Rod outer segment structure: determinants and its effect on the photon response

杆外段结构：决定因素及其对光子响应的影响

• 批准号: 10238092
• 负责人: CLINT L MAKINO
• 金额: $ 52.8万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238092
• 关键词: Address; Affect; Architecture; Bicarbonates; Blindness; Cell membrane; Cells; Cilia; Complex; Cyclic GMP; Cytoplasm; Cytosol; DNA Sequence Alteration; Dependence; Deposition; Diffuse; Diffusion; Distant; Drops; Electron Microscopy; Electrophysiology (science); Excision; Genetic; Goals; Heterogeneity; Image; Ion Channel; Kinetics; Lateral; Lead; Length; Light; Lighting; Location; Membrane; Membrane Proteins; Modeling; Movement; Mus; Mutant Strains Mice; Mutation; Opsin; Photons; Phototransduction; Positioning Attribute; Primates; Property; Proteins; Radial; Reproducibility; Research Priority; Retinal Degeneration; Rhodopsin; Rod; Rod Outer Segments; Role; Salamander; Side; Signal Transduction; Source; Structural Protein; Structure; Surface; Synapses; Testing; Vision; absorption; aqueous; base; biophysical model; cilium biogenesis; design; improved; overexpression; peripherin; photoreceptor degeneration; prevent; quantum; repository; response; retinal neuron; retinal rods; rod outer segment disc; spatiotemporal

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.

视网膜杆构成一个精心制作的纤毛，外节，以提供在昏暗的光线下的视觉。的 杆外部部分包含一堆中空圆盘，其膜填充有视紫红质。杆盘 被穿透椎间盘表面的增厚所打断，可能是为了促进纵向扩散 细胞质中的可溶性物质。外周蛋白-2（又称rds）和rom 1是结构蛋白， 盘缘的形成。供应不足会导致视杆变性和无法修复的失明。我们 假设视杆细胞正常情况下略微过量地表达外周蛋白-2和rom 1，并将多余的蛋白沉积在视杆细胞中， 的increase。但是如果因为突变或者仅仅是每天的表达量不足 波动时，增加的蛋白质可以被牺牲，以转移结构蛋白质到盘缘形成。先前 对突变小鼠的研究揭示了视紫红质表达量对圆盘大小的依赖性， 我们假设视紫红质、外周蛋白和ROM 1的表达水平将单独变化， 将通过电子显微镜检查突变小鼠中的组合以及盘大小和增量长度。 为了作为光子计数器工作，棒必须对吸收的每个光子产生可再现的响应。 但是，在光盘边缘的视紫红质光异构化降低了离子通道附近的cGMP水平， 他们关闭几乎没有延迟，而在光盘中心的光异构化是远离通道，所以 它们仅在cGMP从质膜附近的胞质溶胶向盘中心扩散后才闭合。 因此，在光盘表面上的光致异构化的位置的随机性应有助于光子 反应变异性我们将探讨磁盘大小和增量长度如何影响这种可变性的来源， 生物物理建模和通过单细胞记录，侧照照射指向盘边缘或通过 通过外段的中心，以在距盘边缘的所有可能距离处引发光异构化。 二头肌从基部进入外节，并扩散到其尖端，同时被载体去除 沿着外段，建立轴向浓度梯度。这些圆盘为轴向的 溶解物质的扩散，因此圆盘半径和切口可以改变梯度的陡度。以来 碳酸氢盐加速光响应动力学，在基部的光子响应将比在基部的光子响应快。 tip.我们将评估椎间盘大小、切口、外节长度和碳酸氢盐如何影响光子响应 通过生物物理建模和生物物理建模， 记录单个棒，同时用狭缝照射基部、尖端或整个外节。 视紫红质、外周蛋白-2或rom 1的缺陷改变了视盘结构，并可导致视网膜变性。 疾病该提案探讨了这些蛋白质的相对水平如何影响磁盘大小和增量结构， 以及这些参数如何影响单光子响应再现性。长期目标是了解 圆盘结构的决定因素以及结构如何决定杆的功能特性。