Mao Yingyu, Finnemann Silvia C
Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Larkin Hall, 441 East Fordham Road, 10458, Bronx, NY, USA.
Adv Exp Med Biol. 2016;854:717-23. doi: 10.1007/978-3-319-17121-0_95.
Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 μm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.
哺乳动物眼中视杆光感受器外段的更新涉及到在光照开始后，老化的远端外段碎片（POS）同步进行昼夜性脱落，这些碎片会被相邻的视网膜色素上皮（RPE）迅速从视网膜下间隙清除。RPE细胞中吞噬的POS吞噬体成熟为酸化的吞噬溶酶体，从而完成对POS大分子的酶促降解。在这里，我们使用嗜酸性荧光染料溶酶体示踪剂（LysoTracker）来标记新鲜解剖的活大鼠RPE组织平铺片中的酸性细胞器。我们观察到，所有成像的RPE细胞都含有大量酸化的POS吞噬溶酶体，与光照开始前1小时或光照开始后4小时相比，光照开始后2小时每个细胞中这些吞噬溶酶体的数量显著增加。吞噬缺陷型突变体RCS大鼠RPE中缺乏直径相似（1-2μm）的细胞器，这证实了溶酶体示踪剂实时成像检测到的是POS吞噬溶酶体。在昼夜吞噬爆发期间，RPE/脉络膜中溶酶体膜蛋白LAMP-1没有增加，这表明RPE原位形成POS吞噬溶酶体可能不涉及额外的溶酶体膜生物合成。综上所述，我们报道了一种新的成像方法，该方法可以直接检测POS吞噬体的酸化，并能够对活的RPE组织异位进行POS吞噬作用的快速追踪和定量分析。
