Showing 1 - 15 of 15 results
Optogenetic dissection of RET signaling reveals robust activation of ERK and enhanced filopodia-like protrusions of regenerating axons.
RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.
Rapid and reversible optogenetic silencing of synaptic transmission by clustering of synaptic vesicles.
Acutely silencing specific neurons informs about their functional roles in circuits and behavior. Existing optogenetic silencers include ion pumps, channels, metabotropic receptors, and tools that damage the neurotransmitter release machinery. While the former hyperpolarize the cell, alter ionic gradients or cellular biochemistry, the latter allow only slow recovery, requiring de novo synthesis. Thus, tools combining fast activation and reversibility are needed. Here, we use light-evoked homo-oligomerization of cryptochrome CRY2 to silence synaptic transmission, by clustering synaptic vesicles (SVs). We benchmark this tool, optoSynC, in Caenorhabditis elegans, zebrafish, and murine hippocampal neurons. optoSynC clusters SVs, observable by electron microscopy. Locomotion silencing occurs with tauon ~7.2 s and recovers with tauoff ~6.5 min after light-off. optoSynC can inhibit exocytosis for several hours, at very low light intensities, does not affect ion currents, biochemistry or synaptic proteins, and may further allow manipulating different SV pools and the transfer of SVs between them.
Single-component near-infrared optogenetic systems for gene transcription regulation.
Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.
Nano-positioning and tubuline conformation determine transport of mitochondria along microtubules.
Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules which fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers, similar to GTP-tubulin, that are more straight and rigid. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin step processivity on microtubules consisting of elongated dimers underlies increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin controls mitochondrial motility and as such locally regulates the distribution of mitochondria along axons.
Actin waves transport RanGTP to the neurite tip to regulate non-centrosomal microtubules in neurons.
Microtubule (MT) is the most abundant cytoskeleton in neurons and controls multiple facets of their development. While the MT-organizing center (MTOC) in mitotic cells is typically located at the centrosome, MTOC in neurons switches to non-centrosomal sites. A handful of cellular components have been shown to promote non-centrosomal MT (ncMT) formation in neurons, yet the regulation mechanism remains unknown. Here we demonstrate that the small GTPase Ran is a key regulator of ncMTs in neurons. Using an optogenetic tool that enables light-induced local production of RanGTP, we demonstrate that RanGTP promotes ncMT plus-end growth along the neurite. Additionally, we discovered that actin waves drive the anterograde transport of RanGTP. Pharmacological disruption of actin waves abolishes the enrichment of RanGTP and reduces growing ncMT plus-ends at the neurite tip. These observations identify a novel regulation mechanism of ncMTs and pinpoint an indirect connection between the actin and MT cytoskeletons in neurons.
A time-dependent role for the transcription factor CREB in neuronal allocation to an engram underlying a fear memory revealed using a novel in vivo optogenetic tool to modulate CREB function.
The internal representation of an experience is thought to be encoded by long-lasting physical changes to the brain ("engrams") (Josselyn et al. Nat Rev Neurosci 16:521-534, 2015; Josselyn et al. J Neurosci 37:4647-4657, 2017; Schacter. 2001; Tonegawa et al. Neuron 87:918-931, 2015). Previously, we (Han et al. Science 316:457-460, 2007) and others (Zhou et al. Nat Neurosci 12:1438-1443, 2009) showed within the lateral amygdala (LA), a region critical for auditory conditioned fear, eligible neurons compete against one other for allocation to an engram. Neurons with relatively higher function of the transcription factor CREB were more likely to be allocated to the engram. In these studies, though, CREB function was artificially increased for several days before training. Precisely when increased CREB function is important for allocation remains an unanswered question. Here, we took advantage of a novel optogenetic tool (opto-DN-CREB) (Ali et al. Chem Biol 22:1531-1539, 2015) to gain spatial and temporal control of CREB function in freely behaving mice. We found increasing CREB function in a small, random population of LA principal neurons in the minutes-hours, but not 24 h, before training was sufficient to enhance memory, likely because these neurons were preferentially allocated to the underlying engram. However, similarly increasing CREB activity in a small population of random LA neurons immediately after training disrupted subsequent memory retrieval, likely by disrupting the precise spatial and temporal patterns of offline post-training neuronal activity and/or function required for consolidation. These findings reveal the importance of the timing of CREB activity in regulating allocation and subsequent memory retrieval, and further, highlight the potential of optogenetic approaches to control protein function with temporal specificity in behaving animals.
