Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization

The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells.     

Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photoreceptive processes: The case of Euglena gracilis

In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglenagracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A_498, non fluorescent and B₄₆₂, fluorescent.Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption consists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A₄₉₈ becomes B₄₆₂), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B₄₆₂ form acts as donor for the non-fluorescent A₄₉₈ form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Techniques and applications of three‐dimensional electron microscopy in entomological research

Structural studies using two‐dimensional (2D) images show limitations in understanding the structure and functions of cellular organelle and protein. To overcome the difficulty, over the last few years 3D reconstruction techniques using electron microscopy have been developed at extremely high speed. In this paper, currently available 3D reconstruction techniques of electron microscopy (such as electron tomography, serial section analysis and single particle analysis) are introduced using our data as examples of the application. The 3D structure of mitochondria with the defect of mitochondrial protein in round worm, Caenorhabditis elegans, through electron tomography, the cell–cell interaction in lamina of Drosophila melanogaster by serial‐section using ultramicrotome and high‐voltage electron microscopy and a thin filament related to muscle contraction in Drosophila melanogaster were used for examples of the application. These results through 3D reconstruction reveal the structural changes in a cellular organelle and protein that had not been shown by 2D structure.     

Sensory receptors of the miracidium of Gigantocotyle explanatum (Trematoda:Paramphistomidae)

Five types of sensory receptors are described. Both uniciliated and multiciliated nerve endings occur on the apical papilla. The former structures possibly have a tango- or rheo-receptive function, while the latter may have a chemoreceptive function. A number of uniciliated sensory structures are also present embedded within the intercellular ridges. A pair of unciliated lateral papillae are located in the intercellular ridge separating the first and second tiers of epidermal cells. Each is associated with a number of sheathed unciliate nerve bulbs. A pair of internal “lamellate ciliary organs” are ascribed a photoreceptive function. Each comprises a cylindrical cell body enclosing a large cavity, into which project eight or more cilia bearing a number of concentrically arranged spherical lamellae. A single unicellular “conical organ”, covered with microvilli, projects into an extracellular space, bounded in part by the lateral glands. This structure may represent a second type of photoreceptor, or alternatively may serve as a gyroscopic device.     

Rhodopsin: A Photopigment for Phototaxis in Euglena gracilis

For over 100 years, a major focus of photobiological studies has been the unicellular flagellate, Euglena gracilis, an organism well suited for such investigations by its special complement of organelles that may be considered an ancient, yet complete “visual” system. The possible photoreceptive roles of the cytoplasmic stigma and the photoreceptor (paraflagellar swelling) of E. gracilis are still under debate, because of conflicting interpretations of the results produced so far by the different research groups working on this microorganism. This article deals with our hypothesis, first put forward in the late 1980s, that rhodopsin-like proteins are responsible for photo-detection and that the paraxial rod is involved in the control of flagellar movements. This hypothesis uses oriented dipole and electroconformational coupling mechanisms as the physical phenomena that produce signal transduction. A model for phototaxis is presented.     

Membrane maintenance and electrical properties of photoreceptors of wild-type andrpa (receptor potential absent) mutant blowflies (Calliphora erythrocephala)

Summary The maintenance of photoreceptor cell membranes in the blowfly was investigated in relation to the diurnal cycle, age, and therpa (receptor potential absent) phototransduction mutation. The effect of disturbed membrane assembly on the electrical membrane properties was examined using single-electrode discontinuous current-clamp techniques. In wild-type flies the cross-sectional dimensions of the rhabdomeres were markedly reduced with age, and the quantity of synthetic organelles decreased concurrently, whereas no correlation was found between the diurnal cycle and membrane turnover. Therpa mutation is thought to block the visual transduction cascade in photoreceptor cells and to lead to degeneration of the photoreceptor cell bodies. The volume of rhabdomeres decreased markedly inrpa mutants and the quantity of synthetic organelles was reduced significantly, indicating an imbalance between photoreceptive membrane renewal and degradation. Also, the plasma membrane underwent degenerative changes. The passive electrical properties of photoreceptor cells — resting membrane voltages and input resistances — were only slightly changed from those of wild-type flies, although the photoreceptive membrane did not depolarize in response to light. This indicates no apparent disturbance in the function of the ionic channels in these membranes. Taken together, these results suggest that the photoreceptor cells need a functional phototransduction cascade with its feedback controls to maintain continuous renewal of rhabdomeres, but that the plasma membrane maintains its normal electrochemical properties despite extreme morphological degeneration of photoreceptor cell.     

