Role of Spastin in Apical Domain Control along the Rhabdomere Elongation in Drosophila Photoreceptor

Background

Mutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development.

Methodology/Principal Findings

The spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin''s gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity.

Conclusions/Significance

These results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis.     

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.     

An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

Background

G protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration.

Methodology/Principal Findings

First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo.

Conclusions/Significance

These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors.     

The Green-absorbing Drosophila Rh6 Visual Pigment Contains a Blue-shifting Amino Acid Substitution That Is Conserved in Vertebrates

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10–17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class.Organisms use color vision for survival behaviors such as foraging, mating, and predator avoidance (1–3). Color vision in invertebrates ranges from trichromatic systems capable of detecting UV, blue, and green (e.g. bees and flies) to the highly complex mantis shrimps (stomatopods) having 12 spectrally distinct classes of photoreceptor cells (4). Despite the diversity of invertebrate color vision systems and the large collection of naturally occurring visual pigments, important questions remain concerning the molecular mechanisms that regulate color sensitivity.In both vertebrates and invertebrates, the visual pigment rhodopsin consists of a chromophore (e.g. 11-cis retinal) covalently bound to an opsin apoprotein via a protonated Schiff base. Upon light absorption, the chromophore isomerizes from cis to all-trans, inducing conformational changes in the opsin that produce activated metarhodopsin. Specific interactions between the retinal chromophore and residues in the opsin tune the λ_max of the chromophore. Studies have shown that Glu-113 (bovine position) serves as the retinylidene Schiff base counter-ion in vertebrate visual pigments (5–7). Removing the negative charge of the counter-ion from the binding pocket deprotonates the chromophore and yields a UV-absorbing pigment (5–7). Using sequence alignments, phylogenetic analysis, analysis of the bovine rhodopsin crystal structure (PDB² entry 1U19), and functional experiments, a large number of amino acids involved in the spectral tuning of vertebrate visual pigments have been identified (8).In contrast, the counter-ion for invertebrate rhodopsin remains unknown, and only one spectrally relevant residue has been identified: an amino acid substitution in Drosophila pigments responsible for UV versus visible sensitivity (9). Interestingly, this amino acid substitution (Gly-90 in bovine rhodopsin) coincides with a substitution that mediates UV versus blue sensitivity in several bird species (10, 11). This discovery highlights the value of a cross-phyla comparison of visual pigments as a means to identify structural differences that may regulate color vision in invertebrates.In the present study, we identify an amino acid substitution in Drosophila visual pigments that regulates the color sensitivity of blue- and green-absorbing rhodopsins. For these studies, we employed sequence analysis of invertebrate and vertebrate visual pigments and a functional examination of mutant invertebrate opsins. This amino acid substitution red-shifts the λ_max of the Drosophila Rh1 pigment and reciprocally blue-shifts the λ_max of Rh6 pigment. Interestingly, this site also affects the spectral tuning of vertebrate pigments and corresponds to Ala-292 in bovine rhodopsin (8, 12–16).     

