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Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy.

ABSTRACT: Genetically-encoded tags are useful tools for multicolor and multi-scale cellular imaging. Versatile Interacting Peptide (VIP) tags, such as VIPER, are new genetically-encoded tags that can be used in various imaging applications. VIP tags consist of a coiled-coil heterodimer, with one peptide serving as the genetic tag and the other ("probe peptide") delivering a reporter compatible with imaging. Heterodimer formation is rapid and specific, allowing proteins to be selectively labeled for live-cell and fixed-cell imaging. In this Bio-Protocol, we include a detailed guide for implementing the VIPER technology for imaging receptors on live cells and intracellular targets in fixed cells. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER (Doh et al., 2019a and 2019b).

SUBMITTER: Doh JK

PROVIDER: S-EPMC7360171 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy.

Doh Julia K JK Enns Caroline A CA Beatty Kimberly E KE

Bio-protocol 20191101 21

Genetically-encoded tags are useful tools for multicolor and multi-scale cellular imaging. Versatile Interacting Peptide (VIP) tags, such as VIPER, are new genetically-encoded tags that can be used in various imaging applications. VIP tags consist of a coiled-coil heterodimer, with one peptide serving as the genetic tag and the other ("probe peptide") delivering a reporter compatible with imaging. Heterodimer formation is rapid and specific, allowing proteins to be selectively labeled for live-c ...[more]

PMID: 32665966

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Project description:While the in vivo efficacy of Sn-2 phosphatidylcholine prodrugs incorporated into targeted, non-pegylated lipid-encapsulated nanoparticles was demonstrated in prior preclinical studies, the microscopic details of cell prodrug internalization and trafficking events are unknown. Classic fluorescence microscopy, fluorescence lifetime imaging microscopy, and single-molecule super-resolution microscopy were used to investigate the cellular handling of doxorubicin-prodrug and AlexaFluor™-488-prodrug. Sn-2 phosphatidylcholine prodrugs delivered by hemifusion of nanoparticle and cell phospholipid membranes functioned as phosphatidylcholine mimics, circumventing the challenges of endosome sequestration and release. Phosphatidylcholine prodrugs in the outer cell membrane leaflet translocated to the inner membrane leaflet by ATP-dependent and ATP-independent mechanisms and distributed broadly within the cytosolic membranes over the next 12 h. A portion of the phosphatidylcholine prodrug populated vesicle membranes trafficked to the perinuclear Golgi/ER region, where the drug was enzymatically liberated and activated. Native doxorubicin entered the cells, passed rapidly to the nucleus, and bound to dsDNA, whereas DOX was first enzymatically liberated from DOX-prodrug within the cytosol, particularly in the perinuclear region, before binding nuclear dsDNA. Much of DOX-prodrug was initially retained within intracellular membranes. In vitro anti-proliferation effectiveness of the two drug delivery approaches was equivalent at 48 h, suggesting that residual intracellular DOX-prodrug may constitute a slow-release drug reservoir that enhances effectiveness. We have demonstrated that Sn-2 phosphatidylcholine prodrugs function as phosphatidylcholine mimics following reported pathways of phosphatidylcholine distribution and metabolism. Drug complexed to the Sn-2 fatty acid is enzymatically liberated and reactivated over many hours, which may enhance efficacy overtime.

Dataset Information

Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy.

Publications

Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy.

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