Alternating Cellular Functions by Optogenetic Control of Organelles

Abstract

Organelles play essential roles in cellular homeostasis and various cellular functions in eukaryotic cells. The current experimental strategy to modulate organelle functions is limited due to the dynamic nature and subcellular distribution of organelles in live cells. Optogenetics utilizes photoactivatable proteins to enable dynamic control of molecular activities through visible light. This modality has been rapidly expanded for the dynamic regulation of organelle functions. This chapter describes a method by optical modulation of the mitochondria-lysosome contacts (MLCs). Detailed procedures of transfection, optogenetic MLCs, mitochondrial morphology, and functional analysis are described. Optogenetic control of organelles in live cells offers an innovative paradigm for cell engineering and synthetic biology.

Keywords: Mitochondria-lysosome contacts; Mitochondrial fission; Optogenetics; Regulation of cellular functions; Subcellular manipulation.

Fig. 1

The optogenetics inducing MLCs. (a) Schematic representation of MLCs under blue light exposure. In this system, two light-sensitive proteins, CRY2 and CIB, were strategically tethered to lysosomes and mitochondria, respectively. This anchoring was achieved using specific organelle-targeting transmembrane domains known as LAMP (lysosome-associated membrane protein) and TOM20 (translocase of the outer mitochondrial membrane 20), respectively. Upon illumination with blue light, the light-sensitive proteins, CRY2 and CIB, interacted with each other to form CRY2–CIB complexes. To visualize the expression and localization of these complexes, GFP (Green Fluorescent Protein) and mCherry were utilized as markers, providing fluorescence signals corresponding to the system’s expression in the cells. The SIM images of HeLa cells expressing LAMP–mCherry–CRY2 and TOM20–CIB–GFP without (b) or with blue light exposure for 20 min (c).

Fig. 2

Schematic representation of mitochondria divided into four categories: round, when 1.0 ≤ L/W < 1.5; intermediate, when 1.5 ≤ L/W < 2.0; tubular, when 2.0 ≤ L/W < 5.0; hyperfused, when 5.0 ≤ L/W. “L” stands for length; “W” stands for width; “L/W” stands for dividing the length by the width.

Fig. 3

The real-time OCR (oxygen consumption rate) curves of cells were measured after various treatments. Different points in the OCR curve represent specific stages of the experiment. Here is a breakdown of the OCR curve interpretation for each treatment: basal respiration, OCR before the addition of oligomycin A; maximal mitochondrial respiration capacity, OCR after the injection of FCCP; non-mitochondrial respiration, OCR after the injection of rotenone and antimycin A.