Curated Optogenetic Publication Database

Abstract: Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the relation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here, we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with different optical dimerizers and molecular motors. This method presents a mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.

Abstract: Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the correlation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with other optical dimerizers and molecular motors. This method presents a novel mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.

Abstract: Phosphoinositides are important signaling molecules involved in the regulation of vesicular trafficking. It has been implicated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in insulin-regulated GLUT4 translocation in adipocytes. However, it remains unclear where and how PI(4,5)P2 regulates discrete steps of GLUT4 vesicle translocation in adipocytes, especially on the exocytic arm of regulation. Here, we employed optogenetic tools to acutely control the PI(4,5)P2 metabolism in living cells. By combination of TIRFM imaging, we were able to monitor the temporal-spatial-dependent PI(4,5)P2 regulation on discrete steps of GLUT4 translocation in adipocytes. We found that the plasma membrane localized PI(4,5)P2 is crucial for proper insulin signaling propagation and for insulin-stimulated GLUT4 vesicle translocation in 3T3-L1 adipocytes. Global depletion of PI(4,5)P2 on the cell surface blunted insulin-stimulated Akt phosphorylation and abolished insulin effects in promotion of the docking and fusion of GLUT4 vesicle with the plasma membrane. Furthermore, by development of a novel optogenetic module to selectively modulate PI(4,5)P2 levels on the GLUT4 vesicle docking site, we identified an important regulatory role of PI(4,5)P2 in controlling of vesicle docking process. Local depletion of PI(4,5)P2 at the vesicle docking site promoted GLUT4 vesicle undocking, diminished insulin-stimulated GLUT4 vesicle docking and fusion, but without perturbation of insulin signaling propagation in adipocytes. Our results provide strong evidence that cell surface PI(4,5)P2 plays two distinct functions on regulation of the exocytic trafficking of GLUT4 in adipocytes. PI(4,5)P2 not only regulates the proper activation of insulin signaling in general but also controls GLUT4 vesicle docking process at the vesicle-membrane contact sites.

Abstract: Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.