Immunolocalization of a putative unconventional myosin on the surface of motile mitochondria in locust photoreceptors

Light stimulation of locust (Schistocerca gregaria) photoreceptors results in an actin-dependent translocation of mitochondria towards the photoreceptive microvilli and an antagonistic movement of endoplasmic reticulum towards the cell body. Using immunocytochemical techniques, we have tried to identify myosin-like motors that may drive the light-induced organelle motility. A monoclonal antibody against the motor domain of Acanthamoeba myosin identifies a prominent 110-kDa protein on Western blots of locust retina. Cross-reactivity with two polyclonal anti-myosin antibodies and a monoclonal anti-myosin-I-antibody, together with ATP-dependent binding to actin filaments, provides evidence that the 110-kDa protein is an unconventional myosin. By indirect immunofluorescence, the 110-kDa protein has been localized to both photoreceptors and pigment cells within the retina. In the photoreceptor cells, the 110-kDa protein is bound to the surface of mitochondria. This putative unconventional myosin may thus be a motor protein involved in the light-induced translocation of mitochondria in photoreceptors.     

Morphology of the pineal organ in the salamander Ensatina eschscholtzi

The pineal organ of Ensatina eschscholtzi, a terrestrial and secretive species of salamander of the family Plethodontidae, is a photoreceptive structure lying on the dorsal surface of the diencephalon. The pineal is flattened with a broad lumen and consists of three cell types: photoreceptors, supportive cells, and neurons. Pineal photoreceptors are typical vertebrate photoreceptors and possess outer segment formations which, however, are frequently contorted and disorganized. Sloughing of apical portions of outer segments and vesiculation along the lateral edges of outer segment membrane disks are consistently observed and presumed to represent mechanisms of outer segment membrane recycling. Photoreceptors have basal processes which synapse with neural dendrites. Synapses between photoreceptor basal processes are occasionally observed. All synapses are characterized by synaptic ribbon structures of variable number, size, and configuration. Dense-core vesicles are occasionally observed mingled with clear synaptic vesicles within photoreceptor basal processes. Supportive cells within the pineal function in phagocytosis and recycling of shed outer segment membrane material, and neurons are localized at the lateral margins of the organ. The latter send axons into the ipsilateral side of the dorsal diencephalon. The pineal organ of Ensatina shows marked variation in overall size (cell total), cell type proportions, absolute neuron number, and ratio of photoreceptor number to neuron number for individual pineals. None of these morphological parameters is correlated with body size, sex, or season, and it is assumed that such variability represents significant variation in photosensory capabilities. It is suggested that the pineal organ of Ensatina is a partially degenerate photoreceptive structure.     

Protein translocation across the peroxisomal membrane

Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle. Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different proteins, the majority of which were already identified. However, not much is known regarding the mechanism, of protein translocation across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process that occurs, through a large membrane protein assembly.     

The ER in 3D: a multifunctional dynamic membrane network

The endoplasmic reticulum (ER) is a large, singular, membrane-bound organelle that has an elaborate 3D structure with a diversity of structural domains. It contains regions that are flat and cisternal, ones that are highly curved and tubular, and others adapted to form contacts with nearly every other organelle and with the plasma membrane. The 3D structure of the ER is determined by both integral ER membrane proteins and by interactions with the cytoskeleton. In this review, we describe some of the factors that are known to regulate ER structure and discuss how this structural organization and the dynamic nature of the ER membrane network allow it to perform its many different functions.     

Euglena: the photoreceptor system for phototaxis.

