Engineering adenylate cyclases regulated by near-infrared window light.

Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.

Phosphatidylinositol-3-kinase-atypical protein kinase C signaling is required for Wnt attraction and anterior-posterior axon guidance.

Wnt proteins are conserved axon guidance cues that control growth cone navigation. However, the intracellular signaling mechanisms that mediate growth cone turning in response to Wnts are unknown. We previously showed that Wnt-Frizzled signaling directs spinal cord commissural axons to turn anteriorly after midline crossing through an attractive mechanism. Here we show that atypical protein kinase C (aPKC), is required for Wnt-mediated attraction of commissural axons and proper anterior-posterior (A-P) pathfinding. A PKCzeta pseudosubstrate, a specific blocker of aPKC activity, and expression of a kinase-defective PKCzeta mutant in commissural neurons resulted in A-P randomization in "open-book" explants. Upstream of PKCzeta, heterotrimeric G-proteins and phosphatidylinositol-3-kinases (PI3Ks), are also required for A-P guidance, because pertussis toxin, wortmannin, and expression of a p110gamma kinase-defective construct all resulted in A-P randomization. Overexpression of p110gamma, the catalytic subunit of PI3Kgamma, caused precocious anterior turning of commissural axons before midline crossing in open-book explants and caused dissociated precrossing commissural axons, which are normally insensitive to Wnt attraction, to turn toward Wnt4-expressing cells. Therefore, we propose that atypical PKC signaling is required for Wnt-mediated A-P axon guidance and that PI3K can act as a switch to activate Wnt responsiveness during midline crossing.

Morphogens as conserved axon guidance cues.

Morphogen family proteins are now widely appreciated as axon guidance cues. Because their roles as morphogens are highly conserved across phylogeny, their functional conservation in axon guidance is now being rigorously examined. Recent studies suggest that morphogens are important in shaping topographic projections in chick and Drosophila visual systems, a process that occurs even later in development.

Ryk-mediated Wnt repulsion regulates posterior-directed growth of corticospinal tract.

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Here we show that Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repelled CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity was blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocked the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons.

Bacterial polarity.

Many recent studies have revealed exquisite subcellular localization of proteins, DNA, and other molecules within bacterial cells, giving credence to the concept of prokaryotic anatomy. Common sites for localized components are the poles of rod-shaped cells, which are dynamically modified in composition and function in order to control cellular physiology. An impressively diverse array of mechanisms underlies bacterial polarity, including oscillatory systems, phospho-signaling pathways, the sensing of membrane curvature, and the integration of cell cycle regulators with polar maturation.

Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling.

Commissural neurons in the mammalian dorsal spinal cord send axons ventrally toward the floor plate, where they cross the midline and turn anteriorly toward the brain; a gradient of chemoattractant(s) inside the spinal cord controls this turning. In rodents, several Wnt proteins stimulate the extension of commissural axons after midline crossing (postcrossing). We found that Wnt4 messenger RNA is expressed in a decreasing anterior-to-posterior gradient in the floor plate, and that a directed source of Wnt4 protein attracted postcrossing commissural axons. Commissural axons in mice lacking the Wnt receptor Frizzled3 displayed anterior-posterior guidance defects after midline crossing. Thus, Wnt-Frizzled signaling guides commissural axons along the anterior-posterior axis of the spinal cord.