A Chemical Approach for Profiling Intracellular AKT Signaling Dynamics from Single Cells

Shao, Shiqun; Li, Zhonghan; Cheng, Hong; Wang, Siwen; Perkins, Nicole G; Sarkar, Priyanka; Wei, Wei; Xue, Min

doi:10.1021/jacs.8b08931

Cited by 10 publications

(14 citation statements)

References 24 publications

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“…Multiplexed molecular profiling in single cells in situ holds great promise to reveal cell-to-cell variations, cell-microenvironment interactions and tissue architecture at the single cell level, which are masked by population-based measurements [1,2]. Various methods [3][4][5][6][7][8][9][10] have been developed for multiplexed single-cell analysis. An increasing number of studies have been focused on proteins, for their central roles in biological processes.…”

Section: Introductionmentioning

confidence: 99%

Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Liao

Pham

Mastroeni

et al. 2020

Cells

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The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

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Section: Introductionmentioning

confidence: 99%

Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Liao

Pham

Mastroeni

et al. 2020

Cells

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“…Although these fluorescent peptide sensors allow versatile experimental designs and applications, their use in living cells requires efficient cellular uptake. This limitation can be addressed by introducing cell‐penetrating peptides or through liposome‐based intracellular delivery (Damayanti, Parker, & Irudayaraj, 2013; Kurzawa, Pellerano, & Morris, 2010; Shao et al., 2018).…”

Section: Commentarymentioning

confidence: 99%

AktAR and Akt‐STOPS: Genetically Encodable Molecular Tools to Visualize and Perturb Akt Kinase Activity at Different Subcellular Locations in Living Cells

2022

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The serine/threonine protein kinase Akt integrates diverse upstream inputs to regulate cell survival, growth, metabolism, migration, and differentiation. Mounting evidence suggests that Akt activity is differentially regulated depending on its subcellular location, which can include the plasma membrane, endomembrane, and nuclear compartment. This spatial control of Akt activity is critical for achieving signaling specificity and proper physiological functions, and deregulation of compartment-specific Akt signaling is implicated in various diseases, including cancer and diabetes. Understanding the spatial coordination of the signaling network centered around this key kinase and the underlying regulatory mechanisms requires precise tracking of Akt activity at distinct subcellular compartments within its native biological contexts. To address this challenge, new molecular tools are being developed, enabling us to directly interrogate the spatiotemporal regulation of Akt in living cells. These include, for instance, the newly developed genetically encodable fluorescent-protein-based Akt kinase activity reporter (AktAR2), which serves as a substrate surrogate of Akt kinase and translates Akt-specific phosphorylation into a quantifiable change in Förster resonance energy transfer (FRET). In addition, we developed the Akt substrate tandem occupancy peptide sponge (Akt-STOPS), which allows biochemical perturbation of subcellular Akt activity. Both molecular tools can be readily targeted to distinct subcellular localizations. Here, we describe a workflow to study Akt kinase activity at different subcellular locations in living cells. We provide a protocol for using genetically targeted AktAR2 and Akt-STOPS, along with fluorescence imaging in living NIH3T3 cells, to visualize and perturb, respectively, the activity of endogenous Akt kinase at different subcellular compartments. We further describe a protocol for using chemically inducible dimerization (CID) to control the plasma membranespecific inhibition of Akt activity in real time. Lastly, we describe a protocol for maintaining NIH3T3 cells in culture, a cell line known to exhibit robust Akt activity. In all, this approach enables interrogation of spatiotemporal regulation and functions of Akt, as well as the intricate signaling networks in which it is embedded, at specific subcellular locations.

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“…described a novel chemical method to analyze the intracellular activity of AKT. [ 98 ] AKT plays a crucial role in cell survival, being related to oncogenesis and tumor maintenance. The authors employed a FRET strategy to couple a pair of cyclic peptides targeting two different regions of the protein: (a) the phosphorylated site of Ser474 and (b) a distal epitope of AKT (Figure 3C).…”

Section: Biological Applications Of Fluorescent Cyclic Peptide Probesmentioning

confidence: 99%

“…[97] More recently, Xue et al described a novel chemical method to analyze the intracellular activity of AKT. [98] AKT plays a Figure 3C). The resulting construct displayed a fluorescent signal contingent upon AKT phosphorylation and was used to profile the intracellular dynamics of AKT signaling.…”

Section: Fluorescent Cyclic Peptides Targeting Cell Receptorsmentioning

confidence: 99%

Fluorescent cyclic peptides for cell imaging

2020

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Fluorescent cyclic peptides are excellent chemical scaffolds to build optical agents for molecular imaging. In addition to their favorable physicochemical properties, they can be modified with multiple organic fluorophores and generate useful probes for biological assays targeting specific proteins, namely receptors or enzymes. In this article, we review recent advances in the synthetic approaches for the preparation of fluorescent cyclic peptides as well as some of their applications in biological imaging, from in vitro live‐cell imaging studies to in vivo characterization of preclinical models.

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A Chemical Approach for Profiling Intracellular AKT Signaling Dynamics from Single Cells

Cited by 10 publications

References 24 publications

Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

AktAR and Akt‐STOPS: Genetically Encodable Molecular Tools to Visualize and Perturb Akt Kinase Activity at Different Subcellular Locations in Living Cells

Fluorescent cyclic peptides for cell imaging

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