Cyclophilins: a new family of proteins involved in intracellular folding.

Proteins of the cyclophilin family display two intriguing properties. On the one hand, they are the intracellular receptors for the immunosuppressive drug cyclosporin A (CsA); on the other hand, they function in vitro as enzymes that catalyse slow steps in protein folding. A dissection of the role of CsA in mediating immunosuppression, together with recent studies on the biology of cyclophilins in the absence of this ligand, is providing fundamental insight into the cellular function of this protein family.

A Drosophila mechanosensory transduction channel.

Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, we analyzed Drosophila melanogaster mechanoreceptive mutants for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolished mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs.

A G protein-coupled receptor phosphatase required for rhodopsin function.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors are phosphorylated by kinases that mediate agonist-dependent receptor deactivation. Although many receptor kinases have been isolated, the corresponding phosphatases, necessary for restoring the ground state of the receptor, have not been identified. Drosophila RDGC (retinal degeneration C) is a phosphatase required for rhodopsin dephosphorylation in vivo. Loss of RDGC caused severe defects in the termination of the light response as well as extensive light-dependent retinal degeneration. These phenotypes resulted from the hyperphosphorylation of rhodopsin because expression of a truncated rhodopsin lacking the phosphorylation sites restored normal photoreceptor function. These results suggest the existence of a family of receptor phosphatases involved in the regulation of G protein-coupled signaling cascades.

Arrestin function in inactivation of G protein-coupled receptor rhodopsin in vivo.

Arrestins have been implicated in the regulation of many G protein-coupled receptor signaling cascades. Mutations in two Drosophila photoreceptor-specific arrestin genes, arrestin 1 and arrestin 2, were generated. Analysis of the light response in these mutants shows that the Arr1 and Arr2 proteins are mediators of rhodopsin inactivation and are essential for the termination of the phototransduction cascade in vivo. The saturation of arrestin function by an excess of activated rhodopsin is responsible for a continuously activated state of the photoreceptors known as the prolonged depolarized afterpotential. In the absence of arrestins, photoreceptors undergo light-dependent retinal degeneration as a result of the continued activity of the phototransduction cascade. These results demonstrate the fundamental requirement for members of the arrestin protein family in the regulation of G protein-coupled receptors and signaling cascades in vivo.

Photoreceptor deactivation and retinal degeneration mediated by a photoreceptor-specific protein kinase C.

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in the regulation of a variety of signaling cascades. One member of this family, eye-PKC, is expressed exclusively in the Drosophila visual system. The inaC (inactivation-no-afterpotential C) locus was shown to be the structural gene for eye-PKC. Analysis of the light response from inaC mutants showed that this kinase is required for the deactivation and rapid desensitization of the visual cascade. Light adaptation was also defective in inaC mutant flies. In flies carrying the retinal degeneration mutation rdgB, absence of eye-PKC suppressed photoreceptor cell degeneration. These results indicate that eye-PKC functions in the light-dependent regulation of the phototransduction cascade in Drosophila.

Spectral tuning of rhodopsin and metarhodopsin in vivo.

Color vision is dependent upon the expression of spectrally distinct forms of rhodopsin in different photoreceptor cells. To identify the structural features of rhodopsin that regulate spectral sensitivity and absorption in vivo, we have constructed a series of chimeric Drosophila rhodopsin molecules, derived from a blue- and a violet-sensitive rhodopsin, and used P element-mediated germline transformation to generate transgenic flies that express the modified pigments in the R1-R6 photoreceptor cells of the compound eye. Our analysis of these animals indicates that multiple regions of the opsin protein are involved in regulating rhodopsin spectral sensitivity and that the native and photoactivated forms of rhodopsin can be tuned independently of each other. These results demonstrate the feasibility of designing receptor molecules with specifically modified activated states.

InsP3 receptor is essential for growth and differentiation but not for vision in Drosophila.

