Willem J. DeGrip

Opsin localization and chromophore retinoids identified within the basal brain of the lizard Anolis carolinensis

Since the beginning of this century evidence has accumulated which demonstrates that non-mammalian vertebrates possess photoreceptors situated deep within the brain. While many attempts have been made to localize these sensory cells, studies have either failed or been inconclusive. In this report we have used several experimental approaches to localize the deep brain photoreceptors of the lizard Anolis carolinensis. Using 3 antibodies that bind vertebrate cone opsins, we have immunolabelled cerebrospinal fluid (CSF)-contacting neurons located at the ventricular border within the nucleus ventromedialis of the septum. Western blot analysis indicates that these antibodies recognized a single 40 kD protein in ocular, anterior brain, and pineal extracts. Immunoblots of rodent brain did not show a similar protein band. We have also identified specific retinoids associated with phototransduction (11-cis and all-trans-3,4-didehydroretinaldehyde) within anterior brain extracts. This combined data provides the most detailed analysis of deep brain photoreceptors in any vertebrate. Consequently, we feel Anolis provides an excellent model to study this unexplored sensory system of the vertebrates.

Identification of vertebrate deep brain photoreceptors

Since the beginning of this century evidence has accumulated which demonstrates that nonmammalian vertebrates possess photoreceptors situated deep within the brain. These photoreceptors have been implicated in several different areas of physiology, but in all species examined, they play a critical role in the regulation of circadian and reproductive responses to light. Many attempts have been made to localize these sensory cells over the past 50 years, but until recently all attempts have failed. As a result, this important sensory system remains largely unexplored. Recent attempts to localize these photoreceptors, in a range of vertebrates, using combined antibody and biochemical approaches has met with some success. However, inconsistencies have emerged. Published and preliminary data raise the possibility of several types of encephalic photoreceptor photopigment (cone-like, rod-like or different from both), and depending on species at least two types of photoreceptor cell: CSF-contacting neurons (larval lamprey, reptiles and birds) and classical neurosecretory neurons within the nucleus magnocellularis preopticus (NMPO)(fish and amphibians).

Monitoring of biomass composition from microbiological sources by means of FT‐IR spectroscopy

An FT‐IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm−1. Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the CH stretching vibrational population between 2,984 and 2,780 cm−1. Carbohydrate content was determined using integration over a CO and COC stretching band area between 1,180 and 1,133 cm−1. Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra‐batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT‐IR spectroscopy compared well with the results obtained from elemental analysis. Biotechnol. Bioeng. 2009;103: 123–129.

(1)H and (13)C MAS NMR evidence for pronounced ligand-protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin.

Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven α-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete 1H and 13C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly 13C-labeled 11-cis-retinal. From the labeled pigment, 13C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment. The 1H chemical shifts were assigned by two-dimensional heteronuclear (1H-13C) dipolar correlation spectroscopy with phase-modulated Lee–Goldburg homonuclear 1H decoupling applied during the t1 period. The data indicate nonbonding interactions between the protons of the methyl groups of the retinylidene ionone ring and the protein. These nonbonding interactions are attributed to nearby aromatic acid residues Phe-208, Phe-212, and Trp-265 that are in close contact with, respectively, H-16/H-17 and H-18. Furthermore, binding of the chromophore involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for CH3-16 and CH3-17.

Short and mid-wavelength cone distribution in a nocturnal Strepsirrhine primate (Microcebus murinus).

Strepsirrhines are of considerable interest for understanding the evolution of cone photoreceptors because they represent the most ancestral living primates. The retina of nocturnal Strepsirrhines is reported to contain a single population of medium/long wavelength (MW/LW) cones whereas short wavelength (SW) cones are totally absent. The area centralis of nocturnal Strepsirrhines also lacks the degree of central specialization seen in the fovea of diurnal primates. In this study of a nocturnal Strepsirrhine, the gray mouse lemur (Microcebus murinus), we used specific antibodies that recognize SW and MW/LW opsins to determine the presence of different cone subtypes and their distribution in relation to that of rods and ganglion cells. The results are compared to two diurnal Haplorhine species, a New World (Callithrix jacchus) and an Old World (Macaca fascicularis) monkey. In the mouse lemur, both antibodies to MW/LW cone opsin (COS‐1 and CERN956) label the same population of cones. A small proportion of SW cones is only stained by the JH455 antiserum whereas the monoclonal OS‐2 antibody shows negative staining. These two antibodies label the same SW cone population in other primates. The extracellular matrix of all cones is also labeled by the peanut agglutinin (PNA) lectin. In mouse lemur retinal wholemounts, peak cone density is localized at the area centralis and ranged from 7,500 to 8,000 cones/mm2. SW cones represent less than 0.2 % of the total cone population and are mainly located in the nasal part of the retina. SW cones show an irregular distribution and densities never exceed 49 cones/mm2. The distribution of neurons in the ganglion cell layer shows a distinct centroperipheral gradient with a peak of 28,000 cells/mm2 at the area centralis. Rod distribution shows a centroperipheral gradient with the peak (850,000 rods/mm2) including and extending slightly dorsal to the area centralis. The theoretical spatial resolution of the mouse lemur (4.9 cycles/degree) is slightly lower to that of other nocturnal primates. The densities of rods, cones, and ganglion cell layer neurons represent a compromise between spatial resolution and sensitivity for both photopic and scotopic vision. J. Comp. Neurol. 438:494–504, 2001.

