New issues in phototransduction

Light and dark cycles are one of the principal driving forces for the metabolism of both eukaryotic and prokaryotic organisms, which are the evolutionary closest one to the first living beings. Therefore, is reasonable to think that the alternation between light and dark has influenced the evolution of all the organisms on Earth. Cyanobacteria as well as yeasts have evolved oscillating rhythms of gene expression, also known as circadian rhythms, in order to synchronize their physiological processes with Earth\u2019s day/night cycles. Light cues can also drive unicellular organisms\u2019 motility and growth. However, the light perception of these organisms is limited, and the vast majority of them can express a small number of molecules that can detect a limited range of the light spectrum. On the other hand, metazoan and in particular vertebrates have evolved very specialized and sensitive neurons, rods and cones, which enable visual perception in almost all the lightning conditions that occur during the 24 hours of the day. It has been hypothesized that vertebrates have evolved firstly cone photoreceptors, to perceive light changes during the day (photopic vision). However, cones\u2019 sensitivity is very limited at night and in low illumination conditions their functions as photons detectors are drastically reduced. Rods instead can perceive very dim light signals at night (scotopic vision), even single photons, ensuring an approximate visual stimulus also in poor lighting conditions. It has been proposed that rods are the latest evolved photoreceptors, which have conferred to vertebrates an extraordinary sensitive visual system. The cellular and molecular mechanisms of light and dark adaptation in vertebrates\u2019 photoreceptors have been widely studied and accurately described since the early seventies of the last century. Nonetheless, the study of these fascinating and complex dynamics has left some open questions, regarding both physiological and pathological aspects of photoreceptors metabolism as well as the influence of these alternating mechanisms on phototransduction, the set of enzymatic reactions that transform light stimuli into electrical signals. During the course of my PhD, I have focused my attention on rod\u2019s physiology, in particular on the mechanisms that govern light induced degeneration of photoreceptors, in a Xenopus laevis model of retinitis pigmentosa. Frogs carrying a specific genetic mutation displayed an altered turnover of their cellular body as well as some deficits of phototransduction signaling cascade. In our work, we attempted to estimate how many light induced photoisomerization were necessary to see these alterations. Moreover, I have partially continued the assessment of the response of rods photoreceptors to very localized light stimuli, elicited by mean of a special type of metal coated, tapered optical fibers. Through this technique, we have studied light adaptation of Xenopus laevis\u2019 rods to very confined light stimuli, in order to understand if variations of this process may occur along the rod cells bodies. Furthermore, I have assessed the possible contribution of the circadian rhythms on the phototransduction machinery, by altering the light and dark adaptation cycles of Xenopus frogs. Finally, I have initiated the study of the possible coupling between mechano and phototransduction in rod photoreceptors. This last part is the most fascinating one and made me embrace the idea that sensory neurons are surely specialized cells for one sensory stimulus such as light, sound or chemicals molecules, but others sensory stimuli like temperature variations and small mechanical forces could modulate significantly the perception of the principal stimulus

Speed, adaptation, and stability of the response to light in cone photoreceptors: The functional role of Ca-dependent modulation of ligand sensitivity in cGMP-gated ion channels

The response of cone photoreceptors to light is stable and reproducible because of the exceptional regulation of the cascade of enzymatic reactions that link visual pigment (VP) excitation to the gating of cyclic GMP (cGMP)-gated ion channels (cyclic nucleotide–gated [CNG]) in the outer segment plasma membrane. Regulation is achieved in part through negative feedback control of some of these reactions by cytoplasmic free Ca2+. As part of the control process, Ca2+ regulates the phosphorylation of excited VP, the activity of guanylate cyclase, and the ligand sensitivity of the CNG ion channels. We measured photocurrents elicited by stimuli in the form of flashes, steps, and flashes superimposed on steps in voltage-clamped single bass cones isolated from striped bass retina. We also developed a computational model that comprises all the known molecular events of cone phototransduction, including all Ca-dependent controls. Constrained by available experimental data in bass cones and cone transduction biochemistry, we achieved an excellent match between experimental photocurrents and those simulated by the model. We used the model to explore the physiological role of CNG ion channel modulation. Control of CNG channel activity by both cGMP and Ca2+ causes the time course of the light-dependent currents to be faster than if only cGMP controlled their activity. Channel modulation also plays a critical role in the regulation of the light sensitivity and light adaptation of the cone photoresponse. In the absence of ion channel modulation, cone photocurrents would be unstable, oscillating during and at the offset of light stimuli

NOVEL TECHNIQUES FOR IN VIVO CHARACTERIZATION OF SHORT PEPTIDES AND PROTEINS IN MEMBRANE PERMEABILIZATION AND SIGNAL TRANSDUCTION