Phospholipase C (PLC) is the focal point for two major signal transduction pathways: one initiated by G protein-coupled receptors and the other by tyrosine kinase receptors. Active PLC hydrolyzes phosphatidylinositol bisphosphate (PIP2) into the two second messengers inositol 1,4,5-trisphosphate (InsP3) and diacyl glycerol (DAG). DAG activates protein kinase C, and InsP3 mobilizes calcium from intracellular stores via the InsP3 receptor. Changes in [Ca2+]i regulate the function of a wide range of target proteins, including ion channels, kinases, phosphatases, proteases, and transcription factors (Berridge, 1993). In the mouse, there are three InsP3R genes, and type 1 InsP3R mutants display ataxia and epileptic seizures (Matsumoto et al., 1996). In Drosophila, only one InsP3 receptor (InsP3R) gene is known, and it is expressed ubiquitously throughout development (Hasan and Rosbash, 1992; Yoshikawa et al., 1992; Raghu and Hasan, 1995). Here, we characterize Drosophila InsP3R mutants and demonstrate that the InsP3R is essential for embryonic and larval development. Interestingly, maternal InsP3R mRNA is sufficient for progression through the embryonic stages, but larval organs show asynchronous and defective cell divisions, and imaginal discs arrest early and fail to differentiate. We also generated adult mosaic animals and demonstrate that phototransduction, a model PLC pathway thought to require InsP3R, does not require InsP3R for signaling.

A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila.

Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. Here, we uncover a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism. This process involves the formation of membrane complexes of phosphorylated, activated rhodopsin and its inhibitory protein arrestin, and subsequent clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment. Together, these data define the proapoptotic molecules in Drosophila photoreceptors and indicate a novel signaling pathway for light-activated rhodopsin molecules in control of photoreceptor viability.

Synaptic defects and compensatory regulation of inositol metabolism in inositol polyphosphate 1-phosphatase mutants.

Phosphoinositides function as important second messengers in a wide range of cellular processes. Inositol polyphosphate 1-phosphatase (IPP) is an enzyme essential for the hydrolysis of the 1-phosphate from either Ins(1,4)P2 or Ins(1,3,4)P3. This enzyme is Li+ sensitive, and is one of the proposed targets of Li+ therapy in manic-depressive illness. Drosophila ipp mutants accumulate IP2 in their system and are incapable of metabolizing exogenous Ins(1,4)P2. Notably, ipp mutants demonstrate compensatory upregulation of an alternative branch in the inositol-phosphate metabolism tree, thus providing a means of ensuring continued availability of inositol. We demonstrate that ipp mutants have a defect in synaptic transmission resulting from a dramatic increase in the probability of vesicle release at larval neuromuscular junctions. We also show that Li+ phenocopies this effect in wild-type synapses. Together, these results support a role for phosphoinositides in synaptic vesicle function in vivo and mechanistically question the "lithium hypothesis."

Cytosolic calcium transients: spatial localization and role in Drosophila photoreceptor cell function.

Drosophila phototransduction is a phosphoinositide-mediated and Ca(2+)-regulated signaling cascade ideal for the dissection of feedback regulatory mechanisms. To study the roles of intracellular Ca2+ ([Ca2+]i) in this process, we developed novel techniques for the measurement of [Ca2+]i in intact photoreceptors. We genetically engineered flies that express a UV-specific rhodopsin in place of the normal rhodopsin, so that long wavelength light can be used to image [Ca2+]i changes while minimally exciting the photoreceptor cells. We show that activation with UV generates [Ca2+]i increases that are spatially localized to the rhabdomeres and that are entirely dependent on the influx of extracellular Ca2+. Application of intracellular Ca2+ chelators of varying affinities demonstrates that the Ca2+ influx initially generates a large-amplitude transient that is crucial for negative regulation. Internal Ca2+ stores were revealed by discharging them with thapsigargin. But, in contrast to proposals that IP3-sensitive stores mediate phototransduction, thapsigargin does not mimic or acutely interfere with photoexcitation. Finally, we identify a photoreceptor-specific PKC as essential for normal kinetics of [Ca2+]i recovery.

Specificity in signaling pathways: assembly into multimolecular signaling complexes.

A critical issue in the field of signal transduction is how signaling molecules are organized into different pathways within the same cell. The importance of assembling signaling molecules into architecturally defined complexes is emerging as an essential cellular strategy to ensure specificity and selectivity of signaling. Scaffold proteins function as the pillars of these transduction complexes, bringing together a diversity of signaling components into defined ultramicrodomains of signaling.

The molecular genetics of invertebrate phototransduction.

Phototransduction, the primary event in the processing of visual stimuli, is the conversion of light energy into a change in the ionic permeabilities of the photoreceptor cell membrane. In both vertebrates and invertebrates, this process is carried out through a specialized form of a G-protein-coupled receptor cascade. The mechanisms that mediate visual excitation in the vertebrate photoreceptor have been physiologically and biochemically well characterized, and many aspects of this system have served as prototypes for other transduction cascades. However, there are still many unresolved issues in vertebrate phototransduction. The study of phototransduction in Drosophila offers a unique opportunity to make use of powerful molecular genetic techniques to identify novel transduction molecules, and then to examine the function of these molecules in vivo, in their normal cellular environment. The results of a combination of molecular, genetic, physiological and biochemical studies are beginning to produce a clearer model for the complex mechanisms involved in invertebrate visual transduction.

