Optogenetic techniques enable light-activated control of protein–protein interactions in the cell. This approach has now been used to alter membrane dynamics and induce cellular reorganization. See Letter p.111
The internal organization of cells is finely tuned, and their intracellular dynamics ever-changing. This is particularly apparent in plant, animal and fungal cells, which contain specialized membrane-bound vesicles and organelles in which particular reactions occur. Studies show1 that organelles must be correctly positioned to ensure proper cellular function, but it has so far been difficult or impossible to suddenly and reversibly alter the positions of vesicles and organelles in cells. In this issue, van Bergeijk et al.2 (page 111) describe a technique that provides biologists with tools that can be thought of as light-responsive tweezers, enabling precise and rapid control of organelle positioning and movement in cells.
Organization of the cell's membranes depends on the cytoskeleton, and in particular on cytoskeletal microtubule and microfilament structures, which are involved in intracellular transport. Cells use these cytoskeletal networks, along with dedicated motor proteins such as kinesins, dyneins and myosins3, to distribute and position different organelles in distinct subcellular regions4. For example, organelles called peroxysomes, which break down fatty-acid chains, are often distributed in the region around the nucleus. Early endosomes involved in cellular uptake of various cell-surface proteins are located at the cell periphery, whereas endosomes involved in molecule recycling are located at the cell centre. The mitochondria, which generate ATP molecules, are radially distributed along microtubules in the cytoplasm. Organelle positioning can be even more extreme in highly polarized cells such as neurons and migrating cells, in which specialized functions must be sustained in specific parts of the cell, for example signalling at synaptic junctions or polarized secretion.
Optogenetic tools can regulate rapid and reversible interactions between selected protein domains in specific areas of a cell or tissue, in response to laser illumination5,6,7. In the current study, van Bergeijk et al. used these tools to induce the rapid recruitment of molecular motors to specific membranes. Several optogenetic systems currently exist, and the authors mostly used the 'LOV–ePDZb1' set-up6. This system is based on a modified light–oxygen–voltage (LOV) protein domain. The modified LOV domain changes conformation following illumination with blue light, blocking or unmasking an amino-acid motif through which the modified LOV interacts with another protein domain, ePDZb1.
The authors fused the modified LOV domain to proteins located on the membranes of specific organelles — PEX3 for peroxysomes, Rab11 for recycling endosomes and TOM20 for mitochondria. They fused the ePDZb1 domain to a motor protein, such as the kinesin KIF1A, which transports proteins to the cell periphery, or the BICD2 dynein-recruitment domain, which pulls proteins towards the cell centre. The myosin Vb motor-protein domain was also used to move membranes in neurons called dendrites. Thus, blue light induced indirect tethering of an organelle of interest to a desired motor protein, leading to movement of the organelle around the cell (Fig. 1). Conversely, van Bergeijk and colleagues transiently immobilized organelles and vesicles by fusing ePDZb1 either to the syntaphilin protein, which stably binds to microtubules, or to myosin-Vb, which stably binds the cytoskeleton in non-polarized cells.
Next, the researchers showed that this technique could alter organelle position almost instantaneously, changing the cell's organization in a matter of minutes. The reversibility of the system meant that it was possible to use intermittent cycles of illumination to study both organelle movement and the restoration of normal positioning. Laser illumination could be targeted to spots as small as 250 nanometres wide, as well as to much larger areas, meaning that perturbation of organelle positioning, or sudden immobilization of transport vesicles, could be achieved in specific cellular subdomains.
This system has many applications, as van Bergeijk and co-workers demonstrated. For example, they used local illumination to recruit BICD2 or KIF1A to Rab11-associated recycling endosomes in neuronal cells. This respectively decreased or increased the quantity of recycling endosomes in the growth cone (a structure located at the tip of growing neuronal extensions, and in particular at the extremity of projections called axons). It has been shown8,9 that Rab11 endosomes are involved in axon growth, but the authors' analysis goes further, showing that the presence of recycling endosomes in the growth cone is directly correlated with axon extension.
The authors also used their system to test the 'tug-of-war' model for positioning organelles in neuronal protrusions called dendritic spines. This model states that a balance between stable tethering and motor-driven forces is essential to regulate the positioning of organelles. Using their system, the researchers confirmed this model and defined the precise role of particular motor proteins and anchoring factors in polarized organelle trafficking. In summary, van Bergeijk and colleagues have designed a powerful tool that, with its high spatio-temporal resolution, is a spectacular example of the ability of optogenetics to alter processes in real time in chosen subcellular areas.
Technological revolutions have often provided the tools with which to analyse cellular processes from a different point of view. Examples include the advent of RNA interference, super-resolution fluorescent imaging, and electron microscopy and its subsequent improvements, all of which were instrumental in helping cell biologists to reimagine the cell. The next challenge is not only to improve existing tools, but also to develop additional approaches to asking new questions in a comprehensive and integrated manner.
Optogenetic strategies, including van Bergeijk and co-workers' technique, will have a major part to play here. For example, the quantitative spatio-temporal data that these techniques can generate will be of great use to fields such as systems biology and theoretical modelling. The study of cell biology at the tissue or whole-organism level will similarly benefit from such an approach, because it will be possible to suddenly change the positions and dynamics of specific organelles in particular cell types, and then monitor induced defects. Gene editing now allows us to create modified versions of key cellular regulatory factors10. Combining optogenetic development with gene editing will enable us to control cell organization precisely and to question its role in cellular function. A bright future awaits cell biology.
Bornens, M. Nature Rev. Mol. Cell Biol. 9, 874–886 (2008).
van Bergeijk, P., Adrian, M., Hoogenraad, C. C. & Kapitein, L. C. Nature 518, 111–114 (2015).
Schliwa, M. & Woehlke, G. Nature 422, 759–765 (2003).
de Forges, H., Bouissou, A. & Perez, F. Int. J. Biochem. Cell Biol. 44, 266–274 (2012).
Gautier, A. et al. Nature Chem. Biol. 10, 533–541 (2014).
Strickland, D. et al. Nature Methods 9, 379–384 (2012).
Kennedy, M. J. et al. Nature Methods 7, 973–975 (2010).
Bhuin, T. & Roy, J. K. Cell Tissue Res. 335, 349–356 (2009).
Eva, R. et al. J. Neurosci. 30, 11654–11669 (2010).
Hsu, P. D., Lander, E. S. & Zhang, F. Cell 157, 1262–1278 (2014).