Mobility of Green Fluorescent Protein in Hydrogel-Based Drug-Delivery Systems Studied by Anisotropy and Fluorescence Recovery After Photobleaching

PDZ-domain–containing proteins such as PSD-95 have been implicated in the targeting and clustering of membrane proteins. Biochemical and immunohistochemical studies indicate that PSD-95 recognizes COOH-terminal S/TXV sequences present in Kv1 K+ channels. However, the effect of binding a PDZ domain on a target protein has not been studied in live cells. In the present study, a green fluorescent protein–Kv1.4 fusion protein is used to study the effect of PSD-95 on channel movement. Fluorescence recovery after photobleaching showed that PSD-95 can immobilize K+ channels in the plasma membrane in an all-or-none manner. Furthermore, time lapse imaging showed that channel clusters formed in the presence of PSD-95 are stable in size, shape, and position. As expected from previous reports, two green fluorescent protein–tagged COOH-terminal variants of Kv1.4, Δ15 and V655A, are not clustered by PSD-95. However, coexpression of PSD-95 with V655A, but not Δ15, leads to the appearance of PSD-95 immunoreactivity in the plasma membrane. Furthermore, fluorescence recovery after photobleaching studies show that V655A channels are immobilized by PSD-95. Thus, V655A channels can interact with PSD-95 in a manner that leads to channel immobilization, but not clustering. These experiments document for the first time that PSD-95 immobilizes target proteins. Additionally, the data presented here demonstrate that the structural requirements for protein clustering and immobilization by PSD-95 are distinct.

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Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells—A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid

Pharmaceutics ◽

10.3390/pharmaceutics12090805 ◽

2020 ◽

Vol 12 (9) ◽

pp. 805

Author(s):

Christopher Janich ◽

Daniel Ivanusic ◽

Julia Giselbrecht ◽

Elena Janich ◽

Shashank Reddy Pinnapireddy ◽

...

Keyword(s):

Drug Delivery ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Nucleic Acid ◽

Fluorescent Protein ◽

Cationic Lipid ◽

Nucleic Acid Delivery ◽

Molecular Clone ◽

Green Fluorescent ◽

Hiv 1

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

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The Sudden Recruitment of γ-Tubulin to the Centrosome at the Onset of Mitosis and Its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules

The Journal of Cell Biology ◽

10.1083/jcb.146.3.585 ◽

1999 ◽

Vol 146 (3) ◽

pp. 585-596 ◽

Cited By ~ 238

Author(s):

Alexey Khodjakov ◽

Conly L. Rieder

Keyword(s):

Cell Cycle ◽

Green Fluorescent Protein ◽

Fluorescence Recovery After Photobleaching ◽

Fluorescent Protein ◽

Green Fluorescent Protein Fusion ◽

Protein Fusion ◽

Green Fluorescent ◽

Dynamic Exchange ◽

Two Populations

γ-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a γ-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of γ-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of γ-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of γ-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated γ-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated γ-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional γ-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of γ-tubulin than during interphase.

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In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

The Journal of Cell Biology ◽

10.1083/jcb.200307002 ◽

2004 ◽

Vol 164 (6) ◽

pp. 843-850 ◽

Cited By ~ 21

Author(s):

Stefan Wagner ◽

Simion Chiosea ◽

Maria Ivshina ◽

Jeffrey A. Nickerson

Keyword(s):

Rna Splicing ◽

Fluorescence Recovery After Photobleaching ◽

Fluorescent Protein ◽

Fluorescence Recovery ◽

Exon Junction Complex ◽

Permeabilized Cells ◽

Exon Junction ◽

Rna Export ◽

Green Fluorescent

We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.

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Intracellular Macromolecular Mobility Measured by Fluorescence Recovery after Photobleaching with Confocal Laser Scanning Microscopes

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0496 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4749-4760 ◽

Cited By ~ 152

Author(s):

José Braga ◽

Joana M.P. Desterro ◽

Maria Carmo-Fonseca

Keyword(s):

Aqueous Solution ◽

Diffusion Coefficients ◽

Laser Scanning ◽

Fluorescence Recovery After Photobleaching ◽

Fluorescent Protein ◽

Confocal Laser Scanning ◽

Fluorescence Recovery ◽

Confocal Laser ◽

Wide Range ◽

Green Fluorescent

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22°C reduces the dextran diffusion rates by ∼30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.

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Dual-ligand functionalized carbon nanodots as green fluorescent nanosensors for cellular dual receptor-mediated targeted imaging

The Analyst ◽

10.1039/c9an01530b ◽

2019 ◽

Vol 144 (22) ◽

pp. 6729-6735 ◽

Cited By ~ 1

Author(s):

Fangfang Du ◽

Xuewei Zhao ◽

Wenjing Lu ◽

Zhonghui Guo ◽

Shaomin Shuang ◽

...

Keyword(s):

Drug Delivery ◽

Tumor Cells ◽

Drug Delivery Systems ◽

Therapeutic Agents ◽

Delivery Systems ◽

Carbon Nanodots ◽

Targeted Imaging ◽

Green Fluorescent

The conjugation of dual-ligands to nanoparticles as drug delivery systems that target specific cells is a promising approach for the delivery of therapeutic agents to tumor cells.

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Techniques For The Preparation of Tissue Samples Containing Injectable Polymer Microcapsules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120242 ◽

1985 ◽

Vol 43 ◽

pp. 710-711

Author(s):

G.E. Visscher ◽

R. L. Robison ◽

G. J. Argentieri

Keyword(s):

Drug Delivery ◽

Drug Delivery Systems ◽

Gastrocnemius Muscle ◽

Degradation Process ◽

Delivery Systems ◽

Tissue Reaction ◽

Electron Microscopic ◽

Tissue Samples ◽

Injectable Polymer ◽

Microscopic Techniques

The use of various bioerodable polymers as drug delivery systems has gained considerable interest in recent years. Among some of the shapes used as delivery systems are films, rods and microcapsules. The work presented here will deal with the techniques we have utilized for the analysis of the tissue reaction to and actual biodegradation of injectable microcapsules. This work has utilized light microscopic (LM), transmission (TEM) and scanning (SEM) electron microscopic techniques. The design of our studies has utilized methodology that would; 1. best characterize the actual degradation process without artifacts introduced by fixation procedures and 2. allow for reproducible results.In our studies, the gastrocnemius muscle of the rat was chosen as the injection site. Prior to the injection of microcapsules the skin above the sites was shaved and tattooed for later recognition and recovery. 1.0 cc syringes were loaded with the desired quantity of microcapsules and the vehicle (0.5% hydroxypropylmethycellulose) drawn up. The syringes were agitated to suspend the microcapsules in the injection vehicle.

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