Phototransduction in Drosophila: a paradigm for the genetic dissection of sensory transduction cascades.

A combination of molecular, genetic and physiological studies is providing fundamental insight into the function and regulation of the phototransduction cascade. The availability of Drosophila mutants with defects in visual physiology allows for an in vivo dissection of this complex sensory signal transduction process.

Isolation of a novel visual-system-specific arrestin: an in vivo substrate for light-dependent phosphorylation.

Absorption of a photon of light by rhodopsin triggers mechanisms responsible for excitation as well as regulation of the phototransduction cascade. Arrestins are a family of proteins that appear to be responsible for terminating the active state of G-protein-coupled receptors. One of the major substrates of light-dependent phosphorylation in the visual cascade of Drosophila was purified and partially sequenced. The complete primary structure of the protein was determined by isolating the corresponding gene, which revealed it to be a new isoform of arrestin, Arr2. Arr2 is 401 residues in length, and shares 47% sequence identity with the Drosophila Arr1 protein and 42% with human arrestin. We show that the two Drosophila arrestin genes are differentially regulated, and that Arr2 is a specific substrate for a calcium-dependent protein kinase. This is the first demonstration of in vivo regulation of arrestins in a transduction cascade, and provides a new level of modulation in the function of G-protein-coupled receptors.

The organization of INAD-signaling complexes by a multivalent PDZ domain protein in Drosophila photoreceptor cells ensures sensitivity and speed of signaling.

Phototransduction in Drosophila has emerged as an attractive model system for studying the organization of signaling cascades in vivo. In photoreceptor neurons, the multivalent PDZ protein INAD serves as a scaffold to assemble different components of the phototransduction pathway, including the effector PLC, the light-activated ion channel TRP, and a protein kinase C involved in deactivation of the light response. INAD is required for organizing and maintaining signaling complexes in the rhabdomeres of photoreceptors. This macromolecular organization endows photoreceptors with many of their signaling properties, including high sensitivity, fast activation and deactivation kinetics, and exquisite feedback regulation by small localized changes in [Ca2+]i. Assembly of transduction components into signaling complexes is also an important cellular strategy for ensuring specificity of signaling while minimizing unwanted cross-talk. In this report, we review INAD's role as a signal transduction scaffold and its role in the assembly and localization of photoreceptor complexes.

The biology of vision of Drosophila.

Phototransduction systems in vertebrates and invertebrates share a great deal of similarity in overall strategy but differ significantly in the underlying molecular machinery. Both are rhodopsin-based G protein-coupled signaling cascades displaying exquisite sensitivity and broad dynamic range. However, light activation of vertebrate photoreceptors leads to activation of a cGMP-phosphodiesterase effector and the generation of a hyperpolarizing response. In contrast, activation of invertebrate photoreceptors, like Drosophila, leads to stimulation of phospholipase C and the generation of a depolarizing receptor potential. The comparative study of these two systems of phototransduction offers the opportunity to understand how similar biological problems may be solved by different molecular mechanisms of signal transduction. The study of this process in Drosophila, a system ideally suited to genetic and molecular manipulation, allows us to dissect the function and regulation of such a complex signaling cascade in its normal cellular environment. In this manuscript I review some of our recent findings and the strategies used to dissect this process.

Assembly of the Drosophila phototransduction cascade into a signalling complex shapes elementary responses.

The subcellular compartmentalization of signalling molecules helps to ensure the selective activation of different signal-transduction cascades within a single cell. Although there are many examples of compartmentalized signalling molecules, there are few examples of entire signalling cascades being organized as distinct signalling complexes. In Drosophila photoreceptors, the InaD protein, which consists of five PDZ domains, functions as a multivalent adaptor that brings together several components of the phototransduction cascade into a macromolecular complex. Here we study single-photon responses in several photoreceptor mutant backgrounds, and show that the InaD macromolecular complex is the unit of signalling that underlies elementary responses. We show that the localized activity of this signalling unit promotes reliable single-photon responses as well as rapid activation and feedback regulation. Finally, we use genetic and electrophysiological tools to illustrate how the assembly of signalling molecules into a transduction complex limits signal amplification in vivo.