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Experimental Approaches in Delineating mTOR Signaling
Jiayi Qian1,2, Siyuan Su1,2, Pengda Liu1,2
1Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Abstract:
The mTOR signaling controls essential biological functions including proliferation, growth, metabolism, autophagy, ageing, and others. Hyperactivation of mTOR signaling leads to a plethora of human disorders; thus, mTOR is an attractive drug target. The discovery of mTOR signaling started from isolation of rapamycin in 1975 and cloning of TOR genes in 1993. In the past 27 years, numerous research groups have contributed significantly to advancing our understanding of mTOR signaling and mTOR biology. Notably, a variety of experimental approaches have been employed in these studies to identify key mTOR pathway members that shape up the mTOR signaling we know today. Technique development drives mTOR research, while canonical biochemical and yeast genetics lay the foundation for mTOR studies. Here in this review, we summarize major experimental approaches used in the past in delineating mTOR signaling, including biochemical immunoprecipitation approaches, genetic approaches, immunofluorescence microscopic approaches, hypothesis-driven studies, protein sequence or motif search driven approaches, and bioinformatic approaches. We hope that revisiting these distinct types of experimental approaches will provide a blueprint for major techniques driving mTOR research. More importantly, we hope that thinking and reasonings behind these experimental designs will inspire future mTOR research as well as studies of other protein kinases beyond mTOR.
Insights
The mechanistic target of rapamycin (mTOR) pathway regulates key cellular functions and is implicated in human diseases. This review highlights experimental techniques that have advanced our understanding of mTOR signaling and biology.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- The mechanistic target of rapamycin (mTOR) signaling pathway is crucial for regulating fundamental cellular processes such as proliferation, growth, metabolism, and autophagy.
- Dysregulation of mTOR signaling is associated with numerous human disorders, making it a significant drug target.
- The discovery and elucidation of mTOR signaling have evolved since the isolation of rapamycin in 1975 and the cloning of TOR genes in 1993.
Purpose of the Study:
- To review and summarize the major experimental approaches that have been instrumental in delineating mTOR signaling pathways.
- To provide a blueprint of key techniques that have driven mTOR research over the past decades.
- To inspire future research in mTOR biology and other protein kinase studies by examining the reasoning behind experimental designs.
Main Methods:
- Biochemical immunoprecipitation techniques
- Genetic approaches, including yeast genetics
- Immunofluorescence microscopy
- Hypothesis-driven studies
- Protein sequence and motif analysis
- Bioinformatic approaches
Main Results:
- A comprehensive overview of diverse experimental methodologies employed in mTOR research is presented.
- The review details how various techniques have identified key components and elucidated the intricacies of the mTOR pathway.
- It emphasizes the synergistic role of technique development and foundational studies in advancing mTOR knowledge.
Conclusions:
- Revisiting historical experimental approaches offers valuable insights into the progression of mTOR research.
- The methodologies discussed serve as a guide for current and future investigations into mTOR signaling.
- Understanding the experimental strategies behind mTOR discoveries can foster innovation in the study of protein kinases.
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