Single Particle Tracking Confirms That Multivalent Tat Protein Transduction Domain-induced Heparan Sulfate Proteoglycan Cross-linkage Activates Rac1 for Internalization

Imamura, Junji; Suzuki, Yasuhiro; Gonda, Kohsuke; Roy, Chandra Nath; Gatanaga, Hiroyuki; Ohuchi, Noriaki; Higuchi, Hideo

doi:10.1074/jbc.m110.187450

Cited by 33 publications

(52 citation statements)

References 52 publications

(65 reference statements)

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“…We have also reported that only multivalent TatP (mTatP)-QDs can induce the cross-linking of HSPGs and elicit a signal; in contrast, 2-val-TatP-QDs can bind to HSPGs but cannot induce the cross-linking of HSPGs or elicit intracellular signaling (Fig. 1A) (24). Thus, we employed mTatP-QDs and 2-val-TatP-QDs to study the influence of the HSPG cross-linking.…”

Section: Resultsmentioning

confidence: 99%

“…We have shown previously that the initial binding of TatP to cells occurs through the HS-glycosaminoglycan (GAG) chains of HSPGs on a wide variety of cells, such as HeLa cells; HepG2 cells; and T cells, B cells, and macrophages isolated from healthy donors (24). We have also reported that only multivalent TatP (mTatP)-QDs can induce the cross-linking of HSPGs and elicit a signal; in contrast, 2-val-TatP-QDs can bind to HSPGs but cannot induce the cross-linking of HSPGs or elicit intracellular signaling (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In that study, by repeating short-period observations at different time points up to 15 min, we found (24,25) that HSPGs serve as the cell surface receptors for TatP and that TatP-induced signaling may influence the kinetics of the TatP-HSPG complexes on living cells. However, we suspect that the detailed mechanisms of TatP cell surface pan).…”

mentioning

confidence: 99%

“…However, we suspect that the detailed mechanisms of TatP cell surface pan). The spatial precision of the x and y positions determined from a single QD on the glass surface was ϳ7 nm, as previously described (24). We typically tracked 50 to 150 particles per cell, and we obtained the images from at least three independent experiments.…”

mentioning

confidence: 99%

“…By taking advantage of the brightness and photostability of quantum dots (QDs) (22,23), we have previously investigated the behavior of TatP labeled with QDs in living cells at a single-molecule resolution with high spatial accuracy (24,25). In that study, by repeating short-period observations at different time points up to 15 min, we found (24,25) that HSPGs serve as the cell surface receptors for TatP and that TatP-induced signaling may influence the kinetics of the TatP-HSPG complexes on living cells.…”

mentioning

confidence: 99%

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Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

Suzuki

Roy

Promjunyakul

et al. 2013

Molecular and Cellular Biology

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The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

Suzuki

Roy

Promjunyakul

et al. 2013

Molecular and Cellular Biology

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A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: To enhance the efficiency of recombinant DNA utilization

Mozley

Thompson

Fernandez‐Martell

et al. 2014

Biotechnology Progress

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In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60-70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG-containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl-PEI, was chemically synthesized. Using Design of Experiments-Response Surface Modeling to optimize the transfection process, the function of propyl-PEI was compared to that of unmodified PEI in both parental CHO-S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex cytotoxicity. The combination of propyl-PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process.

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Quantum Dot‐Based Nanotools for Bioimaging, Diagnostics, and Drug Delivery

2016

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Quantum dots (QDs) are highly fluorescent nanocrystals with advanced photophysical and spectral properties: high brightness and stability against photobleaching accompanied by broad excitation and narrow emission spectra. Water-soluble QDs functionalized with biomolecules, such as proteins, peptides, antibodies, and drugs, are used for biomedical applications. The advantages of QD-based approaches to immuno-histochemical analysis, single-molecule tracking, and in vivo imaging (over traditional methods with organic dyes and fluorescent proteins) are explained. The unique spectral properties of QDs offer opportunities for designing systems for multiplexed analysis by multicolor imaging for the simultaneous detection of multiple targets. Conjugation of drug molecules with QDs or their incorporation into QD-based drug-delivery particles makes it possible to monitor real-time drug tracking and carry out image-guided therapy. Because of the tunability of their photophysical properties, QDs emitting in the near-infrared have become an attractive tool for deep-tissue mono- and multiphoton in vivo imaging. We review recent achievements in QD applications for bioimaging, targeting, and drug delivery, as well as challenges related to their toxicity and non-biodegradability. Key and perspectives for further development of advanced QD-based nanotools are addressed.

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Single Particle Tracking Confirms That Multivalent Tat Protein Transduction Domain-induced Heparan Sulfate Proteoglycan Cross-linkage Activates Rac1 for Internalization

Cited by 33 publications

References 52 publications

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: To enhance the efficiency of recombinant DNA utilization

Quantum Dot‐Based Nanotools for Bioimaging, Diagnostics, and Drug Delivery

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