Curated Optogenetic Publication Database

Abstract: Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.

Abstract: Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

Abstract: Cellular functioning relies on active transport of organelles by molecular motors. To explore how intracellular organelle distributions affect cellular functions, several optogenetic approaches enable organelle repositioning through light-inducible recruitment of motors to specific organelles. Nonetheless, robust application of these methods in cellular populations without side effects has remained challenging. Here, we introduce an improved toolbox for optogenetic control of intracellular transport that optimizes cellular responsiveness and limits adverse effects. To improve dynamic range, we employed improved optogenetic heterodimerization modules and engineered a photosensitive kinesin-3, which is activated upon blue light-sensitive homodimerization. This opto-kinesin prevented motor activation before experimental onset, limited dark-state activation, and improved responsiveness. In addition, we adopted moss kinesin-14 for efficient retrograde transport with minimal adverse effects on endogenous transport. Using this optimized toolbox, we demonstrate robust reversible repositioning of (endogenously tagged) organelles within cellular populations. More robust control over organelle motility will aid in dissecting spatial cell biology and transport-related diseases.

Abstract: Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays, in which surface-immobilized motor proteins drive microtubule propulsion, are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, bio-computation and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ~5-fold enrichment within 6 seconds upon illumination. Subsequently, proteins are released with a half-life of 13 seconds when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of non-uniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.

Abstract: Cells depend on the proper positioning of their organelles, suggesting that active manipulation of organelle positions can be used to explore spatial cell biology and to restore cellular defects caused by organelle misplacement. Recently, blue-light dependent recruitment of specific motors to selected organelles has been shown to alter organelle motility and positioning, but these approaches lack rapid and active reversibility. The light-dependent interaction of phytochrome B with its interacting factors has been shown to function as a photoswitch, dimerizing under red light and dissociating under far-red light. Here we engineer phytochrome domains into photoswitches for intracellular transport that enable the reversible interaction between organelles and motor proteins. Using patterned illumination and live-cell imaging, we demonstrate that this system provides unprecedented spatiotemporal control. We also demonstrate that it can be used in combination with a blue-light dependent system to independently control the positioning of two different organelles. Precise optogenetic control of organelle motility and positioning will provide a better understanding of and control over the spatial biology of cells.

Abstract: To establish and maintain their complex morphology and function, neurons and other polarized cells exploit cytoskeletal motor proteins to distribute cargoes to specific compartments. Recent studies in cultured cells have used inducible motor protein recruitment to explore how different motors contribute to polarized transport and to control the subcellular positioning of organelles. Such approaches also seem promising avenues for studying motor activity and organelle positioning within more complex cellular assemblies, but their applicability to multicellular in vivo systems has so far remained unexplored. Here, we report the development of an optogenetic organelle transport strategy in the in vivo model system Caenorhabditis elegans. We demonstrate that movement and pausing of various organelles can be achieved by recruiting the proper cytoskeletal motor protein with light. In neurons, we find that kinesin and dynein exclusively target the axon and dendrite, respectively, revealing the basic principles for polarized transport. In vivo control of motor attachment and organelle distributions will be widely useful in exploring the mechanisms that govern the dynamic morphogenesis of cells and tissues, within the context of a developing animal.

Abstract: Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.