Optogenetic Control of Molecular Motors and Organelle Distributions in Cells

SummaryIntracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival, and apoptosis. Here, we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon exposure to blue light, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven toward the cell periphery upon light-induced recruitment of kinesin, or toward the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible, and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells

U0126 Protects Cells against Oxidative Stress Independent of Its Function as a MEK Inhibitor

U0126 is a potent and selective inhibitor of MEK1 and MEK2 kinases. It has been widely used as an inhibitor for the Ras/Raf/MEK/ERK signaling pathway with over 5000 references on the NCBI PubMed database. In particular, U0126 has been used in a number of studies to show that inhibition of the Raf/MEK/ERK pathway protects neuronal cells against oxidative stress. Here, we report that U0126 can function as an antioxidant that protects PC12 cells against a number of different oxidative-stress inducers. This protective effect of U0126 is independent of its function as a MEK inhibitor, as several other MEK inhibitors failed to show similar protective effects. U0126 reduces reactive oxygen species (ROS) in cells. We further demonstrate that U0126 is a direct ROS scavenger in vitro, and the oxidation products of U0126 exhibit fluorescence. Our finding that U0126 is a strong antioxidant signals caution for its future usage as a MEK inhibitor and for interpreting some previous results

Optical activation of TrkA signaling

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 (CRY2) and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports the survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling. During embryonic development, nerve growth factor (NGF) plays a critical role in supporting neuronal differentiation, survival, and plasticity 1. In adults, NGF supports neural maintenance and repair, and deficits in NGF signaling have been implicated in several neurodegenerative disorders including Alzheimer's and Parkinson's diseases 2-4. Aberrantly elevated activity of NGF is also involved in chronic inflammatory and. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity

The activation of opto-Raf or opto-AKT protects undifferentiated and differentiated PC12 cells against hydrogen peroxide.

<p>(<b>A</b>) Transfected PC-12 cells with CIB1-GFP-CAAX only, opto-AKT, or opto-Raf were first illuminated with 0.2 mW/cm² blue light for 1 hour before being subjected to 200 μM of hydrogen peroxide. The blue light (480 nm) was kept on throughout the duration of the experiment. Cell death was probed <i>via</i> PI staining. (<b>B</b>) Undifferentiated PC12 cells transfected with opto-Raf or opto-AKT demonstrated less cell death after they were exposed to 200 μM of hydrogen peroxide for 2 hours. The red channel was thresholded (cut-off at 30,000 arbitrary units) to represent PI-stained dead cells. Scale bar = 50 μm. (<b>C</b>) Hydrogen peroxide treatment to undifferentiated PC12 cells at 200 μM for 2 hours showed that the activation of opto-AKT and opto-Raf exerted a prominent protective effect compared to CIB1-GFP-CAAX control. (<b>D</b>) Hydrogen peroxide treatment to differentiated PC12 cells at 200 μM for 2 hours showed that the activation of opto-AKT and opto-Raf exerted a prominent protective effect compared to CIB1-GFP-CAAX control. In (<b>C</b>) and (<b>D</b>), the dark controls showed comparable cell death to CIB1-GFP-CAAX singly transfected controls. For all the results, each set of data comprises of 3 sets of experiments with 1000–3000 cells each. Data is represented as mean +/- standard deviation.</p

The Timing of Raf/ERK and AKT Activation in Protecting PC12 Cells against Oxidative Stress

<div><p>Acute brain injuries such as ischemic stroke or traumatic brain injury often cause massive neural death and irreversible brain damage with grave consequences. Previous studies have established that a key participant in the events leading to neural death is the excessive production of reactive oxygen species. Protecting neuronal cells by activating their endogenous defense mechanisms is an attractive treatment strategy for acute brain injuries. In this work, we investigate how the precise timing of the Raf/ERK and the AKT pathway activation affects their protective effects against oxidative stress. For this purpose, we employed optogenetic systems that use light to precisely and reversibly activate either the Raf/ERK or the AKT pathway. We find that preconditioning activation of the Raf/ERK or the AKT pathway immediately before oxidant exposure provides significant protection to cells. Notably, a 15-minute transient activation of the Raf/ERK pathway is able to protect PC12 cells against oxidant strike that is applied 12 hours later, while the transient activation of the AKT pathway fails to protect PC12 cells in such a scenario. On the other hand, if the pathways are activated after the oxidative insult, i.e. postconditioning, the AKT pathway conveys greater protective effect than the Raf/ERK pathway. We find that postconditioning AKT activation has an optimal delay period of 2 hours. When the AKT pathway is activated 30min after the oxidative insult, it exhibits very little protective effect. Therefore, the precise timing of the pathway activation is crucial in determining its protective effect against oxidative injury. The optogenetic platform, with its precise temporal control and its ability to activate specific pathways, is ideal for the mechanistic dissection of intracellular pathways in protection against oxidative stress.</p></div

Optogenetic activation of opto-Raf and opto-AKT protects PC12 cells against phototoxicity.

<p>(<b>A</b>) The design scheme of opto-Raf. The CIB1 domain is anchored to the plasma membrane via a CAAX motif. Upon light stimulation, CIB1-CRY2 interaction should recruit cytoplasmic CRY2-mCherry-Raf1 to the plasma membrane and subsequently activate the downstream ERK pathway. In the dark, CIB1-CRY2 should spontaneously dissociate and return Raf1 to the cytoplasm to inactivate the Raf/ERK pathway. (<b>B</b>) CIB1-GFP-CAAX was clearly located at the plasma membrane as indicated by the green fluorescence image. Blue light rapidly recruited cytosolic CRY2-mCherry-Raf1 to the plasma membrane within 15 seconds (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153487#pone.0153487.s010" target="_blank">S1 Movie</a>)</b>. Scale bar = 20 μm. (<b>C</b>) Western blot analysis shows phosphor-ERK upon 10, 30 and 60 minutes of blue light activation of opto-Raf. No band was observed when the culture was kept in the dark. (<b>D</b>) The design scheme of opto-AKT, similar to that of opto-Raf. Blue light should activate opto-AKT and the pathway. (<b>E</b>) Blue light illumination induced rapid recruitment of CRY2-mCherry-AKT from cytosol to the plasma membrane within 20 seconds (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153487#pone.0153487.s011" target="_blank">S2 Movie</a>)</b>. Scale bar = 20 μm. (<b>F</b>) Western blot analysis of phosphorylated AKT confirms light-induced activation of CRY2-mCherry-AKT at 160kDa. The endogenous AKT band at 60kDa is not affected by the blue light stimulation. (<b>G</b>) Cells transfected with CIB1-GFP-CAAX show phototoxicity at high blue light intensities. Comparable cell death could be seen when the intensities were at 0 and 0.2 mW/cm², while significant cell death was observed when the cells were subjected to 1.5 mW/cm² of blue light. Scale bar = 100 μm. (<b>H-I</b>) Quantification of cell death rates for cells transfected with CIB1-GFP-CAAX only (<b>H</b>) or CIB1-GFP-CAAX and CRY2-mCherry (<b>I</b>) that were exposed to blue light at 0, 0.2, 0.8 and 1.5 mW/cm² for 24 hours. Untransfected cells in the same culture were used as controls. Each set of data comprises of 5 sets of experiments with 2000–5000 cells each. Data is represented as mean +/- standard deviation.</p

Optical Activation of TrkA Signaling

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer’s disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor’s intracellular kinase domain. As TrkA signaling is highly dynamic, mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein <i>Arabidopsis</i> cryptochrome 2 and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling