Epitope masking of rhabdomeric rhodopsin during endocytosis-induced retinal degeneration.
Epitope masking of rhabdomeric rhodopsin during endocytosis-induced retinal degeneration.
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作者：
N. Orem;P. Dolph
目的研究视紫红质在内吞作用介导的视网膜变性中的命运。方法将果蝇在完全黑暗中饲养，并在解剖和固定视网膜之前将其置于光照下24 h。在超微冷冻切片机上切割1 μ m冷冻切片，然后用视紫红质或抑制蛋白特异性抗体染色。光感受器细胞特异性蛋白质的定位通过共聚焦显微镜确定。结果果蝇在经历内吞介导的视网膜变性过程中，在变性过程的早期表现出明显的横纹肌视紫红质丢失。使用不同的免疫试剂，遗传背景，和光处理，我们已经发现，结合arrestin的视紫红质掩蔽的C-末端单克隆抗体表位，并导致视紫红质免疫反应性的损失。只有当视紫红质从质膜中耗尽时，才会发生免疫反应性横纹肌视紫红质的损失，使得在横纹肌内发现与抑制蛋白处于化学计量水平。结论：当视紫红质和arrestin水平相等时，arrestin与视紫红质的结合导致视紫红质C-末端抗体表位的掩蔽。由于掩蔽只能发生在大部分视紫红质已经从横纹肌耗尽后，因此可以得出结论，在内吞诱导的视网膜变性期间，大部分视紫红质定位于小斑点中的细胞体。这些数据表明视紫红质在细胞质中处于极高的局部浓度。数据进行了讨论的背景下，在视网膜变性疾病的感光细胞凋亡的模型。
PURPOSE To determine the fate of rhodopsin during endocytosis-mediated retinal degeneration. METHODS Drosophila stocks were raised in complete darkness and shifted to light for 24 h prior to dissection and fixation of retinas. 1 microm frozen sections were cut on an ultracryomicrotome, then stained with antibodies specific for rhodopsin or arrestin. Localization of photoreceptor cell-specific proteins was determined by confocal microscopy. RESULTS Flies that are in the process of undergoing endocytosis-mediated retinal degeneration exhibit an apparent loss of rhabdomeric rhodopsin at early times during the degenerative process. Using different immunological agents, genetic backgrounds, and light treatments, we have found that the binding of arrestin to rhodopsin masked the C-terminal monoclonal antibody epitope and resulted in the loss of rhodopsin immunoreactivity. The loss of immunoreactive rhabdomeric rhodopsin only occurred when rhodopsin was depleted from the plasma membrane such that it was found within the rhabdomere at stoichiometric levels with arrestin. CONCLUSIONS When rhodopsin and arrestin are found at equal levels, binding of arrestin to rhodopsin results in the masking of the antibody epitope on the C-terminus of rhodopsin. Since masking can only occur after most of the rhodopsin has been depleted from the rhabdomere, it can be concluded that during endocytosis-induced retinal degeneration, much of the rhodopsin is localized to the cell body in small puncta. These data suggest that rhodopsin is at extremely high local concentrations in the cytoplasm. The data are discussed in the context of a model for photoreceptor cell apoptosis in retinal degenerative disorders.