My scientific interest is focused on the field of cellular electrical activity, ranging from the study of intracellular enzymatic processes to the characterization of new generation of drugs. For this purpose I also used the most powerful techniques of investigation, including patch-clamp technique, fluorescence imaging, and surface plasmon resonance (SPR) spectroscopy. Moreover, to shed light on complex molecular mechanisms, unconventional strategies were employed, requiring sometimes the realization of specific devices not commercially available. In particular my PhD thesis includes two different scientific projects: the biophysical characterization of antimicrobial peptides and the modulation of visual phototransduction in vertebrate cones. In the first project, the patch-clamp technique was employed to study the pore forming properties of synthetic cecropin-melittin hybrid peptide (CM15), alamethicin F50/5 and its synthetic analog [L-Glu(OMe)7,18,19] under strict physiological conditions. These short peptides selectively permeabilize the bacteria plasma membrane leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. I pursued this study by recording the ion current through the channels formed by these peptides, once inserted in the membrane of photoreceptor rod outer segment membrane (OS) isolated from frog retinae. The peptides were applied to (and removed from) the extracellular OS side in ~50 ms with a computer-controlled microperfusion system, so that the ion channel characteristics (as its selectivity, blockade and gating) and the dynamics of pore formation could be precisely assessed. On the basis of the electrophysiological recordings obtained, it was demonstrated that, different than alamethicins, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate that CM15 form pores according to a toroidal model. Moreover, in order to understand the divalent-cation dependency of [L-Glu(OMe)7,18,19] binding to the lipid bilayer at the molecular level, the electrophysiological experiments were paralleled with experiments employing SPR spectroscopy. Results indicate the presence of Ca2+ in the external solution increases the probability of formation of smaller and more stable [L-Glu(OMe)7,18,19] pores. The second project of this thesis concerns the investigation of the physiological role of the neuronal calcium sensor zGCAP3 in the photrasduction cascade. This study was pursued through the simulation of an over expression and a knock-down of this protein, by delivering it, or its monoclonal antibody, into zebrafish cone cytosol, while recording their photoresponses with the patch-clamp technique. The intracellular protein delivery was attained via the patch pipette, by ejecting the proteins out of a tube inserted into the pipette lumen. A microperfusion system was employed to apply the desired exogenous molecules with a precise timing. However, the long tapered shape of the pipette shank make it very difficult to perfuse efficiently the cell with this strategy. For this reason a pressure polishing setup was assembled to enlarge the patch pipette shank, through the calibrated combination of heat and air pressure. This allowed to insert quartz or plastic tubes in the pipette lumen very close to its tip. In order to obtain a substantial and specific silencing of the zGCAP3s in zebrafish cones, surface plasmon resonance experiments were performed to allow the selection of a monoclonal antibody with strong affinity for zGCAP3 and low cross interaction with other components of the phototransduction cascade. Results showed that the perfusion with GCAP3 did not altered significantly the light response, while he anti-zGCAP3 incorporation in the cytosol caused the progressive photoresponse fall, followed by the progressive fall of saturating flash response amplitude, probably due to the progressive GC inhibition. The unexpected lack of an effect of zGCAP3 incorporation in the cone cytosol, suggests that the endogenous number of zGCAP3 is saturating, i.e. their number is equal or above the number of their target molecules (guanylate cyclase), therefore any further increase of zGAP3 in the cytosol is uneffective

Biophysical mechanisms of membrane perturbation and signal transduction produced by proteins and peptides

