József Orbán

Tyrosine hydroxylase positive perisomatic rings are formed around various amacrine cell types in the mammalian retina

Dopaminergic neurons of the central nervous system are mainly found in nuclei of the midbrain and the hypothalamus that provide subcortical and cortical targets with a rich and divergent innervation. Disturbance of signaling through this system underlies a variety of deteriorating conditions such as Parkinsons disease and schizophrenia. Although retinal dopaminergic signaling is largely independent of the above circuitry, malfunction of the retinal dopaminergic system has been associated with anomalies in visual adaptation and a number of retinal disorders. Dopamine (DA) is released mainly in a paracrine manner by a population of tyrosine hydroxylase expressing (TH+) amacrine cells (AC) of the mammalian retina; thus DA reaches virtually all retinal cell types by diffusion. Despite this paracrine release, however, the so called AII ACs have been considered as the main targets of DA signaling owing to a characteristic and robust ring‐like TH+ innervation to the soma/dendritic‐stalk area of AII cells. This apparent selectivity of TH+ innervation seems to contradict the divergent DAergic signaling scheme of other brain loci. In this study, however, we show evidence for intimate proximity between TH+ rings and somata of neurochemically identified non‐AII cells. We also show that this phenomenon is not species specific, as we observe it in popular mammalian animal models including the rabbit, the rat, and the mouse. Finally, our dataset suggests the existence of further, yet unidentified post‐synaptic targets of TH+ dendritic rings. Therefore, we hypothesize that TH+ ring‐like structures target the majority of ACs non‐selectively and that such contacts are wide‐spread among mammals. Therefore, this new view of inner retinal TH+ innervation resembles the divergent DAergic innervation of other brain areas through the mesolimbic, mesocortical, and mesostriatal signaling streams.

Nucleotide dependent differences between the α-skeletal and α-cardiac actin isoforms

The thermodynamic properties of the actin filaments prepared from cardiomyocytes were investigated with differential scanning calorimetry. This method could distinguish between the alpha-cardiac and alpha-skeletal components of the actin filaments polymerised from ADP-actin monomers by their different melting temperatures (T(m)). Similar separation was not possible with filaments polymerised from ATP-actin monomers. Further analyses revealed that the activation energy (E(act)) was greater for filaments of alpha-skeletal actin than for alpha-cardiac actin monomers when the filaments were polymerised from ADP-actin monomers. These results showed that the alpha-cardiac actin filaments were thermodynamically less stable than the filaments of alpha-skeletal actin and their difference was nucleotide dependent. Based on these results and considering previous observations it was concluded that the existence of two actin isoforms and their nucleotide dependent conformational differences are part of the tuning regulatory mechanism by which the cardiac muscle cells can maintain their biological function under pathological conditions.

The effect of pH ont he thermal stability of a-actin isoforms

SummaryThe effect of pH was characterised on the thermal stability of magnesium saturated skeletal and cardiac α-actin isoforms with differential scanning calorimetry (DSC) at pH 7.0 and 8.0. The calorimetric curves were further analysed to calculate the enthalpy and transition entropy changes. The activation energy was also determined to describe the energy consumption of the initiation of the thermal denaturation process. Although the difference in Tmvalues is too small to interpret the difference between the a-actin isoforms, the values of the activation energy indicated that the α-skeletal actin is probably more stable compared to the α-cardiac actin. The difference in the activation energies indicated that lowering the pH can produce a more stable protein matrix in both cases of the isoforms. The larger range of the difference in the values of the activation energies suggested that the α-cardiac actin is probably more sensitive to the change of the pH compared to the α -skeletal actin.

