracked by confocal microscopy, where colocalization of fluorescent markers implies an interaction or co-compartmentalization. However, this type of analysis is inherently low-throughput, is limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we have developed a localization sensor composed of a quenched and attachable SNAP-tag substrate (SNAPSwitch). SNAPSwitch enables quantitative detection of protein, nucleic acid and nanoparticle trafficking to locations of interest within live cells using flow cytometry. Using this approach, we followed the trafficking of DNA complexes travelling from endosomes into the cytosol and to the nucleus. We also show that antibody targeted to the transferrin (CD71) or hyaluronan (CD44) receptor is initially sorted into different compartments following endocytosis. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track the localization of materials in cells.

scholarly journals Binding Affinity, Cellular Uptake, and Subsequent Intracellular Trafficking of the Nano-Gene Vector P123-PEI-R13

2016 ◽  
Vol 2016 ◽  
pp. 1-8
Author(s):  
Yaguang Zhang ◽  
Hongmei Shu ◽  
Jing Hu ◽  
Min Zhang ◽  
Junweng Wu ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cellular Uptake ◽  
Intracellular Trafficking ◽  
Transfection Efficiency ◽  
Cross Linking ◽  
Low Molecular Weight ◽  
Gene Vector ◽  
Dna Complexes ◽  
Dynamic Source

A nano-gene vector PEI-P123-R13 was synthesized by cross-linking low molecular weight PEI with P123 and further coupling bifunctional peptide R13 to the polymer for targeting tumor and increasing cellular uptake. The binding assessment of R13 toαvβ3 positive cells was performed by HRP labeling. The internalization pathways of P123-PEI-R13/DNA complexes were investigated based on the effect of specific endocytic inhibitors on transfection efficiency. The mechanism of intracellular trafficking was investigated based on the effect of endosome-lysosome acidification inhibitors, cytoskeleton, and dynein inhibitors on transfection efficiency. The results indicated that the bifunctional peptide R13 had the ability of binding toαvβ3 positive cellsin vitro. The modification of P123-PEI-R13 with R13 made it display new property of internalization. P123-PEI-R13/DNA complexes were conducted simultaneously via clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and possible energy-independent route. After internalization, P123-PEI-R13/DNA complexes could escape from the endosome-lysosome system because of its acidification and further took microtubule as the track and dynein as the dynamic source to be transported toward the microtubule (+) end, to wit nucleus, under the action of microfilament, and with the aid of intermediate filament.

scholarly journals Intracellular trafficking of cationic liposome–DNA complexes in living cells

Soft Matter ◽  
2012 ◽  
Vol 8 (30) ◽  
pp. 7919 ◽  
Author(s):  
Stefano Coppola ◽  
Laura C. Estrada ◽  
Michelle A. Digman ◽  
Daniela Pozzi ◽  
Francesco Cardarelli ◽  
...  
Keyword(s):  
Intracellular Trafficking ◽  
Cationic Liposome ◽  
Living Cells ◽  
Dna Complexes

scholarly journals Missing pieces in understanding the intracellular trafficking of polycation/DNA complexes

2009 ◽  
Vol 139 (2) ◽  
pp. 88-93 ◽  
Author(s):  
You-Yeon Won ◽  
Rahul Sharma ◽  
Stephen F. Konieczny
Keyword(s):  
Intracellular Trafficking ◽  
Dna Complexes

The Role of Circulating Neutrophils in Increased Procoagulant Activity after Acute Ischemic Stroke

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3682-3682
Author(s):  
Zhipeng Yao ◽  
Tao Li ◽  
Muhua Cao ◽  
Ruishuang Ma ◽  
Xiaoming Wu ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Platelet Aggregation ◽  
Confocal Microscopy ◽  
Acute Ischemic Stroke ◽  
Procoagulant Activity ◽  
Coagulation Cascade ◽  
Coagulation Activation ◽  
Dna Complexes ◽  
Fibrin Formation

