Tohoku University and Nippon Medical School develop technology to manipulate proteins in living cells using light

A research group led by Associate Professor Toshiyuki Kowada and Professor Shin Mizukami of the Institute of Multidisciplinary Research for Advanced Materials at Tohoku University, and Professor Hayashi Yamamoto of the Institute of Advanced Medical Sciences at Nippon Medical School has announced that they have developed a technique for rapid and quantitative manipulation of protein localization in living cells using molecules that change color and structure in response to light (photochromic compounds). This technique allows for the reversible manipulation of protein localization under violet and green light. They confirmed that the technique could be used to reproduce mitophagy (selective degradation of damaged mitochondria). The technique is expected to be used in the clarification of the mechanisms of diseases, including neurodegenerative diseases such as Parkinson's disease. These results were published in the June 18 issue of the international journal Nature Chemical Biology.

The processes of life are controlled by the interactions of intracellular molecules such as proteins, and the manipulation of these interactions contributes to understanding of the mechanisms of these molecules. Artificial methods to control intracellular proteins include the optogenetic approach (optogenetics) using photoresponsive proteins and the CID (chemically induced dimerization) approach using low-molecular-weight compounds. Although they control dimerization and dissociation, both approaches face challenges, such as the need for continuous light irradiation in the photogenetic approach and the large protein size in the CID approach. Moreover, the recently developed caged compound-based CID system has the problem that dimerization and dissociation cannot be repeated.

In this study, the research group developed a "photochromic CID method" that allows for repetitive regulation of protein dimerization and dissociation with light at two different wavelengths. The method uses two different protein tags (eDHFR and HaloTag) and a photochromic dimerizer (pcDH) to dimerize and dissociate the protein tags. When pcDH is added to cells expressing HaloTag (34 kDa) intracellularly, pcDH and HaloTag quickly form a covalent bond. pcDH exists in a trans form (E form) before light exposure and when it is exposed to green light. In this form, pcDH has a low affinity to eDHFR, and HaloTag and eDHFR do not form a dimer. Meanwhile, irradiation with violet light converts pcDH to the cis form (Z form), which has an increased binding affinity to eDHFR, causing dimerization of eDHFR and HaloTag. They confirmed that the photochromic CID system could be used for reversible and repetitive control of dimerization/dissociation with violet and green light.

To demonstrate the effectiveness of this method, eDHFR fused with a near-infrared fluorescent protein was expressed in the cytoplasm of human-derived cells, and HaloTag fused with an orange fluorescent protein was expressed in the plasma membrane, mitochondrial outer membrane, and endoplasmic reticulum membrane. Moreover, pcDH was added to the cell culture medium to label cells using HaloTag. When the cells were exposed to violet light and observed under a fluorescence microscope, the eDHFR fusion protein in the cytoplasm rapidly migrated to the plasma membrane, mitochondrial outer membrane, and endoplasmic reticulum membrane where HaloTag was expressed. When the cells were then irradiated with green light, the eDHFR fusion protein diffusing back into the cytoplasm was observed. When the cells were exposed to blue light (445 nm), which has a wavelength between green light (555 nm) and violet light (405 nm), the localization was only at an intermediate level, confirming that quantitative manipulation is possible using combinations of violet and green light with various intensity ratios. The reaction durations required for dimerization by violet light and dissociation by green light were 0.78 s and less than 0.3 s (image acquisition limit), respectively, confirming the feasibility of fast control.

To further validate the effectiveness of the method for medical and physiological research, it was used to reproduce mitophagy by photoinduction. In the early stages of mitophagy, PINK1 kinase accumulates on damaged mitochondria, and the Parkin protein eventually migrates to the outer membrane before later stages of mitophagy occur. However, the amount of PINK1 accumulation and duration of engagement required for mitophagy were unknown.

Therefore, the research group performed an experiment in which cells expressing a fusion protein of eDHFR and the catalytic domain of PINK1 in the cytoplasm and HaloTag on the mitochondrial outer membrane were treated with pcDH and exposed to violet light followed by green light for a certain duration (1, 3, and 10 minutes). Thus, mitophagy was successfully reproduced. Furthermore, there was a marked difference in the degrees of migration of the PINK1 and Parkin proteins between 3 and 10 minutes, indicating that mitophagy requires an engagement duration of 10 minutes or longer. Mitophagy is robust against interfering factors such as transient environmental changes.

Yamamoto said, "Currently, I focus my research on the direction of what happens when the state of proteins in cells is changed and work with Dr. Mizukami on specific applications."

Mizukami said, "We expect this technology to become a powerful tool for understanding various life phenomena and pathogenic mechanisms at the molecular level. Since various parameters can be freely controlled by using light with two different wavelengths, we will be able to simultaneously induce the quantitative and qualitative changes to see the responses of the cells."

Journal Information
Publication: Nature Chemical Biology
Title: Quantitative control of subcellular protein localization with a photochromic dimerizer
DOI: 10.1038/s41589-024-01654-w