Showing 1 - 10 of 10 results
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.
Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase.
Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle.
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.
Optogenetic Control of Endoplasmic Reticulum-Mitochondria Tethering.
The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.
Optogenetic control of the Dab1 signaling pathway.
The Reelin-Dab1 signaling pathway regulates development of the mammalian brain, including neuron migrations in various brain regions, as well as learning and memory in adults. Extracellular Reelin binds to cell surface receptors and activates phosphorylation of the intracellular Dab1 protein. Dab1 is required for most effects of Reelin, but Dab1-independent pathways may contribute. Here we developed a single-component, photoactivatable Dab1 (opto-Dab1) by using the blue light-sensitive dimerization/oligomerization property of A. thaliana Cryptochrome 2 (Cry2). Opto-Dab1 can activate downstream signals rapidly, locally, and reversibly upon blue light illumination. The high spatiotemporal resolution of the opto-Dab1 probe also allows us to control membrane protrusion, retraction and ruffling by local illumination in both COS7 cells and in primary neurons. This shows that Dab1 activation is sufficient to orient cell movement in the absence of other signals. Opto-Dab1 may be useful to study the biological functions of the Reelin-Dab1 signaling pathway both in vitro and in vivo.
Light-induced Notch activity controls neurogenic and gliogenic potential of neural progenitors.
Oscillations in Notch signaling are essential for reserving neural progenitors for cellular diversity in developing brains. Thus, steady and prolonged overactivation of Notch signaling is not suitable for generating neurons. To acquire greater temporal control of Notch activity and mimic endogenous oscillating signals, here we adopted a light-inducible transgene system to induce active form of Notch NICD in neural progenitors. Alternating Notch activity saved more progenitors that are prone to produce neurons creating larger number of mixed clones with neurons and progenitors in vitro, compared to groups with no light or continuous light stimulus. Furthermore, more upper layer neurons and astrocytes arose upon intermittent Notch activity, indicating that dynamic Notch activity maintains neural progeny and fine-tune neuron-glia diversity.
Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor.
Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.
Optogenetic Inhibitor of the Transcription Factor CREB.
Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events.
Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides.
Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently inhibited kinases in the light but were sterically blocked from kinase interaction in the dark. Photoactivatable inhibitors for cyclic-AMP dependent kinase (PKA) and myosin light chain kinase (MLCK) are described, together with studies that shed light on proper positioning of the peptides in the LOV domain. These inhibitors altered endogenous signaling in living cells and produced light-dependent changes in cell morphodynamics.
Optical control of mammalian endogenous transcription and epigenetic states.
The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.