Pigmented epithelium induces complete retinal reconstitution from dispersed embryonic chick retinae in reaggregation culture.

Reaggregation of dispersed retinal cells of the chick embryo leads to histotypic retinospheroids in which the laminar organization remains incomplete: photoreceptors form rosettes which are surrounded by constituents of the other retinal layers. Here, for the first time, a complete arrangement of layers is achieved in cellular spheres (stratoids), provided that fully dispersed retinal cells are younger than embryonic day E6, and are reaggregated in the presence of a monolayer of retinal pigmented epithelium (RPE). A remarkable mechanism of stratoid formation from 1 to 15 days in vitro is revealed by the establishment of a radial Müller glia scaffold and of photoreceptors. During the first two days of reaggregation on RPE, rosettes are still observed. At this stage immunostaining with vimentin and F11 antibodies for radial Müller glia reveal a disorganized pattern. Subsequently, radial glia processes organize into long parallel fibre bundles which are arranged like spokes to stabilize the surface and centre of the stratoid. The opsin–specific antibody CERN 901 detects photoreceptors as they gradually build up an outer nuclear layer at the surface. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.

Early development of the retina and pineal complex in the sea lamprey: Comparative immunocytochemical study

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and γ‐aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA‐negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium‐sized to large larvae, PCNA‐immunoreactive (‐ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late‐proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA‐ir perikaryon was observed in the retina of larval lampreys, although a few GABA‐ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA‐ir ganglion cells occur in the pineal organ of 15–17 mm larvae, and their number increases during the larval period. The only GABA‐ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods. J. Comp. Neurol. 442:250–265, 2002.

PHOTOEXCITATION OF RHODOPSIN: CONFORMATION CHANGES IN THE CHROMOPHORE, PROTEIN AND ASSOCIATED LIPIDS AS DETERMINED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— The visual pigment rhodopsin is the major membrane protein in the rod photoreceptor membrane. Rhodopsins function is to transduce the light induced isomerization (ll‐cis to all‐trans) of its internally located retinylidene chromophore into transient expression of signal sites at the surface of the protein. Fourier transform infrared (FTIR) difference spectroscopy has been used to study all of the steps in the photobleaching sequence of rhodopsin. Early protein alterations involving the peptide backbone and aspartic and/or glutamic carboxyl groups were detected which increase upon lumirhodopsin formation and spread to water exposed carboxyl groups by metarhodopsin II. The intensified and frequency shifted hydrogen‐out‐of‐plane vibrations of the chromophore that are present in bathorhodopsin are absent in lumirhodopsin. This indicates that by lumirhodopsin, the chromophore has relaxed relative to its more strained all‐frans form in bathorhodopsin. Finally, the transition to metarhodopsin II is found to involve perturbation of the acyl tail region of unsaturated phospholipid molecules possibly in response to small changes in the shape of the rhodopsin.

Fourier transform infrared study of photoreceptor membrane. I. Group assignments based on rhodopsin delipidation and reconstitution

Fourier transform infrared spectroscopy has been used to study the structure of bovine photoreceptor membrane. Rhodopsin appears to contain an extensive alpha-helical structure which is arranged predominantly perpendicular to the membrane plane. Spectra of delipidated rhodopsin and rhodopsin membranes reconstituted from dioleyl-phosphatidylcholine were compared with native photoreceptor membrane from rod outer segments in order to facilitate peak assgnments. It is concluded that spectroscopic peaks characteristic of several protein and lipid groups can be assigned. We also find delipidation leads to alteration of the rhodopsin structure which is resorted upon reconstitution. Membranes both suspended in 2H2O and dehydrated were compared in order to detect possible conformational differences. Dehydration does not appear to grossly alter rhodopsin structure, although it may affect delipidated rhodopsin.

Evidence for rhodopsin refolding during the decay of Meta II.

Fourier transform infrared difference spectroscopy (FTIR) reveals that the Meta II intermediate of the rhodopsin bleaching cascade is structurally distorted relative to rhodopsin. In addition to previously detected alterations in the state of carboxyl groups, a small part of the protein back-bone undergoes a conversion from alpha-helical to beta-type structure. All of these changes partially reverse during Meta II decay. This evidence together with FTIR studies of earlier photointermediates indicates that of the known photointermediates the protein structure of Meta II is the most distorted. It is concluded that light causes rhodopsin to convert into a conformationally distorted form (Meta II), which subsequently refolds into a more rhodopsin-like conformation (opsin).