Retinal rods construct an elaborate cilium, the outer segment, in order to provide for vision in dim light. The rod outer segment contains a stack of hollow disks whose membranes are packed with rhodopsin. Rod disks are punctuated by an incisure(s) that penetrates the disk surface, presumably to promote longitudinal diffusion of soluble substances in the cytoplasm. Peripherin-2 (aka rds) and rom1 are structural proteins critical to the formation of the disk rim. An inadequate supply causes a rod degeneration and irreparable blindness. We hypothesize that the rod normally expresses peripherin-2 and rom1 in slight excess and deposits the surplus in the incisure. But if expression levels should be inadequate because of a mutation or even just daily fluctuations, the incisure can be sacrificed to divert the structural proteins to disk rim formation. Previous studies on mutant mice revealed a dependence of disk size on the amount of rhodopsin expressed, so to test our hypothesis, the expression levels of rhodopsin, peripherin- and rom1 will be varied singly and in combinations in mutant mice and disk size and incisure length will be examined by electron microscopy. To operate as a photon counter, the rod must generate reproducible responses to each photon absorbed. But a rhodopsin photoisomerization at the edge of a disk reduces the cGMP levels near ion channels causing them to close with little delay, whereas a photoisomerization at the disk center is distant from the channels, so they close only after cGMP diffuses from the cytosol near the plasma membrane towards the disk center. Hence, randomness in the location of photoisomerization on the disk surface should contribute to photon response variability. We will explore how disk size and incisure length affect this source of variability by biophysical modeling and by single cell recording with side-on illumination directed to the disk rims or passing through the center of the outer segment to elicit photoisomerizations at all possible distances from the disk rim. Bicarbonate enters the outer segment at its base and diffuses to its tip with concomitant removal by carriers along the outer segment, setting up an axial concentration gradient. The disks create a barrier for the axial diffusion of soluble substances, so disk radius and incisures can alter the steepness of the gradient. Since bicarbonate accelerates photoresponse kinetics, photon responses at the base will be faster than those at the tip. We will assess how disk size, incisures, outer segment length and bicarbonate affect photon response variability due to randomness in the axial location of photoisomerization by biophysical modeling and by recording single rods while illuminating either the base, tip or the entire outer segment with a slit. Deficiencies in rhodopsin, peripherin-2 or rom1 alter disk structure and can cause a degenerative retinal disease. This proposal explores how relative levels of these proteins influence disk size and incisure structure, and how these parameters affect single photon response reproducibility. The long term goal is to understand the determinants of disk structure and how structure determines the rod's functional properties.
Proper disk formation in rod outer segments is essential for vision in dim lighting and for preventing rod degeneration. Here, we investigate how rod outer segment disk structure changes with the expression levels of opsin and several critical structural proteins in mutant mice in order to explore the role of the incisure as a depository for structural proteins when expression exceeds the demand required for proper disk formation. We further test by biophysical modeling and by single cell recording, the effects of disk structure on single photon response reproducibility, a feature of signaling that is critical for rods in their function as photon counters.