The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Light converts rhodopsin, the prototypical G protein-coupled receptor, into a form capable of activating G proteins. Recent work has shown that the light-activated state of different rhodopsins can possess different molecular properties, especially different abilities to activate G protein. For example, bovine rhodopsin is ∼20-fold more effective at activating G protein than parapinopsin, a non-visual rhodopsin, although these rhodopsins share relatively high sequence similarity. Here we have investigated possible structural aspects that might underlie this difference. Using a site-directed fluorescence labeling approach, we attached the fluorescent probe bimane to cysteine residues introduced in the cytoplasmic ends of transmembrane helices V and VI in both rhodopsins. The fluorescence spectra of these probes as well as their accessibility to aqueous quenching agents changed dramatically upon photoactivation in bovine rhodopsin but only moderately so in parapinopsin. We also compared the relative movement of helices V and VI upon photoactivation of both rhodopsins by introducing a bimane label and the bimane-quenching residue tryptophan into helices VI and V, respectively. Both receptors showed movement in this region upon activation, although the movement appears much greater in bovine rhodopsin than in parapinopsin. Together, these data suggest that a larger conformational change in helices V and VI of bovine rhodopsin explains why it has greater G protein activation ability than other rhodopsins. The different amplitude of the helix movement may also be responsible for functional diversity of G protein-coupled receptors.Rhodopsin, the photosensitive G protein-coupled receptor (GPCR),³ is responsible for transmitting a light signal into an intracellular signaling cascade through activation of G protein in visual and non-visual photoreceptor cells. Rhodopsin consists of a protein moiety (opsin, comprising seven transmembrane α-helical segments) combined with a chromophore (11-cis retinal) that acts as the light-sensitive ligand. Photoisomerization of the 11-cis retinal to the all-trans form induces structural changes in the protein moiety that then enable it to couple with and activate the G protein.The crystal structure of inactive bovine rhodopsin has been extensively investigated (1–3). Recently, a crystal structure of inactive invertebrate squid rhodopsin was also solved (4), and crystal structures of the inactive form of β-adrenergic receptors and A2 adenosine receptor have been reported (5–7). Remarkably, all of these crystal structures exhibit a very similar arrangement for the seven transmembrane helices (4, 8). Together, these facts suggest that the architecture for the inactive form is conserved among rhodopsin-like GPCRs.The structural features of an activated GPCR are much less defined. Thus, a variety of biochemical and biophysical methods, including cross-linking methods (9, 10) and site-directed spin and fluorescence labeling methods (10–13), have been employed to identify the dynamic and structural changes involved in forming the activated state. The data from these studies consistently suggest that some kind of movement of helix VI is involved in the formation of the active state of the rhodopsins. In particular, the cytoplasmic end of helix VI has been proposed to rotate and/or tilt toward helix V (10–13). Remarkably, the recent crystal structures of bovine opsin are consistent with the widely accepted helix motion model. Both the structures of opsin (the ligand-free form of rhodopsin that has partial G protein activation ability) and a complex of opsin with a peptide derived from the G protein C terminus show a movement of helix VI toward helix V, compared with the dark state rhodopsin structure (14, 15). Studies of β-adrenergic and muscarinic receptors also show that agonist binding promotes movement of helix VI toward helix V in these receptors (16, 17). Because the region between the cytoplasmic ends of helices V and VI in various GPCRs is a main site of interaction with G proteins (18), it is possible that movement of helices V and VI leads to formation of a conformation capable of interacting with G protein (19).Together, these studies imply that the active state conformation of GPCRs may be similar. However, a detailed comparison of the active-state conformation for two different GPCRs has never been precisely undertaken in the same laboratory using the same methods.In this context we have been investigating rhodopsins with different functional properties to determine whether their active states have different conformations. Our goal was to determine whether any functional or structural differences in the active states of these GPCRs could be detected under the exact same experimental conditions.Previously, we have found that several rhodopsins, such as an invertebrate rhodopsin and a vertebrate non-visual rhodopsin parapinopsin (20, 21), can be activated not only by light but also by exogenous all-trans retinal acting as a full agonist (22). This is in contrast to vertebrate visual rhodopsins, including bovine rhodopsin, which cannot fully form the active state by direct binding of all-trans retinal (23), although all-trans retinal can fully activate some rhodopsin mutants (24). Other invertebrate rhodopsin (25) and the circadian photoreceptor melanopsin (26) can also bind all-trans retinal directly.Interestingly, the active form of the all-trans retinal-activated rhodopsins exhibit some striking differences in their spectroscopic and biochemical properties compared with vertebrate visual rhodopsins (27). In particular, the efficiency of bovine rhodopsin for activating G protein is ∼20∼50-fold higher than that of parapinopsin and invertebrate rhodopsin. This difference could be related to the difference in position of a specific amino acid residue counterion that is essential for rhodopsin to absorb visible light, namely one at position 113 or 181 (28).