Toward quantitative modeling of intracellular signal transduction: the next future of cell biology

Signal transduction is a mechanism by which the cell responds to the extracellular input such as growth factor and finally outputs the phenotype to adapt to the constantly changing environment. The signal transduction is comprised of a diverse array of chemical reactions that take place within the cell. Several decades of extensive investigation have unveiled the complicated network of the signal transduction pathway including feedback/feedforward regulations. However, it is still unclear how the dynamics of the signal transduction system affects cellular function such as cell cycle progression, differentiation, and apoptosis.

To address this issue, we aim to clarify the mechanisms underlying the cell fate determination by the following three technical approaches: "Visualization", "quantification" and "manipulation" of the signal transduction system in living cells by fluorescence imaging. We employ mammalian cultured cells, fission yeast, nematodes.

 

1. Visualization

We use the biosensor based on the principle of fluorescence resonance energy transfer (FRET) and Kinase Translocation Reporter (KTR) to visualize the activity of the signal transduction in a living cell. With these approaches, we would like to reveal the mechanism of how continuous and analogous signaling systems generate irreversible and digital phenomena such as cell cycle progression and apoptosis.

Topics: Dynamics of ERK and Akt and the role in cell cycle progression; Dynamics of p38 and JNK and cell death; Simultaneous imaging of nerve activity and behavior in C. elegans.

Ref: Miura H, Cell Rep, 2018; Aoki K, Dev Cell, 2017; Maryu G, CSF, 2016; Aoki K, Mol Cell, 2013; Komatsu N, MBoC, 2011

2. Quantification

We extract information of signaling activities and reaction parameters of signal transduction with fluorescence imaging and image analysis. We are developing technologies to quantify the number of molecules and protein-protein interactions (dissociation constants) of endogenous protein molecules by combining CRISPR / Cas9 gene editing technology with fluorescence cross-correlation spectroscopy. This technique enables to measure the reaction parameters of the signal transduction system at the single cell level without antibodies.

Topics: Quantification of intracellular concentrations and dissociation constants of cell cycle-related factors in fission yeast and cultured cells, Information extraction from time-lapse images.

Ref: Komatsubara A, JBC, 2019; Kondo Y, PLoS Comp, 2018; Sadaie W, MCB, 2014; Fujita Y, FEBS J, 2014; Aoki K, PNAS, 2011

3. Manipulation

We develop perturbation methods of intracellular signal transduction by small compounds and light. We are focusing on CRY2-CIB system and PhyB-PIF system, which respond to blue light and red/near-infrared light, respectively. The causal relationship between that signal transduction pathway and cell function can be more directly verified by these manipulation techniques.  

Topics: Improvement of the genetically encoded PhyB-PIF system; Light manipulation of C. elegans behavior using PhyB-PIF system

Ref: Uda Y, PNAS, 2017; Aoki K, Dev Cell, 2017; Aoki K, Mol Cell, 2013