Live-cell measurements of kinase activity in single cells using translocation reporters

Abstract

Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter's distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, we present a protocol to execute and analyze live-cell microscopy experiments using KTRs. We describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. We provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in ∼10 d.

Caliban: Accurate cell tracking and lineage construction in live-cell imaging experiments with deep learning

“…To solve this assignment problem, one constructs a cost function for a possible pairing across frames, which is traditionally based on each object's location and appearance features (brightness, size, etc.) 28 . The guiding intuition is that objects are unlikely to move large distances or have distinct changes in appearance from frame-to-frame if the frame rate is sufficiently high.…”

“…Integration of deep learning into live-cell imaging analysis pipelines achieve performance boosts by combining the improved segmentation accuracy of deep learning with conventional object tracking algorithms 13,[25][26][27] . These algorithms include linear programming 28 and the Viterbi algorithm 29 ; both have seen extensive use on live-cell imaging data. While useful, these object tracking algorithms have limitations.…”

Optical activation of TrkB receptors

“…We employed the nucleus export of kinase translocation reporter (KTR) to report the activation of MAPK/ERK pathway. The KTR technology was developed to report phosphorylationregulated nucleocytoplasmic translocation [28], where the relative intensity of cytoplasmic versus nuclear fluorescence is used as a proxy for kinase activity in living cells [29]. On the other hand, subcellular translocation of the pleckstrin homology domain of Akt (AktPH) was used to indicate the activation of PI3K/Akt pathway, where AktPH-GFP can be recruited to the cell membrane upon the phosphorylation of Akt [30].…”

EllipTrack: A Global-Local Cell-Tracking Pipeline for 2D Fluorescence Time-Lapse Microscopy

“…In a typical 2D fluorescence time-lapse microscopy experiment, cells are labeled with fluorescent markers for the signals of interest and imaged periodically under a microscope to generate a movie. A computational tool called a "cell tracker" is then applied to the movie to track cells over time and extract signals from each cell (Kudo et al, 2017). Due to its ability to visualize molecular activities in living cells in real time, time-lapse microscopy has been used to study a wide range of biological questions.…”