⁴ Further biochemical analyses using chimeric mutants combining rhodopsins with lower and higher G protein activation abilities suggested that the difference in G protein activation ability was because of a structural difference in transmembrane helices in the active states but not because of difference in amino acid sequence of G protein interaction site (29) (Fig. 1, A–C). In addition, the active states of parapinopsin and the invertebrate rhodopsin are thermally stable and can be reconverted to the inactive state by subsequent light absorption, showing photo-regenerable or bistable nature (21, 28), unlike the active state of bovine rhodopsin, which is thermally unstable and cannot revert to the inactive state by subsequent light absorption (30).Open in a separate windowFIGURE 1.Molecular properties and sites of fluorescent probe attachment for bovine rhodopsin and parapinopsin. A, sequence alignment of bovine rhodopsin and parapinopsin. Amino acid residues to which cysteine and fluorescence label were introduced are marked with red. The amino acid residues identical and similar between bovine rhodopsin and parapinopsin are shown with white characters with black and gray background, respectively. Bovine rhodopsin and parapinopsin show 41% sequence identity and 61% similarity. In this paper the residue number of parapinopsin is described by using the bovine rhodopsin numbering system. B and C, comparison of G protein activation ability of rhodopsin and parapinopsin wild type (WT) proteins and loop-replaced mutants. In these mutants the second and/or third cytoplasmic loop was swapped between the two receptors. ParaL2 and ParaL3 indicate mutants of bovine rhodopsin in which second and third loops were replaced with the corresponding loop of parapinopsin, respectively. RhoL2 and RhoL3 indicate mutants of parapinopsin in which the second and third loops were replaced with the corresponding loops of bovine rhodopsin, respectively. ParaL2L3 and RhoL2L3 are mutants of bovine rhodopsin and parapinopsin in which both the second and third loops were swapped, respectively. See Terakita et al. (29) for more details. Data are presented as the means ± S.E. of three separate experiments except for mutants RhoL3, RhoL2L3, and ParaL2L3 (n = 2). D, model of bovine rhodopsin. Amino acid residues which were mutated to cysteine to enable attachment of the fluorescent probe bimane or mutated to tryptophan are indicated. Positions 226, 227, 244, 250, and 251 in the crystal structure of the dark state of bovine rhodopsin (PDB code 1GZM) are shown. E, reaction of the mBBr label with a sulfhydryl group. The mutants labeled with mBBr are named by the number of the residue and the suffix B₁. F, reaction of the PDT-bimane with a sulfhydryl group. The mutants labeled with PDT-bimane are named by the number of the residue and the suffix B₂. The disulfide linkage between the label and protein can be cleaved using Tris(2-carboxyethyl)phosphine (32).In this study we used site-directed fluorescence labeling (13, 31) to compare the structural features of active states of bovine rhodopsin with lamprey parapinopsin, a UV-sensitive non-visual pigment in the pineal organs (21). Parapinopsin shows relatively high sequence similarity (∼60%) to bovine rhodopsin, yet it has a greatly reduced ability to activate G protein (see Fig. 1, A–C) (21, 28). Using established protocols, we introduced cysteine residues into the cytoplasmic ends of helices V and VI, the region proposed to rearrange upon activation in GPCRs (11, 12, 14, 18). We then site-specifically labeled these cysteines with the small, non-polar fluorescent probe, bimane, and used the spectral properties of these bimane probes to act as reporter groups for environmental changes around their site of attachment upon formation of the photoactivated state for both rhodopsins.In addition, we measured changes in the relative proximity of the cytoplasmic ends of helix VI to helix V in both rhodopsin and parapinopsin using the tryptophan-induced-quenching of bimane (TrIQ-bimane) fluorescence method (31, 32). TrIQ-bimane measures the efficiency of intramolecular fluorescence quenching of bimane caused by tryptophan (Trp), which occurs in a distance-dependent manner. The goal of this study was to determine whether the helices in both receptors moved in the same way during formation of the active state. Our results show that whereas movement of helix VI relative to helix V occurs during formation of the active state for both parapinopsin and bovine rhodopsin, the “amplitude” of the movement is markedly different between the two rhodopsins.     

Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels

Background

Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies.

Principal Findings

We describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies.

Significance

We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.     

Gogo Receptor Contributes to Retinotopic Map Formation and Prevents R1-6 Photoreceptor Axon Bundling

Background

Topographic maps form the basis of neural processing in sensory systems of both vertebrate and invertebrate species. In the Drosophila visual system, neighboring R1–R6 photoreceptor axons innervate adjacent positions in the first optic ganglion, the lamina, and thereby represent visual space as a continuous map in the brain. The mechanisms responsible for the establishment of retinotopic maps remain incompletely understood.

Results

Here, we show that the receptor Golden goal (Gogo) is required for R axon lamina targeting and cartridge elongation in a partially redundant fashion with local guidance cues provided by neighboring axons. Loss of function of Gogo in large clones of R axons results in aberrant R1–R6 fascicle spacing. Gogo affects target cartridge selection only indirectly as a consequence of the disordered lamina map. Interestingly, small clones of gogo deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Using single photoreceptor type rescue, we show that Gogo expression exclusively in R8 cells is sufficient to mediate targeting of all photoreceptor types in the lamina. Upon lamina targeting and cartridge selection, R axons elongate within their individual cartridges. Interestingly, here Gogo prevents bundling of extending R1-6 axons.

Conclusion

Taken together, we propose that Gogo contributes to retinotopic map formation in the Drosophila lamina by controlling the distribution of R1–R6 axon fascicles. In a later developmental step, the regular position of R1–R6 axons along the lamina plexus is crucial for target cartridge selection. During cartridge elongation, Gogo allows R1–R6 axons to extend centrally in the lamina cartridge.     

Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors.

Methodology/Principal Findings

Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc''s gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors.

Conclusions/Significance

In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development.     

Demonstration of a sensory rhodopsin in eubacteria

We report the first sensory rhodopsin observed in the eubacterial domain, a green light-activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all-trans retinal to form a pink pigment (lambda max 543 nm) with a photochemical reaction cycle of 110 ms half-life (pH 6.8, 18 degrees C). Co-expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane-embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS-PAGE and autofluorography of 3H-labelled all-trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix-helix interactions with membrane-embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.     

The Retromer Complex Is Required for Rhodopsin Recycling and Its Loss Leads to Photoreceptor Degeneration

Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.     

All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.

The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 × longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a β-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

     

Distinct polymer physics principles govern chromatin dynamics in mouse and Drosophila topological domains

Background

In higher eukaryotes, the genome is partitioned into large "Topologically Associating Domains" (TADs) in which the chromatin displays favoured long-range contacts. While a crumpled/fractal globule organization has received experimental supports at higher-order levels, the organization principles that govern chromatin dynamics within these TADs remain unclear. Using simple polymer models, we previously showed that, in mouse liver cells, gene-rich domains tend to adopt a statistical helix shape when no significant locus-specific interaction takes place.

Results

Here, we use data from diverse 3C-derived methods to explore chromatin dynamics within mouse and Drosophila TADs. In mouse Embryonic Stem Cells (mESC), that possess large TADs (median size of 840 kb), we show that the statistical helix model, but not globule models, is relevant not only in gene-rich TADs, but also in gene-poor and gene-desert TADs. Interestingly, this statistical helix organization is considerably relaxed in mESC compared to liver cells, indicating that the impact of the constraints responsible for this organization is weaker in pluripotent cells. Finally, depletion of histone H1 in mESC alters local chromatin flexibility but not the statistical helix organization. In Drosophila, which possesses TADs of smaller sizes (median size of 70 kb), we show that, while chromatin compaction and flexibility are finely tuned according to the epigenetic landscape, chromatin dynamics within TADs is generally compatible with an unconstrained polymer configuration.

Conclusions

Models issued from polymer physics can accurately describe the organization principles governing chromatin dynamics in both mouse and Drosophila TADs. However, constraints applied on this dynamics within mammalian TADs have a peculiar impact resulting in a statistical helix organization.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1786-8) contains supplementary material, which is available to authorized users.     

Identification of small-molecule allosteric modulators that act as enhancers/disrupters of rhodopsin oligomerization

The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5 mM. The rod visual pigments, G protein–coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein–coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear. Here, we address this gap in knowledge by studying rod phototransduction. As the rod outer segment is known to adjust its size proportionally to overexpression or reduction of rhodopsin expression, genetic perturbation of rhodopsin cannot be used to resolve this question. Therefore, we turned to high-throughput screening of a diverse library of 50,000 small molecules and used a novel assay for the detection of rhodopsin dimerization. This screen identified nine small molecules that either disrupted or enhanced rhodopsin dimer contacts in vitro. In a subsequent cell-free binding study, we found that all nine compounds decreased intrinsic fluorescence without affecting the overall UV-visible spectrum of rhodopsin, supporting their actions as allosteric modulators. Furthermore, ex vivo electrophysiological recordings revealed that a disruptive, hit compound #7 significantly slowed down the light response kinetics of intact rods, whereas compound #1, an enhancing hit candidate, did not substantially affect the photoresponse kinetics but did cause a significant reduction in light sensitivity. This study provides a monitoring tool for future investigation of the rhodopsin signaling cascade and reports the discovery of new allosteric modulators of rhodopsin dimerization that can also alter rod photoreceptor physiology.     

Genetic interaction of centrosomin and bazooka in apical domain regulation in Drosophila photoreceptor

Background

Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes'' role in photoreceptor morphogenesis.

Methodology/Principal Findings

Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn''s gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs).

Conclusions/Significance

These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor.     

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.     

Bili Inhibits Wnt/β-Catenin Signaling by Regulating the Recruitment of Axin to LRP6

Background

Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ß-catenin pathway.

Methodology/Principal Findings

In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a β-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/β-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/β-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation.

Conclusions

These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/β-catenin pathway.     

The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells

Background

When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response.

Results

In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization.

Significance

We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.