Focusing light inside live tissue using reversibly switchable bacterial phytochrome as a genetically encoded photochromic guide star.
Focusing light deep by engineering wavefronts toward guide stars inside scattering media has potential biomedical applications in imaging, manipulation, stimulation, and therapy. However, the lack of endogenous guide stars in biological tissue hinders its translations to in vivo applications. Here, we use a reversibly switchable bacterial phytochrome protein as a genetically encoded photochromic guide star (GePGS) in living tissue to tag photons at targeted locations, achieving light focusing inside the tissue by wavefront shaping. As bacterial phytochrome-based GePGS absorbs light differently upon far-red and near-infrared illumination, a large dynamic absorption contrast can be created to tag photons inside tissue. By modulating the GePGS at a distinctive frequency, we suppressed the competition between GePGS and tissue motions and formed tight foci inside mouse tumors in vivo and acute mouse brain tissue, thus improving light delivery efficiency and specificity. Spectral multiplexing of GePGS proteins with different colors is an attractive possibility.
Potassium channel-based optogenetic silencing.
Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.
Light Control of the Tet Gene Expression System in Mammalian Cells.
Gene expression and its network structure are dynamically altered in multicellular systems during morphological, functional, and pathological changes. To precisely analyze the functional roles of dynamic gene expression changes, tools that manipulate gene expression at fine spatiotemporal resolution are needed. The tetracycline (Tet)-controlled gene expression system is a reliable drug-inducible method, and it is used widely in many mammalian cultured cells and model organisms. Here, we develop a photoactivatable (PA)-Tet-OFF/ON system for precise temporal control of gene expression at single-cell resolution. By integrating the cryptochrome 2-cryptochrome-interacting basic helix-loop-helix 1 (Cry2-CIB1) light-inducible binding switch, expression of the gene of interest is tightly regulated under the control of light illumination and drug application in our PA-Tet-OFF/ON system. This system has a large dynamic range of downstream gene expression and rapid activation/deactivation kinetics. We also demonstrate the optogenetic regulation of exogenous gene expression in vivo, such as in developing and adult mouse brains.
Light-activated protein interaction with high spatial subcellular confinement.
Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications.
Discovery of long-range inhibitory signaling to ensure single axon formation.
A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. Here, the authors offer in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer's disease.
Optogenetic control of endogenous Ca(2+) channels in vivo.
Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.
Photo-activatable Cre recombinase regulates gene expression in vivo.
Techniques allowing precise spatial and temporal control of gene expression in the brain are needed. Herein we describe optogenetic approaches using a photo-activatable Cre recombinase (PA-Cre) to stably modify gene expression in the mouse brain. Blue light illumination for 12 hours via optical fibers activated PA-Cre in the hippocampus, a deep brain structure. Two-photon illumination through a thinned skull window for 100 minutes activated PA-Cre within a sub-millimeter region of cortex. Light activation of PA-Cre may allow permanent gene modification with improved spatiotemporal precision compared to standard methods.
Light generation of intracellular Ca(2+) signals by a genetically encoded protein BACCS.
Ca(2+) signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca(2+) channel switch (BACCS), as an optogenetic tool for generating Ca(2+) signals. BACCS opens Ca(2+)-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca(2+) concentration more rapidly, such that Ca(2+) elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca(2+). Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca(2+) signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.
Optogenetic control of PIP3: PIP3 is sufficient to induce the actin-based active part of growth cones and is regulated via endocytosis.
Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is highly regulated in a spatiotemporal manner and plays multiple roles in individual cells. However, the local dynamics and primary functions of PIP3 in developing neurons remain unclear because of a lack of techniques for manipulating PIP3 spatiotemporally. We addressed this issue by combining optogenetic control and observation of endogenous PIP3 signaling. Endogenous PIP3 was abundant in actin-rich structures such as growth cones and "waves", and PIP3-rich plasma membranes moved actively within growth cones. To study the role of PIP3 in developing neurons, we developed a PI3K photoswitch that can induce production of PIP3 at specific locations upon blue light exposure. We succeeded in producing PIP3 locally in mouse hippocampal neurons. Local PIP3 elevation at neurite tips did not induce neurite elongation, but it was sufficient to induce the formation of filopodia and lamellipodia. Interestingly, ectopic PIP3 elevation alone activated membranes to form actin-based structures whose behavior was similar to that of growth-cone-like "waves". We also found that endocytosis regulates effective PIP3 concentration at plasma membranes. These results revealed the local dynamics and primary functions of PIP3, providing fundamental information about PIP3 signaling in neurons.