Department of Biology, Saint Joseph's University, Philadelphia, Pennsylvania, USA.
Vis Neurosci. 2021 Sep 15;38:E013. doi: 10.1017/S0952523821000122.
Retinomotor movements include elongation and contraction of rod and cone photoreceptors, and mass migration of melanin-containing pigment granules (melanosomes) of the retinal pigment epithelium (RPE) within the eyes of fish, frogs, and other lower vertebrates. Eyes of these animals do not contain dilatable pupils; therefore the repositioning of the rods and cones and a moveable curtain of pigment granules serve to modulate light intensity within the eye. RPE from sunfish (Lepomis spp.) can be isolated from the eye and dissociated into single cells, allowing in vitro studies of the cytoskeletal and regulatory mechanisms of organelle movement. Pigment granule aggregation from distal tips of apical projections into the cell body can be triggered by the application of underivatized cAMP, and dispersion is effected by cAMP washout in the presence of dopamine. While the phenomenon of cAMP-dependent pigment granule aggregation in isolated RPE was described many years ago, whether cAMP acts through the canonical cAMP-PKA pathway to stimulate motility has never been demonstrated. Here, we show that pharmacological inhibition of PKA blocks pigment granule aggregation, and microinjection of protein kinase A catalytic subunit triggers pigment granule aggregation. Treatment with a cAMP agonist that activates the Rap GEF, Epac (Effector protein activated by cAMP), had no effect on pigment granule position. Taken together, these results confirm that cAMP activates RPE pigment granule motility by the canonical cAMP-PKA pathway. Isolated RPE cells labeled with antibodies against PKA RIIα and against PKA-phosphorylated serine/threonine amino acids show diffuse, punctate labeling throughout the RPE cell body and apical projections. Immunoblotting of RPE lysates using the anti-PKA substrate antibody demonstrated seven prominent bands; two bands in particular at 27 and 64 kD showed increased levels of phosphorylation in the presence of cAMP, indicating their phosphorylation could contribute to the pigment granule aggregation mechanism.
视蛋白运动包括鱼类、青蛙和其他低等脊椎动物眼睛中的棒状和圆锥状光感受器的伸长和收缩，以及视网膜色素上皮（RPE）中含有黑色素的色素颗粒（黑素小体）的大规模迁移。这些动物的眼睛没有可扩张的瞳孔；因此，棒状和圆锥状的重新定位以及可移动的色素颗粒幕布可以调节眼睛内的光强度。太阳鱼（Lepomis spp.）的 RPE 可以从眼睛中分离出来并解离成单个细胞，从而可以在体外研究细胞器运动的细胞骨架和调节机制。未衍生的 cAMP 的应用可以触发来自顶端突起远端尖端的色素颗粒聚集，而多巴胺存在时通过 cAMP 冲洗可以实现分散。虽然多年前就描述了分离的 RPE 中 cAMP 依赖性色素颗粒聚集的现象，但 cAMP 是否通过经典的 cAMP-PKA 途径作用以刺激运动性尚未得到证明。在这里，我们表明 PKA 的药理学抑制阻断了色素颗粒聚集，并且蛋白激酶 A 催化亚基的微注射触发了色素颗粒聚集。用激活 Rap GEF、Epac（cAMP 激活的效应蛋白）的 cAMP 激动剂处理对色素颗粒位置没有影响。总之，这些结果证实 cAMP 通过经典的 cAMP-PKA 途径激活 RPE 色素颗粒运动。用针对 PKA RIIα 和 PKA 磷酸化丝氨酸/苏氨酸氨基酸的抗体标记的分离的 RPE 细胞在整个 RPE 细胞体和顶端突起中显示弥散、点状标记。用抗 PKA 底物抗体对 RPE 裂解物进行免疫印迹显示出七个明显的条带；特别是在 cAMP 存在下，两条在 27 和 64 kDa 的条带显示出更高水平的磷酸化，表明它们的磷酸化可能有助于色素颗粒聚集机制。
