Quantum Dot–Fluorescent Protein FRET Probes for Sensing Intracellular pH

Dennis, Allison M.; Rhee, Won Jong; Sotto, David C.; Dublin, Steven N.; Bao, Gang

doi:10.1021/nn2038077

Cited by 317 publications

(229 citation statements)

References 36 publications

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“…From several FRET-based sensors designed to measure endosomal pH (16,(30)(31)(32), we used a derivative of pHlameleon (16) incorporated into the viral membrane. Unlike other approaches, this strategy avoided chemical modifications of the virus, which could compromise its ability to undergo fusion.…”

Section: Discussionmentioning

confidence: 99%

“…1D), the ability to determine the pH reliably in late endosomes was limited by cell autofluorescence. This problem can be alleviated by incorporating a larger number of pHlameleon-based sensors into the viral membrane and/or by implementing alternative labeling strategies (32). Further improvements in sensitivity and temporal resolution of pH measurements could shed light on biogenesis of acidic compartments.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes

Padilla‐Parra

Matos

Kondo

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

104

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Diverse enveloped viruses enter host cells through endocytosis and fuse with endosomal membranes upon encountering acidic pH. Currently, the pH dynamics in virus-carrying endosomes and the relationship between acidification and viral fusion are poorly characterized. Here, we examined the entry of avian retrovirus that requires two sequential stimuli-binding to a cognate receptor and low pH-to undergo fusion. A genetically encoded sensor incorporated into the viral membrane was used to measure the pH in viruscarrying endosomes. Acid-induced virus fusion was visualized as the release of a fluorescent viral content marker into the cytosol. The pH values in early acidic endosomes transporting the virus ranged from 5.6 to 6.5 but were relatively stable over time for a given vesicle. Analysis of viral motility and luminal pH showed that cells expressing the transmembrane isoform of the receptor (TVA950) preferentially sorted the virus into slowly trafficking, less acidic endosomes. In contrast, viruses internalized by cells expressing the GPI-anchored isoform (TVA800) were uniformly distributed between stationary and mobile compartments. We found that the lag times between acidification and fusion were significantly shorter and fusion pores were larger in dynamic endosomes than in more stationary compartments. Despite the same average pH within mobile compartments of cells expressing alternative receptor isoforms, TVA950 supported faster fusion than TVA800 receptor. Collectively, our results suggest that fusion steps downstream of the low-pH trigger are modulated by properties of intracellular compartments harboring the virus.confocal imaging | nano-pH-meter | single virus tracking | FRET M any enveloped viruses use endocytosis and vesicular trafficking to enter host cells, where acidification of an endosomal lumen serves as a trigger for viral fusion (1, 2). Viral fusion proteins are fine-tuned to respond to different pH values. Those that are activated at less acidic pH (∼6.0) are thought to mediate fusion with early endosomes, whereas those with a lower pH threshold (∼5.0) appear to direct the virus entry from late endosomes (1, 3). However, recent evidence implies that factors other than low pH, such as specific endosome-resident lipids, can determine the intracellular compartments from which the viral capsid is released into the cytosol (2, 4, 5). Progress in understanding the complex regulation of virus-endosome fusion has been hindered by poor accessibility of intracellular compartments and lack of direct techniques for monitoring this process in situ.Single-virus imaging is a powerful tool for gaining critical insights into virus entry (reviewed in ref. 6), but detection of virus-endosome fusion (here defined as the release of viral content into the cytoplasm) is technically challenging (7,8). To understand the relationship between the pH trigger and virus-endosome fusion, it is essential to visualize these events in real time. A ratiometric method developed by the Zhuang group (9, 10) permits monitoring endos...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes

Padilla‐Parra

Matos

Kondo

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

104

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“…In bioanalytical applications, the great advantage of FRET is the ability to turn QD PL ''on'' or ''off'' in response to biorecognition events (e.g., ligand-receptor binding, enzyme activity, DNA hybridization) or other physicochemical stimuli (e.g., pH 131,132 In each case, the underlying idea is that a donor/acceptor is added or removed from the vicinity of a FRETpaired QD, either physically (e.g., association or dissociation) or through a change in its resonance (i.e., a large spectral shift). FRET-based sensing has been thoroughly reviewed elsewhere, 4,16 and we have limited ourselves to a few more recent examples here.…”

Section: Bioanalysis and Bioimaging With Quantum Dotsmentioning

confidence: 99%

Quantum Dots in Bioanalysis: A Review of Applications across Various Platforms for Fluorescence Spectroscopy and Imaging

2013

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Semiconductor quantum dots (QDs) are brightly luminescent nanoparticles that have found numerous applications in bioanalysis and bioimaging. In this review, we highlight recent developments in these areas in the context of specific methods for fluorescence spectroscopy and imaging. Following a primer on the structure, properties, and biofunctionalization of QDs, we describe select examples of how QDs have been used in combination with steady-state or time-resolved spectroscopic techniques to develop a variety of assays, bioprobes, and biosensors that function via changes in QD photoluminescence intensity, polarization, or lifetime. Some special attention is paid to the use of Förster resonance energy transfer-type methods in bioanalysis, including those based on bioluminescence and chemiluminescence. Direct chemiluminescence, electrochemiluminescence, and charge transfer quenching are similarly discussed. We further describe the combination of QDs and flow cytometry, including traditional cellular analyses and spectrally encoded barcode-based assay technologies, before turning our attention to enhanced fluorescence techniques based on photonic crystals or plasmon coupling. Finally, we survey the use of QDs across different platforms for biological fluorescence imaging, including epifluorescence, confocal, and twophoton excitation microscopy; single particle tracking and fluorescence correlation spectroscopy; super-resolution imaging; near-field scanning optical microscopy; and fluorescence lifetime imaging microscopy. In each of the above-mentioned platforms, QDs provide the brightness needed for highly sensitive detection, the photostability needed for tracking dynamic processes, or the multiplexing capacity needed to elucidate complex systems. There is a clear synergy between advances in QD materials and spectroscopy and imaging techniques, as both must be applied in concert to achieve their full potential.

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“…Studies including cell experiments often focus only on the proof that intracellular measurements are generally possible. Monitoring the behavior of the targeted ion concentration, however, is often excluded or only done over very short times [93,94]. Instead, it is important to also focus on the long-term monitoring of the dynamics of the lysosomal pH upon induced ion deregulation.…”

Section: Theranostics As Biomedical Approach For Polyelectrolyte Multmentioning

confidence: 99%

Intracellular delivery and sensing based on polyelectrolyte multilayer capsules

Nazarenus¹,

Gil²,

Parak³

2015

Nano Based Drug Delivery

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Quantum Dot–Fluorescent Protein FRET Probes for Sensing Intracellular pH

Cited by 317 publications

References 36 publications

Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes

Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes

Quantum Dots in Bioanalysis: A Review of Applications across Various Platforms for Fluorescence Spectroscopy and Imaging

Intracellular delivery and sensing based on polyelectrolyte multilayer capsules

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