Characterization of connexin36 gap junctions in the human outer retina

Retinal connexins (Cx) form gap junctions (GJ) in key circuits that transmit average or synchronize signals. Expression of Cx36, -45, -50 and -57 have been described in many species but there is still a disconcerting paucity of information regarding the Cx makeup of human retinal GJs. We used well-preserved human postmortem samples to characterize Cx36 GJ constituent circuits of the outer plexiform layer (OPL). Based on their location, morphometric characteristics and co-localizations with outer retinal neuronal markers, we distinguished four populations of Cx36 plaques in the human OPL. Three of these were comprised of loosely scattered Cx36 plaques; the distalmost population 1 formed cone-to-rod GJs, population 2 in the mid-OPL formed cone-to-cone GJs, whereas the proximalmost population 4 likely connected bipolar cell dendrites. The fourth population (population 3) of Cx36 plaques conglomerated beneath cone pedicles and connected dendritic tips of bipolar cells that shared a common presynaptic cone. Overall, we show that the human outer retina displays a diverse cohort of Cx36 GJ that follows the general mammalian scheme and display a great functional diversity.

Noncooperative stabilization effect of phalloidin on ADP.BeFx- and ADP.AlF4-actin filaments

Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actin-bound ATP is hydrolyzed to ADP and P i. We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4. The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x- or ADP.AlF 4-actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4-bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.

FASCIN and alpha-actinin can regulate the conformation of actin filaments.

BACKGROUND Actin filament bundling proteins mediate numerous processes in cells such as the formation of cell membrane protrusions or cell adhesions and stress fiber based locomotion. Among them alpha-actinin and fascin are the most abundant ones. This work characterizes differences in molecular motions in actin filaments due to the binding of these two actin bundling proteins. METHODS We investigated how alpha-actinin and fascin binding modify the conformation of actin filaments by using conventional and saturation transfer EPR methods. RESULTS The result characteristic for motions on the microsecond time scale showed that both actin bundling proteins made the bending and torsional twisting of the actin filaments slower. When nanosecond time scale molecular motions were described the two proteins were found to induce opposite changes in the actin filaments. The binding of one molecule of alpha-actinin or fascin modified the conformation of numerous actin protomers. CONCLUSION As fascin and alpha-actinin participates in different cellular processes their binding can serve the proper tuning of the structure of actin by establishing the right conformation for the interactions with other actin binding proteins. Our observations are in correlation with the model where actin filaments fulfill their biological functions under the regulation by actin-binding proteins. GENERAL SIGNIFICANCE Supporting the general model for the cellular regulation of the actin cytoskeleton we showed that two abundant actin bundling proteins, fascin and alpha-actinin, alter the conformation of actin filaments through long range allosteric interactions in two different ways providing the structural framework for the adaptation to specific biological functions.

Connexin36 Expression in the Mammalian Retina: A Multiple-Species Comparison

Much knowledge about interconnection of human retinal neurons is inferred from results on animal models. Likewise, there is a lack of information on human retinal electrical synapses/gap junctions (GJ). Connexin36 (Cx36) forms GJs in both the inner and outer plexiform layers (IPL and OPL) in most species including humans. However, a comparison of Cx36 GJ distribution in retinas of humans and popular animal models has not been presented. To this end a multiple-species comparison was performed in retinas of 12 mammals including humans to survey the Cx36 distribution. Areas of retinal specializations were avoided (e.g., fovea, visual streak, area centralis), thus observed Cx36 distribution differences were not attributed to these species-specific architecture of central retinal areas. Cx36 was expressed in both synaptic layers in all examined retinas. Cx36 plaques displayed an inhomogenous IPL distribution favoring the ON sublamina, however, this feature was more pronounced in the human, swine and guinea pig while it was less obvious in the rabbit, squirrel monkey, and ferret retinas. In contrast to the relative conservative Cx36 distribution in the IPL, the labels in the OPL varied considerably among mammals. In general, OPL plaques were rare and rather small in rod dominant carnivores and rodents, whereas the human and the cone rich guinea pig retinas displayed robust Cx36 labels. This survey presented that the human retina displayed two characteristic features, a pronounced ON dominance of Cx36 plaques in the IPL and prevalent Cx36 plaque conglomerates in the OPL. While many species showed either of these features, only the guinea pig retina shared both. The observed similarities and subtle differences in Cx36 plaque distribution across mammals do not correspond to evolutionary distances but may reflect accomodation to lifestyles of examined species.