Abstract Introduction: Acute ischemic stroke (AIS) is a hypoxic ischemic disorder associated with a sterile inflammatory reaction. Neuronal injury from ischemic stroke is aggravated by invading peripheral polymorphonuclear cells (PMNs). Neutrophils not only have a remarkable neurotoxic effect from the release of proteolytic enzymes, but also foster coagulation cascade by exposing membrane phosphatidylserine (PS), aggregating to platelets and releasing neutrophil extracellular traps (NETs). However, the role of circulating neutrophils in hypercoagulation state after AIS remains unclear. Our aims were to determine the procoagulant role of circulating neutrophils after AIS, and to elucidate the mechanism of neutrophils-induced thrombophilia. Methods: 73 newly diagnosed AIS patients and 35 risk factors matched controls were enrolled. Patient blood samples were collected at 6 h, 12 h, 24 h, 3 d, and 7 d after the onset of clinical symptoms. PS exposure on neutrophils and neutrophil-platelet aggregation was measured by flow cytometry and confocal microscopy. The percentage of NETs-releasing PMNs was quantified by confocal microscopy. Double-stranded DNA and myeloperoxidase-DNA complexes were also measured as in vivo markers of NETosis. Specifically, dsDNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Myeloperoxidase-DNA complex was measured using a capture ELISA.The procoagulant activity (PCA) of neutrophils was measured by clotting time and purified coagulation complex assays. Plasma levels of coagulation activation were evaluated by thrombin-antithrombin experiment. Results: The initial levels of PS+ neutrophils and neutrophil-platelet aggregation were 2.1- and 1.8-fold higher, respectively, in AIS than controls. Specifically, PS+ neutrophils peaked at 24 hours and returned to their initial levels at 3 days while neutrophil-platelet aggregation elevated at initial and remained high for the later time points. Patient neutrophils supported significantly shortened clotting times and increases in Xase activity and thrombin formation compared to controls (P < 0.001 for all). Interestingly, treatment with lactadherin, a PS antagonist, effectively restored patient PCA to control levels. The amounts of NETs+ cells, dsDNA and myeloperoxidase-DNA complexes were elevated at 3 days after stroke and positively correlated with thrombin generation. Furthermore, cultured endothelial cells were intensely activated by neutrophils of stroke patients and subsequently supported massive fibrin formation. Moreover, blockade of NET formation with DNAse I inhibited fibrin formation by approximately 60%. In a subanalysis, 27 patients with early infections were matched with 27 patients without infections according to S100B peak levels. These two patient subgroups showed significant differences in the temporal pattern of PS+ neutrophils, neutrophil-platelet aggregation, NETs%, markers of NET formation, and coagulation activation. The levels of coagulation markers were significantly higher in patients with early infections than in patients without (P < 0.05 for all). Conclusions: The thrombophilic susceptibility could be partly due to the activation of neutrophils after ischemic stroke. AIS patients with early infections are more prone to thrombosis. Our studies identify PS exposure on neutrophils and formation of NET as potentially novel therapeutic targets in the treatment of AIS. Figure 1. The changes in the number of neutrophils, PS+ neutrophils, and neutrophil-platelet aggregation (NPA) measured within 7 days of stroke. *P < 0.05 vs. control. Figure 1. The changes in the number of neutrophils, PS+ neutrophils, and neutrophil-platelet aggregation (NPA) measured within 7 days of stroke. *P < 0.05 vs. control. Disclosures No relevant conflicts of interest to declare.

scholarly journals A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Laura I. FitzGerald ◽  
Luigi Aurelio ◽  
Moore Chen ◽  
Daniel Yuen ◽  
Joshua J. Rennick ◽  
...  
Keyword(s):  
Immune Responses ◽  
Intracellular Trafficking ◽  
Cellular Membrane ◽  
Live Cells ◽  
Quantitative Detection ◽  
Membrane Material ◽  
Dna Complexes ◽  
Fluorescent Markers ◽  
Molecular Sensor ◽  
In Cells

Abstract Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

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