Arshavsky's Research Projects

Our laboratory studies molecular and cellular mechanisms of vision. We are interested in many functional aspects of the vertebrate photoreceptor cell, which is a sensory neuron responsible for detection and primary processing of information entering the eye in the form of photons of light. The basic functions of photoreceptors are to capture photons, to generate a second messenger signal, to translate this signal into a change in electrical activity and, finally, to transmit this information to the secondary neurons in the retina through modulation of synaptic release. Because the function of this cell is so well defined and because it is uniquely suitable for study using modern multi-disciplinary approaches, the photoreceptor is an almost unmatched model system for addressing fundamental issues in molecular and cellular neuroscience, as well as in cell signaling in general. At this time we are pursuing three major experimental directions.

I. REGULATION OF G PROTEIN SIGNALING

Our first direction is to understand how the duration of the light-evoked response is regulated on the molecular level. The photoresponse should not only be sensitive but also rapid, allowing the photoreceptor to respond to the next light event. As such, our cones can detect the flickering of a computer monitor operating at a 60-hertz frequency. The molecular mechanisms behind this phenomenon utilize the regulation of G protein signaling in the phototransduction cascade illustrated below. Vision begins when a molecule of visual pigment, a GPCR, becomes excited by light. Photoexcited visual pigment activates multiple molecules of transducin, a photoreceptor-specific member of the G protein family. Each activated transducin stimulates the activity of its effector, cGMP phosphodiesterase, which leads to the decrease in intracellular levels of cGMP. This ultimately leads to a reduction in the conductivity of the cGMP-gated ion channels on the photoreceptor plasma membrane, producing a stereotypical electrical response known as the photoresponse.


Rapid photoresponse recovery requires rapid inactivation of all phototransduction proteins. We are particularly interested in mechanisms regulating the lifetime of activated transducin. Like any other G protein, transducin inactivates upon the hydrolysis of GTP bound to its alpha subunit. However, the intrinsic rate of this reaction is only 1-2 turnovers per minute, nearly 100-fold too slow to account for the recovery rate of real photoresponses. The dim flash responses of mammalian rods reach their response peak in about 150 ms and subsequently recover with a time constant of about 200 ms (see Figure below).

These flash responses were recorded from a rod of a wild type mouse (WT) and a rod of a mouse lacking the GTPase activating complex (R9AP knockout) by Marie Burns and her colleagues at the University of California-Davis.

A Studies conducted over the past decade revealed that photoreceptors contain several proteins engaged in activating the slow intrinsic GTPase activity of transducin to a physiologically fast rate. The key role in this process belongs to the GTPase activating protein complex consisting of the ninth member of Regulators of G protein Signaling family (RGS9), the type 5 b subunit of G proteins (Gb5) and the RGS9 Anchor Protein (R9AP), which anchors the complex on the surface of the photoreceptor membranes. Furthermore, the ability of this complex to activate transducin GTPase is further potentiated by the effector protein for transducin, the g-subunit of cGMP phosphodiesterase. Work in this direction allowed us to pinpoint several principles in cell signaling, which extend beyond visual signaling pathways. For example, we developed the concept of affinity adapters, which are protein domains or subunits that direct regulatory proteins to their specific physiological targets in different signaling pathways, and found a case where such an adapter regulates the specificity of RGS-G protein interactions in the brain. Another example regards the functional role of the N-terminal domain of RGS9, termed DEP. DEP homology domains are present in over 60 proteins found in the human genome, but in most cases their function remains unknown. We have shown that the binding of RGS9 to R9AP in photoreceptors occurs via the DEP domain and that this binding is essential for RGS9 delivery to photoreceptor outer segments. Finally, our studies of the membrane anchor, R9AP, resulted in the discovery of its brain-specific homolog, R7BP, which regulates the intracellular localization patterns of brain-specific RGS9 homologues. The studies of R7BP are now continued in the laboratory of our alumnus, Kirill Martemyanov at the University of Minnesota.

The challenge of our current experiments is to define the role of each individual protein subunit of the GTPase activating complex in regulating its catalytic activity, substrate specificity as well as its delivery to photoreceptor outer segments. We are also interested in exploring several The challenge of our current experiments is to define the role of each individual protein component of the GTPase activating complex in regulating its catalytic activity and substrate specificity, as well as its delivery to photoreceptor outer segments. We are also interested in understanding how the speed of photoresponse recovery affects visual perception mediated by both rod- and cone-driven pathways. This is a subject of ongoing behavioral studies of mutant mice lacking individual components of the GTPase activating complex conducted in collaboration with Robert Barlow at the SUNY Upstate Medical University.

II. LIGHT-DEPENDENT TRANSLOCATION OF SIGNALING PROTEINS BETWEEN\
THE FUNCTIONAL COMPARTMENTS OF PHOTORECEPTORS


A fascinating property of photoreceptors is their ability to adjust the protein complement of their outer segment, the cellular compartment where phototransduction takes place, to optimize their ability to function at various levels of ambient illumination. Studies conducted by our lab and others revealed that at least four signaling proteins — arrestin, transducin, recoverin and Grb-14 — translocate between the outer and inner segments in a light-dependent manner.

