An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Summary

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Abstract

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Introduction

Cell-to-cell communications play critical roles in embryonic patterning in developmental processes. In vertebrate embryos, the metameric structures called somites are formed along the anterior-posterior body axis with a precise temporal accuracy under the control of a time-keeping clock, called the segmentation clock¹. During this process, a group of presomitic mesoderm (PSM) cells are periodically converted into somites in a synchronous manner. This process involves synchronized oscillatory gene expression and PSM cells that oscillate in phase form the same somites. The period of the oscillatory gene expression is around 2 to 3 h in mice and a....

Protocol

1. Generation of Stable Cell Lines by the Tol2 System

1. Transfect plasmid vectors (Figure 1A) of Tol2-based optogenetic modules together with the transposase (Tol2) expression vector (pCAGGS-mT2TP) into C2C12 cells. In all steps, culture cells with DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin at 37 °C (Table 1), in the presence of 5% CO₂, otherwise denoted.
   1. Count trypsinized cells with a cell-counter, and plate 5 x 10⁴ C2C12 cells per well in a 12-well plate one day before transfection.
   2. Co-transfect 0.375 μg of Tol2-....

Results

We adapted the LightOn system^11,12, which enables photo-induced gene expression in mammalian cells, to the study of genetic oscillators with 2- to 3-h periodicity. This system comprises of two parts: the photo-inducible transcriptional activator hGAVPO and a UAS-promoter cassette to drive transcription of arbitrary genes of interest. To accelerate the pulsatile kinetics of photo-induced gene expression, the polyA sequence in the U.......

Discussion

We showed a method to control gene expression dynamics with a periodicity of 2 to 3 h. This time-scale is much shorter than the ones in other conventional systems, including the Tet-On system and the original LightOn system. Key parameters to reach the ultradian time-scales are half-lives of photo-induced molecular products, mRNAs, and proteins. These kinetic parameters may depend on cell types and species. For tuning the kinetics, replacing Hes1 3'UTR sequences with others is a straight-forward way, because it does .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FACS	Becton, Dickinson and Company	FACSAriaII SORP
Camera	Andor	iKon M-934
Microscope	Olympus	IX-81 ZDC
PMT device	Churitsu eletric corp.	CL24B-LIC/B
Blue LED illuminator	OptoCode	LEDB-SBOXH
DMEM	Nacalai	08459-35
Penicillin-streptomycin	Nacalai	26253-84
Fetal bovine serum	Sigma	172012
KRYSTAL24 (black 24 well plate )	Hi-tech	303012
D-Luciferin Potassium Salt	Nacalai	20028-24
Light meter	LI-COR Biosciences	LI-250A
anti-HA-Peroxidase antibody	Roche	clone 3F10
anti-Actin-Peroxidase antibody	Wako	clone 2F3