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

基本信息

批准号：
10650776
负责人：
CLINT L MAKINO
金额：
$ 56.73万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
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/10650776
关键词：
Acceleration Address Affect Architecture Bicarbonates Blindness Cell membrane Cells Cilia Complex Cyclic GMP Cytoplasm Cytosol DNA Sequence Alteration Dependence Deposition Diffuse Diffusion Disparity 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 Penetration 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 repository response retinal neuron retinal rods rod outer segment disc spatiotemporal

项目摘要

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.

视网膜杆构造了一个精美的纤毛，即外部段，以便在昏暗的光线下提供视觉。这杆外部段包含一堆空心盘，其膜上充满了视紫红质。杆盘被穿透盘表面的切口打断，大概是为了促进纵向扩散细胞质中的可溶性物质。外围蛋白-2（又名RDS）和ROM1是对蛋白质至关重要的结构蛋白磁盘边缘的形成。供应不足会导致杆变性和无法弥补的失明。我们假设杆通常以轻微的过量表达外围蛋白-2和rom1，并将剩余沉积在切口。但是，如果由于突变甚至每天都不是不足的表达水平波动，可以牺牲切口将结构蛋白转移到盘状形成。以前的关于突变小鼠的研究表明，磁盘大小对表达的视紫红质的量的依赖性，因此要测试我们的假设，视紫红质，外周蛋白和ROM1的表达水平将单独进行，并在将通过电子显微镜检查突变小鼠和磁盘大小和切口长度的组合。要作为光子计数器操作，杆必须对每个光子吸收的每个光子产生可重复的响应。但是磁盘边缘的视紫红质光异构化会降低导致离子通道附近的CGMP水平它们几乎不会延迟关闭，而磁盘中心的光异构化距离频道很远，因此仅在CGMP从质膜附近的细胞质扩散到磁盘中心之后，它们才关闭。因此，磁盘表面光异构化位置的随机性应有助于光子响应变异性。我们将探讨磁盘大小和切口长度如何通过生物物理建模和通过侧面照明指向磁盘轮辋或通过的单细胞记录穿过外部段的中心，在距磁盘边缘的所有可能距离处引起光异构化。碳酸氢盐进入其底部的外部段，并以载体伴随的去除扩散到其尖端沿外部段，设置轴向浓度梯度。磁盘为轴向创造障碍可溶性物质的扩散，因此磁盘半径和切口会改变梯度的陡度。自从碳酸氢盐加速了光响应动力学，基部的光子反应将快于提示。我们将评估磁盘大小，切口，外部段长度和碳酸氢盐如何影响光子响应通过生物物理建模和通过记录单杆，同时用缝隙照亮底座，尖端或整个外部段。视紫红质，外围蛋白-2或ROM1的缺陷改变磁盘结构，可能导致视网膜变性疾病。该建议探讨了这些蛋白质的相对水平如何影响盘的大小和切入结构，以及这些参数如何影响单个光子响应可重复性。长期目标是了解磁盘结构的决定因素以及结构如何确定杆的功能特性。