One experimental approach which we developed in order to analyze protein translocation quantitatively is particularly useful in these studies. This approach takes advantage of the layered organization of the photoreceptors in the vertebrate retina. It is based on serial tangential sectioning of flat-mounted frozen retina followed by the Western blot analysis of proteins of interest in individual sections.

Schematic diagram depicting the method of serial tangential sectioning of the photoreceptor layer. Marker proteins, rhodopsin (confined to the outer segments), cytochrome C (present in high concentrations in the mitochondria-rich ellipsoid region of the inner segment) and synaptophysin (present in pre-synaptic vesicles), allow the assignment of proteins to specific subcellular compartments of the rod cell.

Applying serial tangential sectioning with Western blotting to the analysis of subcellular transducin distributions in the rods of dark- and light-adapted rats, we found that ~90% of transducin undergoes the light-dependent translocation within only ~ 10 minutes. Collaborating with the laboratory of Edward Pugh, we then found that transducin translocation is adaptive because the reductionin the outer segment transducin concentration is accompanied by a corresponding reduction in the signal amplification of the phototransduction cascade. This mechanism extends the operating range of photoreceptor sensitivity upon exposures to bright background illumination.

Quantitative analysis of transducin distribution throughout the subcellular compartments of rat rods. A. Western blots of serial tangential sections from dark-adapted (Dark) and illuminated (Light) rat retinas. Rhodopsin (Rho), transducin subunits (G a t and G b t) and cytochrome C (Cyt) -specific protein bands were detected. B. Densitometric profiles of each of the proteins analyzed in the Western blots shown above in A expressed as a % of the total protein in all sections analyzed. C. Cartoon showing the distribution of transducin subunits in the rod photoreceptor cell in dark and light-adapted retinas.

Schematic Representation of Transducin, Arrestin, and Recoverin Subcellular Distribution in the Dark- and Light-Adapted Rod

Our current efforts are devoted to understanding the functional consequences of the translocation of each of these proteins. For example, we have found that light-driven transducin translocation away from rod outer segments helps rods adapt to bright light because the reduction in the outer segment transducin concentration is accompanied by a corresponding reduction in the signal amplification of the phototransduction cascade. This mechanism extends the operating range of photoreceptor sensitivity upon exposure to bright background illumination. Similar functions are proposed for arrestin and recoverin, although direct evidence in support of this hypothesis remains to be obtained. We also explore the idea that translocation of signaling proteins may underlie broader aspects of photoreceptors’ ability to function under conditions of bright light exposure, including energy rationing and neuroprotection from the adverse effect of constant illumination.

 

Another area of interest is to understand the molecular and cellular mechanisms underlying protein translocation. We are investigating how these events are triggered by light, how steep concentration gradients of signaling proteins are maintained between the photoreceptor compartments, and what are the contributions of intracellular diffusion and molecular motor-driven mechanisms in each case.

III. PROTEIN TRAFFICKING AND TARGETING IN PHOTORECEPTORS

The highly compartmentalized structure of photoreceptor cells makes them a very attractive model for studying the patterns of protein trafficking and targeting in a specialized neuron. Our recent studies aiming to understand how the GTPase activating complex is delivered to rod outer segments revealed that the central role in the underlying mechanism belongs to R9AP. Surprisingly, we could not find any specific outer segment targeting information encoded in R9AP’s amino acid sequence and instead showed that it is randomly packaged into vesicles bound for all intracellular destinations. But since rhodopsin-bearing vesicles headed for the outer segment are so abundant in this cell, the vast majority of R9AP molecules passively winds up in the outer segment. Using the animal model of transgenic Xenopus, we further showed that any membrane-associated protein construct lacking or deprived of a specific intracellular targeting sequence is also directed to the outer segment (see Figure below). Our current work is devoted to understanding the relationship between this “default” trafficking pathway and targeted outer segment protein delivery utilized by other proteins like rhodopsin.

b5

Cytochrome b5 (green) normally localizes in the ER, but without its targeting sequence is redirected tothe outer segment (right).

 

Another direction of our work is to understand the role of the actin/spectrin cytoskeleton in supporting the structural and functional organization of the outer segment. Our colleagues at Duke, Krish Kizhatil and Vann Bennett, discovered that the plasma membrane of the outer segment is decorated by ankyrin G, whereas the plasma membrane of the rest of the photoreceptor cell is decorated by ankyrin B. They further demonstrated that ankyrin G forms a tight complex with the beta-subunit of the cGMP-gated channel. In collaboration with these investigators, we demonstrated that this interaction plays a critical role in the channel delivery to the outer segment from biosynthetic membranes located outside this compartment. We are now searching for additional ankyrin G-binding proteins in photoreceptors in order to analyze the relationships among this ankyrin-dependent protein trafficking pathway, the classical rhodopsin pathway and the default pathway described above.

ankyrin
A mutation in the ankyrin G binding site on the cGMP-gated channel prevents its normal localization in the plasma membrane of the outer segment (left) and instead leads to channel accumulation in the biosynthetic membranes of the inner segment.