Markus Covert, Doctoral Dissertation Advisor (AC)
Techniques for Studying Decoding of Single Cell Dynamics.
Frontiers in immunology
2019; 10: 755
Cells must be able to interpret signals they encounter and reliably generate an appropriate response. It has long been known that the dynamics of transcription factor and kinase activation can play a crucial role in selecting an individual cell's response. The study of cellular dynamics has expanded dramatically in the last few years, with dynamics being discovered in novel pathways, new insights being revealed about the importance of dynamics, and technological improvements increasing the throughput and capabilities of single cell measurements. In this review, we highlight the important developments in this field, with a focus on the methods used to make new discoveries. We also include a discussion on improvements in methods for engineering and measuring single cell dynamics and responses. Finally, we will briefly highlight some of the many challenges and avenues of research that are still open.
View details for PubMedID 31031756
Live-cell measurements of kinase activity in single cells using translocation reporters
2018; 13 (1): 155–69
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.
View details for PubMedID 29266096