My primary research interest is focused on the field of cellular electrical activity, ranging from the ion channels that generates it, up to the study of intracellular processes regulating it, and new generation of drugs. For this purpose, during my Ph.D. I have learnt and improved different cutting-edge techniques, i.e. the patch-clamp technique, the fluorescence imaging, and the synthesis and use of model membranes. Moreover, to explore particular aspects of these molecular mechanisms and to overcome the issues raised during the investigations, non-conventional strategies were employed, even requiring the development of specific devices not commercially available. In summary, my Ph.D. thesis is focused on two projects: the biophysical characterization of a particular class of membrane active peptides, and the modulation of visual phototransduction in vertebrate cones. In the first project, I investigated the mechanism of membrane perturbation of cell-penetrating and antimicrobial peptides using the patch-clamp technique. Cell-penetrating peptides (CPPs) are short peptides that are able to cross the cell membrane via energy-dependent and/or independent mechanisms, with low toxicity and without the use of specific receptors. This ability is preserved even when CPPs are conjugated with a large cargo, thus representing an innovative pharmacological tool for the diffusion of large and hydrophilic drugs into the cells. Despite the mechanism of cellular uptake is still debated in literature, it has been proved that it can occur by either direct translocation or endocytosis. In the latter case, though, the cargo-peptide complex often remains trapped inside the endocytic vesicles and is not able to reach its therapeutic target. A possible solution to this problem could be found in another class of small peptides, similar to CPPs, i.e. the antimicrobial peptides (AMPs). AMPs are 12-50 amino acids long peptides, which represent an essential part in the innate immune system in most organisms. Indeed, they are among the first defensive molecules released during infections and their activity is direct thorough the membrane of bacteria, causing its destruction and consequently the death of the pathogen. Therefore, the ability of AMPs to disrupt biological membranes could be exploited to improve the CPPs escape from the endocytic vesicles in addition to, of course, their application as a novel class of antibiotics. The idea is to conjugate the CPP with a molecule that possess an antimicrobial activity, which can destroy the vesicle membrane, and help the complex to reach its target once it has been internalized in the cell. On this ground, the first project I carried out regards the study of a novel chimeric peptide, CM18-Tat11, composed of the antimicrobial peptide CM18 (a cecropin-mellitin hybrid peptide) linked to the cell-penetrating peptide Tat11 (derived from the basic domain of HIV-1 Tat protein). In particular, I investigated the membrane perturbing activity of this peptide (and of its elements) using the patch-clamp technique and operating under strictly physiological conditions. This study has been carried out by recording the ion current flowing through the channels formed by these peptides (if any), once inserted in the membrane of Chinese hamster ovary (CHO) cells. In these experiments, the peptides were applied to (and removed from) the extracellular CHO membrane in ~50 ms with a computer-controlled microperfusion system. Therefore, besides assessing ion channel characteristics, the dynamics of pore formation and disaggregation could be precisely evaluated as well. I found that CM18-Tat11 produces a large and irreversible plasma membrane lysis, at concentration where CM18 and Tat11 give instead a nearly reversible membrane permeabilization and no perturbation, respectively. Furthermore, using the same method, I studied the biophysical characteristic of another antimicrobial peptide, called CM12, which sequence was obtained from the optimization of CM18. When applied on CHO, CM12 and CM18 produce voltage-independent membrane permeabilization, and no single-channel events were detected at low peptides concentration. These results indicate that both peptides form pores according to a toroidal model, in which the lipid layer bends continuously through the pore so that the core is formed by both lipid head groups and the peptides. Finally, I have studied the single-channels properties generated by the pore-forming peptide alamethicin (Alm) F50/5 and its [L-Glu(OMe)7,18,19] analog inserted in a natural membrane and in giant unilamellar vesicles (GUVs). The possibility to compare the channel activity in the precisely controlled lipid environment of GUVs, with the one recorded in a natural membrane, will open new possibilities in the biophysical characterization of the pores. The second project of this thesis is focused on the study of the physiological role of the calcium sensor GCAP3 (guanylate cyclase activated protein 3) in the phototransduction cascade in zebrafish. I pursued this study simulating the over expressions and the knockdown of this protein, through the delivery of zGCAP3, or of its monoclonal antibody, into zebrafish cone cytoplasm, while recording their photorensponses with the patch-clamp technique. The proteins were administered inside the cone via the patch pipette thanks to an intracellular perfusion system developed in this thesis. This system allows the delivery of exogenous molecules inside the cell with a controlled timing, by expelling them with a small teflon tube inserted into the pipette lumen controlled by a microperfusion apparatus. Results indicated that the increase in the concentration in zGCAP3 did not altered significantly the light response, while the perfusion with the antibody anti-zGCAP3 caused the progressive fall of the dark current, together with the progressive slowing down of the flash response kinetics. The surprising lack of an effect of zGCAP3 incorporation, suggests that the endogenous number of zGCAP3 is saturating, therefore any further increase of this sensor is ineffective. However, the effects of the antibody can be explained as an inhibition of the target enzyme of zGCAP3, which is the guanylate cyclase (GC). Finally, no experiments mentioned above would have been accomplished without the development of a “pressure-polishing” system, which makes it possible to modify the geometry of the patch-clamp pipette. The pipette shank (the final part of the pipette) is, in fact, very long and tapered, thus generating a high resistance to the passage of ions and molecules, and making very difficult to perfuse efficiently the cell with the internal perfusion. The pressure polishing setup I developed enlarged the patch pipette shank, using a calibrated combination of heat and air pressure. These pipettes minimized errors in membrane potential control and allowed the insertion of teflon tubes in the pipette lumen very close to its tip

