Quantum Dots Tracking Endocytosis and Transport of Proteins Displayed by Mammalian Cells

Zhang, Meng-Qian; Wang, Zhi-Gang; Fu, Dan-Dan; Zhang, Jumei; Liu, Hao-Yang; Liu, Shu Lin; Pang, Dai‐Wen

doi:10.1021/acs.analchem.2c00411

Cited by 10 publications

(11 citation statements)

References 35 publications

(47 reference statements)

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“…A protocol was developed to track BODIPY C12-labeled VLDLs’ from a zebrafish embryo yolk sack, and utilizing live fluorescence imaging with the embryos immobilized in agar allowed for the study of the biogenesis, transfer, uptake, and fate of individual endogenous single EVs from the yolk to the other parts of the fish [ 53 ]. Diving deeper into the endocytosis mechanism, by attaching a HaloTag to a platelet-derived growth factor receptor (PDGFR) and expressing the construct in Martin–Darby canine kidney cells, the movement of the growth factor being endocytosed can be accurately tracked [ 54 ]. By associating the HaloTag with a quantum dot, the endocytosis mechanism was visualized through spinning-disk confocal microscopy [ 54 ].…”

Section: Inner and Extracellular Transportmentioning

confidence: 99%

“…Diving deeper into the endocytosis mechanism, by attaching a HaloTag to a platelet-derived growth factor receptor (PDGFR) and expressing the construct in Martin–Darby canine kidney cells, the movement of the growth factor being endocytosed can be accurately tracked [ 54 ]. By associating the HaloTag with a quantum dot, the endocytosis mechanism was visualized through spinning-disk confocal microscopy [ 54 ]. Live-cell imaging revealed that the HaloTag entered the cells, where the protein moved along microfilaments and microtubules to the lysosomes around the nucleus [ 54 ].…”

Section: Inner and Extracellular Transportmentioning

confidence: 99%

“…By associating the HaloTag with a quantum dot, the endocytosis mechanism was visualized through spinning-disk confocal microscopy [ 54 ]. Live-cell imaging revealed that the HaloTag entered the cells, where the protein moved along microfilaments and microtubules to the lysosomes around the nucleus [ 54 ]. From there, the endocytosed proteins followed one of two paths.…”

Section: Inner and Extracellular Transportmentioning

confidence: 99%

“…From there, the endocytosed proteins followed one of two paths. The first path is the recycling of the PDGFR by circulating endosomes around the nucellus back to the plasma membrane [ 54 ]. The second path includes full endocytosis, leading to degradation.…”

Section: Inner and Extracellular Transportmentioning

confidence: 99%

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Unravelling the Mystery inside Cells by Using Single-Molecule Fluorescence Imaging

Zalejski,

Sun,

Sharma

2023

J. Imaging

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Live-cell imaging is a powerful technique to study the dynamics and mechanics of various biological molecules like proteins, organelles, DNA, and RNA. With the rapid evolution of optical microscopy, our understanding of how these molecules are implicated in the cells’ most critical physiological roles deepens. In this review, we focus on how spatiotemporal nanoscale live-cell imaging at the single molecule level allows for profound contributions towards new discoveries in life science. This review will start by summarizing how single-molecule tracking has been used to analyze membrane dynamics, receptor–ligand interactions, protein–protein interactions, inner- and extra-cellular transport, gene expression/transcription, and whole organelle tracking. We then move on to how current authors are trying to improve single-molecule tracking and overcome current limitations by offering new ways of labeling proteins of interest, multi-channel/color detection, improvements in time-lapse imaging, and new methods and programs to analyze the colocalization and movement of targets. We later discuss how single-molecule tracking can be a beneficial tool used for medical diagnosis. Finally, we wrap up with the limitations and future perspectives of single-molecule tracking and total internal reflection microscopy.

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Section: Inner and Extracellular Transportmentioning

confidence: 99%

Section: Inner and Extracellular Transportmentioning

confidence: 99%

Section: Inner and Extracellular Transportmentioning

confidence: 99%

Section: Inner and Extracellular Transportmentioning

confidence: 99%

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Unravelling the Mystery inside Cells by Using Single-Molecule Fluorescence Imaging

Zalejski,

Sun,

Sharma

2023

J. Imaging

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“…Among them, HaloTag technology offers a facile and rapid approach to install a bioorthogonal handle on POIs in a highly selective fashion. − HaloTag protein is an engineered haloalkane dehalogenase that covalently reacts with synthetic ligands comprising a chloroalkane linker with different functional modalities, such as fluorophores, ,− drugs, or resins . The covalent conjugation between HaloTag protein and the chloroalkane ligand is highly specific and irreversible in physiological conditions. − Therefore, it is desirable to utilize the HaloTag system to develop a protein-immobilized bioprobe for multiple applications.…”

Section: Introductionmentioning

confidence: 99%

Coumarin-Conjugated Macromolecular Probe for Sequential Stimuli-Mediated Activation

Jiang,

Li,

Ren

et al. 2023

Bioconjugate Chem.

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Protein bioconjugation has emerged as one of the most valuable tools for the development of protein-based biochemical assays. Here, we report a fluorescent macromolecular material, RF16_Halo, in which the coumarin derivative RF16 is specifically conjugated onto HaloTag protein to achieve a dual-stimuli-mediated fluorescence response. RF16 is first obtained by installing a H 2 O 2 -sensitive boron cage onto the C7 hydroxy moiety of the coumarin fluorophore with a HaloTag ligand attaching to the pH-labile 1,3dioxane moiety. Upon stimulation, RF16_Halo exhibits a sequential fluorescence response to H 2 O 2 /pH at both liquid and solid interfaces. The fluorescence of the RF16_Halo-based protein film increases linearly toward H 2 O 2 with a higher sensitivity when compared with that of RF16. Subsequently, the H 2 O 2 -cleaved RF16_Halo presents a pH-dependent fluorescence decrease under acidic conditions. Such a stimulus-responsive fluorescence "off−on−off" multimode enables RF16_Halo to be applied as a sequential logic circuit. In addition, we evaluate the fluorescence labeling ability of RF16 to intracellular IRE1_Halo protein and demonstrate that RF16 containing the HaloTag ligand could be precisely retained in cells to track IRE1_Halo protein. Hence, we provide a unique structural design strategy to construct a fluorescence dual-responsive macromolecular probe for information encryption and protein tracking in cells.

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HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing

2023

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Visualizing the structure and dynamics of biomolecules is critical to understand biological function, and requires methods to fluorescently label targets of interest in their cellular context. Self‐labelling proteins, which combine a genetically encoded tag with a small‐molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations of fluorescent proteins. Among them, the HaloTag protein is the most broadly used, showing fast specific labelling with a small, easy to functionalize and cell‐permeant ligand. Synthetic chemistry and protein engineering have provided a portfolio of powerful imaging tools exploiting HaloTag, along with general methods to optimize and adapt them to specific applications. Here, we provide an overview of fluorescent reporters based on the HaloTag protein for imaging and biosensing, highlighting engineering strategies and general applications.

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Quantum Dots Tracking Endocytosis and Transport of Proteins Displayed by Mammalian Cells

Cited by 10 publications

References 35 publications

Unravelling the Mystery inside Cells by Using Single-Molecule Fluorescence Imaging

Unravelling the Mystery inside Cells by Using Single-Molecule Fluorescence Imaging

Coumarin-Conjugated Macromolecular Probe for Sequential Stimuli-Mediated Activation

HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing

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