Linda V. Chadwell

Identification of a Novel Drosophila Opsin Reveals Specific Patterning of the R7 and R8 Photoreceptor Cells

The function of the compound eye is dependent upon a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant files, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes.

Honeybee Blue- and Ultraviolet-Sensitive Opsins: Cloning, Heterologous Expression in Drosophila, and Physiological Characterization

The honeybee (Apis mellifera) visual system contains three classes of retinal photoreceptor cells that are maximally sensitive to light at 440 nm (blue), 350 nm (ultraviolet), and 540 nm (green). We performed a PCR-based screen to identify the genes encoding the Apis blue- and ultraviolet (UV)-sensitive opsins. We obtained cDNAs that encode proteins having a high degree of sequence and structural similarity to other invertebrate and vertebrate visual pigments. The Apis blue opsin cDNA encodes a protein of 377 amino acids that is most closely related to other invertebrate visual pigments that are thought to be blue-sensitive. The UV opsin cDNA encodes a protein of 371 amino acids that is most closely related to the UV-sensitive Drosophila Rh3 and Rh4 opsins. To test whether these novel Apis opsin genes encode functional visual pigments and to determine their spectral properties, we expressed them in the R1–6 photoreceptor cells of blindninaE mutant Drosophila, which lack the major opsin of the fly compound eye. We found that the expression of either the Apis blue- or UV-sensitive opsin in transgenic flies rescued the visual defect of ninaEmutants, indicating that both genes encode functional visual pigments. Spectral sensitivity measurements of these flies demonstrated that the blue and UV visual pigments are maximally sensitive to light at 439 and 353 nm, respectively. These maxima are in excellent agreement with those determined previously by single-cell recordings fromApis photoreceptor cells and provide definitive evidence that the genes described here encode visual pigments having blue and UV sensitivity.

Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain.

RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.

The Growth-Suppressive Function of the Polycomb Group Protein Polyhomeotic Is Mediated by Polymerization of Its Sterile Alpha Motif (SAM) Domain

Background: The mechanism by which Sterile Alpha Motifs (SAMs) self-associate and polymerize to control protein function is unknown. Results: SAM polymerization in Polyhomeotic, a Polycomb group protein, is controlled by an unstructured linker sequence in Polyhomeotic. Conclusion: Polyhomeotic growth suppressive function is enhanced by increasing SAM polymerization. Significance: Functions of other SAM domain-containing proteins could be manipulated through their unstructured linkers. Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.

Heterologous Expression of Limulus Rhodopsin

Invertebrates such as Drosophila or Limulus assemble their visual pigment into the specialized rhabdomeric membranes of photoreceptors where phototransduction occurs. We have investigated the biosynthesis of rhodopsin from the Limulus lateral eye with three cell culture expression systems: mammalian COS1 cells, insect Sf9 cells, and amphibian Xenopus oocytes. We extracted and affinity-purified epitope-tagged Limulus rhodopsin expressed from a cDNA or cRNA from these systems. We found that all three culture systems could efficiently synthesize the opsin polypeptide in quantities comparable with that found for bovine opsin. However, none of the systems expressed a protein that stably bound 11-cis-retinal. The protein expressed in COS1 and Sf9 cells appeared to be misfolded, improperly localized, and proteolytically degraded. Similarly, Xenopus oocytes injected with Limulus opsin cRNA did not evoke light-sensitive currents after incubation with 11-cis-retinal. However, injecting Xenopus oocytes with mRNA from Limulus lateral eyes yielded light-dependent conductance changes after incubation with 11-cis-retinal. Also, expressing Limulus opsin cDNA in the R1-R6 photoreceptors of transgenic Drosophila yielded a visual pigment that bound retinal, had normal spectral properties, and coupled to the endogenous phototransduction cascade. These results indicate that Limulus opsin may require one or more photoreceptor-specific proteins for correct folding and/or chromophore binding. This may be a general property of invertebrate opsins and may underlie some of the functional differences between invertebrate and vertebrate visual pigments.

A model system for analyzing somatic mutations in Drosophila melanogaster

Presently there are no good assays for comparing somatic mutation frequencies and spectra between different vertebrate and invertebrate organisms. Here we describe a new lacZ mutation reporter system in D. melanogaster, which complements existing systems in the mouse. The results obtained with the new model indicate two-to threefold higher frequencies of spontaneous mutations than in the mouse, with most of the mutations characterized as large genome rearrangements.

p53 Binding Prevents Phosphatase-mediated Inactivation of Diphosphorylated c-Jun N-terminal Kinase

Background: The p53 and c-Jun N-terminal kinase (JNK) pathways both act to initiate an apoptotic response to genotoxic stress. Results: The DNA binding domain of p53 binds to diphosphorylated JNK and prevents its dephosphorylation. Conclusion: p53 potentiates the level of JNK activity. Significance: Changes in p53 levels may coordinate the timing of an apoptotic response through regulation of JNK activity. c-Jun N-terminal kinase (JNK) is a serine/threonine phosphotransferase whose sustained activation in response to genotoxic stress promotes apoptosis. In Drosophila, the normally rapid JNK-dependent apoptotic response to genotoxic stress is significantly delayed in Dmp53 (Drosophila p53) mutants. Likewise, the extent of JNK activity after UV irradiation is dependent on p53 in murine embryonic fibroblasts with loss of p53 resulting in diminished JNK activity. Together, these results suggest that p53 potentiates the JNK-dependent response to genotoxic stress; however, the mechanism whereby p53 stimulates JNK activity remains undefined. Here, we demonstrate that both Drosophila and human p53 can directly stimulate JNK activity independently of p53-dependent gene transcription. Furthermore, we demonstrate that both the Drosophila and human p53 orthologs form a physical complex with diphosphorylated JNK (DPJNK) both in vivo and in vitro, suggesting that the interaction is evolutionarily conserved. Focusing on human p53, we demonstrate that the interaction maps to the DNA binding domain (hp53DBD). Intriguingly, binding of p53DBD alone to DPJNK prevented its inactivation by MAPK phosphatase (MKP)-5; however, JNK was still able to phosphorylate c-Jun while in a complex with the p53DBD. Apparent dissociation constants for the p53DBD·DPJNK (274 ± 14 nm) and MKP-5·DPJNK (55 ± 8 nm) complexes were established; however, binding of MKP-5 and p53 to JNK was not mutually exclusive. Together, these results suggest that stress-dependent increases in p53 levels potentiate JNK activation by preventing its rapid dephosphorylation by MKPs and that the simultaneous activation of p53 and JNK may constitute a “fail-safe” switch for the JNK-dependent apoptotic response.