Ion transport modeling for retinal rod photoreceptor cells

"July 2010.""A Thesis presented to the Faculty of the Graduate School at the University of Missouri In Partial Fulfillment of the Requirements for the Degree Master of Science."Thesis supervisor: Dr. Jinglu Tan.In this study, a mathematical model is developed to describe the ion transport activities associated with the response of rod photoreceptor to light stimulus. In the model, the cell body is modeled as two capacitors connected via the connecting cilium. Roles of different ion channels during a photoreceptor light response are analyzed, and the relations between changes in ion concentration and response currents are assessed. Methods are developed for computing the membrane potential from ion concentrations and relating the material and electrical resistances. The steady state under different conditions can be uniquely defined with only three measured values. The model can effectively describe the rod photoreceptor response to different light stimuli. Model simulation of the a-wave for progressive narrowing of the connecting cilium corresponds well with published literature on hereditary retinal degeneration of Abyssinian cats. Reductions in amplitude and changes in the a-wave waveform are observed in different stages of the disease. Changes in the receptor response amplitude may not be measurable till the conductance of the connecting cilium is reduced to a comparable magnitude of the ion channels. The model can provide quantitative information of ionic activities, changes in ion concentrations and membrane voltage in the outer segment and the inner compartment. The ionic environment is found to be different between the outer segment and the inner compartment. During receptor response, changes in the outer segment appear to be stronger and quicker than those in the inner compartment. Reductions in the connecting cilium transport can reset the dark resting state.Includes bibliographical references (pages 87-91)

The Limit of Photoreceptor Sensitivity: Molecular Mechanisms of Dark Noise in Retinal Cones

Detection threshold in cone photoreceptors requires the simultaneous absorption of several photons because single photon photocurrent is small in amplitude and does not exceed intrinsic fluctuations in the outer segment dark current (dark noise). To understand the mechanisms that limit light sensitivity, we characterized the molecular origin of dark noise in intact, isolated bass single cones. Dark noise is caused by continuous fluctuations in the cytoplasmic concentrations of both cGMP and Ca2+ that arise from the activity in darkness of both guanylate cyclase (GC), the enzyme that synthesizes cGMP, and phosphodiesterase (PDE), the enzyme that hydrolyzes it. In cones loaded with high concentration Ca2+ buffering agents, we demonstrate that variation in cGMP levels arise from fluctuations in the mean PDE enzymatic activity. The rates of PDE activation and inactivation determine the quantitative characteristics of the dark noise power density spectrum. We developed a mathematical model based on the dynamics of PDE activity that accurately predicts this power spectrum. Analysis of the experimental data with the theoretical model allows us to determine the rates of PDE activation and deactivation in the intact photoreceptor. In fish cones, the mean lifetime of active PDE at room temperature is ∼55 ms. In nonmammalian rods, in contrast, active PDE lifetime is ∼555 ms. This remarkable difference helps explain why cones are noisier than rods and why cone photocurrents are smaller in peak amplitude and faster in time course than those in rods. Both these features make cones less light sensitive than rods

Vertebrate vision : about physical determinants of photoreceptor sensitivity and kinetics

Rod and cone photoreceptors transform information about incoming light into neural signals with broadly similar molecular mechanisms. Yet their sensitivity, response kinetics and adaptation properties are quite different as rods mediate dim-light vision and cones function mainly under daylight. This thesis 1. addresses the functional differences between rods and cones as well as mammalian and non-mammalian photoreceptors and 2. provides novel findings regarding the existence and regulation of rod-cone interactions at the photoreceptor level. Rod and cone photoresponses to brief flashes of light were recorded with electroretinogram (ERG) from isolated rodent and amphibian retinas. Various phototransduction models were used to compare their relevant parameters over a range of adapting conditions. The study focused on how the following physical factors shape and limit photoreceptor function: operating temperature, thermal stability of the amphibian long wavelength sensitive (A1-)visual pigment, outer segment dimensions, morphology and electrical connections between adjacent rods and cones. Mammalian rod photoreceptors generate faster photoresponses but light-adapt less efficiently than amphibian rods. In the rodent and anuran rods studied in this thesis, the main differences could be accounted for by the higher operating temperature and smaller outer segment size of the rodent photoreceptors. Additionally, the slender outer segments of the mammalian rods enabled sufficient quantal responses and high quantum catch despite the observed desensitizing effect of warming. Long wavelength -sensitive cone photoreceptors have been hypothesized to be desensitized by thermal excitation of their visual pigment molecules. However, it has been shown experimentally only in amphibian cones that utilize the A2-chromophore. The relative stability of the A1-based cone pigments - used by all terrestrial vertebrates - has remained unclear, as well as its role in limiting cone function. In this study, thermal isomerization rate of the long wavelength sensitive (A1-)visual pigment was estimated to play at most a minor role in regulating cone sensitivity of the frog Rana temporaria. Finally, ERG light responses originating in mouse cone photoreceptors were found to be suppressed in the dark-adapted retina, apparently through direct electrical coupling between rods and cones. The results indicated this coupling is weakened by moderate background light, explaining a long known phenomenon of unknown origin: light-induced growth of cone flash responses in mammalian ERG. This is indicative of a previously unknown mechanism of retinal adaptation