Nansi Jo Colley

The cyclophilin homolog ninaA is required in the secretory pathway

In Drosophila, the major rhodopsin Rh1 is synthesized in endoplasmic reticulum (ER)-bound ribosomes of the R1-R6 photoreceptor cells and is then transported to the rhabdomeres where it functions in phototransduction. Mutations in the cyclophilin homolog ninaA lead to a 90% reduction in Rh1 opsin. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We now show that mutations in the ninaA gene severely inhibit opsin transport from the ER, leading to dramatic accumulations of ER cisternae in the photoreceptor cells. These results demonstrate that ninaA functions in the ER. Interestingly, ninaA and Rh1 also colocalize to secretory vesicles, suggesting that Rh1 may require ninaA as it travels through the distal compartments of the secretory pathway. These results are discussed in relation to the possible role of cyclophilins in protein folding and intracellular protein trafficking.

Normal Phototransduction in Drosophila Photoreceptors Lacking an InsP3 Receptor Gene

The Drosophila light-sensitive channels TRP and TRPL are prototypical members of an ion channel family responsible for a variety of receptor-mediated Ca(2+) influx phenomena, including store-operated calcium influx. While phospholipase Cbeta is essential, downstream events leading to TRP and TRPL activation remain unclear. We investigated the role of the InsP(3) receptor (InsP(3)R) by generating mosaic eyes homozygous for a deficiency of the only known InsP(3)R gene in Drosophila. Absence of gene product was confirmed by RT-PCR, Western analysis, and immunocytochemistry. Mutant photoreceptors underwent late onset retinal degeneration; however, whole-cell recordings from young flies demonstrated that phototransduction was unaffected, quantum bumps, macroscopic responses in the presence and absence of external Ca(2+), light adaptation, and Ca(2+) release from internal stores all being normal. Using the specific TRP channel blocker La(3+) we demonstrated that both TRP and TRPL channel functions were unaffected. These results indicate that InsP(3)R-mediated store depletion does not underlie TRP and TRPL activation in Drosophila photoreceptors.

Two-step process for photoreceptor formation in Drosophila.

The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1–R8). But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulators and sends its projection to different layers of the optic lobes. Here, we show that the spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1–R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process. First, during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain. Second, terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties.

Calnexin Is Essential for Rhodopsin Maturation, Ca2+ Regulation, and Photoreceptor Cell Survival

In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. Here, we demonstrate a multifunctional role for calnexin as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration that is enhanced by light, suggesting that calnexins function as a Ca2+ buffer is important for photoreceptor cell survival. Our results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration.

Evidence for light perception in a bioluminescent organ

Here we show that bioluminescent organs of the squid Euprymna scolopes possess the molecular, biochemical, and physiological capability for light detection. Transcriptome analyses revealed expression of genes encoding key visual transduction proteins in light-organ tissues, including the same isoform of opsin that occurs in the retina. Electroretinograms demonstrated that the organ responds physiologically to light, and immunocytochemistry experiments localized multiple proteins of visual transduction cascades to tissues housing light-producing bacterial symbionts. These data provide evidence that the light-organ tissues harboring the symbionts serve as extraocular photoreceptors, with the potential to perceive directly the bioluminescence produced by their bacterial partners.

Calnexin Deficiency Leads to Dysmyelination

Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx−/−) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx−/− mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.

Regulation of phototransduction responsiveness and retinal degeneration by a phospholipase D–generated signaling lipid

Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)–triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld null flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.

Role of Asparagine-linked Oligosaccharides in Rhodopsin Maturation and Association with Its Molecular Chaperone, NinaA

Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites forN-linked glycosylation (N20I and N196I). We show that Asn20 is the sole site for glycosylation. The Rh1N20I protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1N20I; therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1N20I/NinaA interaction. We further show that Rh1N20I interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.

Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway.

The Gos28 SNARE protein mediates intra-Golgi transport of rhodopsin and is required for photoreceptor survival.

Background: The Golgi SNARE, Gos28, plays important roles in vesicular transport during protein trafficking. Results: Mutations in gos28 lead to defective rhodopsin trafficking and retinal degeneration, which is rescued by human Gos28 expressed in transgenic flies. Conclusion: Drosophila Gos28 functions as a t-SNARE in medial- to trans-Golgi transport of rhodopsin during its biosynthesis. Significance: Gos28 represents a novel locus in neurodegeneration. SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28s SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.