The photoreceptor structures (eyespot-paraflagellar body-flagellum) for Euglena phototaxis were investigated by electron microscopy. The paraflagellar body--the photoreceptor--is a highly ordered crystalline lamellar structure. Optical diffraction of the electron micrographs and resulting filtered images of the paraflagellar body suggest that it is formed of rods in a helical arrangement. The action spectra for phototaxis, the in situ spectrum by microspectrophotometry of the paraflagellar body, and flavin analysis of the organism indicate that the photoreceptor molecule is a flavoprotein. The phototaxis action spectrum is similar to the spectrum for O2 evolution and implies that similar molecules participate in the photo processes. As a result, a photochemical scheme is suggested in which a photo-excited flavin and a cytochrome participate in the photoprocess. The photochemistry and photoreceptor structures for Euglena phototaxis are likened to a photoneuro sensory cell.     

TPR-containing proteins control protein organization and homeostasis for the endoplasmic reticulum

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into a number of functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein–protein interactions, supporting interactions with a wide range of ligands or substrates. Seven TPR-containing proteins have thus far been shown to localize to the ER and control protein organization and homeostasis within this multifunctional organelle. Here, we discuss the roles of these proteins in controlling ER processes and organization. The crucial roles that TPR-containing proteins play in the ER are highlighted by diseases or defects associated with their mutation or disruption.     

NMR structure and MD simulations of the AAA protease intermembrane space domain indicates peripheral membrane localization within the hexaoligomer

We have determined the solution NMR structure of the intermembrane space domain (IMSD) of the human mitochondrial ATPase associated with various activities (AAA) protease known as AFG3-like protein 2 (AFG3L2). Our structural analysis and molecular dynamics results indicate that the IMSD is peripherally bound to the membrane surface. This is a modification to the location of the six IMSDs in a model of the full length yeast hexaoligomeric homolog of AFG3L2 determined at low resolution by electron cryomicroscopy [1]. The predicted protein–protein interaction surface, located on the side furthest from the membrane, may mediate binding to substrates as well as prohibitins.     

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid‐rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB‐suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB‐suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.     

Morphological investigations of the Euglena gracilis isolated paraflagellar body

By means of scanning electron microscopy we have investigated the morphology of the photoreceptive-locomotory apparatus of the flagellate Euglena gracilis. As can be seen from the micrographs, there is a strong connection between the paraflagellar rod and the paraflagellar body; structural and functional details are discussed, and a simple model of the photoreceptive apparatus is given.     

Light induction of the Euglena chloroplast protein synthesis elongation factors: relative effectiveness of different wavelength ranges

The abilities of different wavelength ranges of light to promote the increase in the activities of the Euglena chloroplast protein synthesis elongation factors (EFs) during chloroplast biogenesis have been determined. Blue light was far more effective than either green light or red light in increasing the level of chloroplast EF-G, a nuclear encoded gene product. This observation suggests that the induction of EF-Gchl is under the control of the blue photoreceptor that has been identified in Euglena. Blue light was also the most effective wavelength range in facilitating the increase in EF-Ts, a nuclear gene product, and EF-Tu, a chloroplast gene product. However, red light and surprisingly green light were also effective. These results are not consistent with either of the known blue or blue/red photoreceptor systems in Euglena being the sole component involved in the light induction of these two factors and suggest that a green photoresponse may also be important in the development of the chloroplast. The specific activity of the Euglena mitochondrial protein biosynthetic translocase (EF-Gmt) decreased in cells exposed to light. Blue light caused an immediate decline in EF-Gmt activity; whereas, there was a temporal delay in the decrease in EF-Gmt activity when cells were exposed to either red or green light.     

Structure and function in the euglenoid eyespot apparatus: The fine structure,and response to environmental changes

Summary The microanatomy of the eyespot apparatus of Euglena gracilis Z was examined with the electron microscope. The stigma was found to be a membrane-bounded organelle showing no close homology with the chloroplast or any other organelle. The structure and pigment content of the stigma both diminish with extended hetrotrophic growth, and quickly regain normal dimensions upon exposure to light. Synthesis of the red pigment is particularly sensitive to inhibition by chloramphenicol, whereas construction of the structure itself is specifically inhibited by cycloheximide.The paraflagellar body appears to consist of two sets of parallel 80 Å striations intersecting at 60°. It is within the flagellar membrane, but separated from the axoneme by another structure, the paraflagellar rod. This elongated structure has an ordered substructure which appears as intersecting sets of parallel striations; part of its basal portion projects as a circular flange which makes contact with the paraflagellar body.     

The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.