Tsuiki Eiko, Fujita Akikazu, Ohsaki Yuki, Cheng Jinglei, Irie Toshiaki, Yoshikawa Kiwamu, Senoo Haruki, Mishima Kazuaki, Kitaoka Takashi, Fujimoto Toyoshi
Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Invest Ophthalmol Vis Sci. 2007 Jun;48(6):2858-67. doi: 10.1167/iovs.06-0768.
To investigate the effect of light stimulation on lipid droplets (LDs) and LD proteins in the retinal pigment epithelium (RPE).
Dark-adapted mouse eyes were exposed to intense flashes of light, and ARPE-19 cells were treated with all-trans-retinol. The two specimens were labeled with BODIPY493/503 for LDs and with antibodies for three LD proteins: adipocyte differentiation-related protein (ADRP), TIP47, and Rab18. The labeling intensity in fluorescence microscopy was quantified by image analysis. Localization of mutated TIP47 was also examined. Immunoelectron microscopy was performed for ADRP in mouse RPE. Expression of TIP47 in ARPE-19 cells was knocked down by RNA interference (RNAi), and its effect on retinyl ester storage was measured by HPLC.
Both flashes of light on mouse eyes and all-trans-retinol on ARPE-19 cells caused rapid translocation of TIP47 from the cytosol to LDs, whereas ADRP distributed constitutively in LDs. The density of LDs did not show visible changes by any treatment. The localization of TIP47 to LDs was abolished when either the amino-terminal or the carboxyl-terminal half of the molecule was deleted, but was enhanced by a short deletion in the carboxyl terminus. Manipulation of TIP47 expression by RNAi or cDNA transfection did not affect the retinyl ester amounts in ARPE-19 cells significantly.
All-trans-retinol generated by photobleaching in the retina induces rapid translocation of TIP47 to LDs in the RPE.
研究光刺激对视网膜色素上皮（RPE）中脂滴（LDs）及LD蛋白的影响。
将暗适应的小鼠眼睛暴露于强光闪烁下，并用全反式视黄醇处理ARPE - 19细胞。这两个样本用BODIPY493/503标记LDs，并用针对三种LD蛋白的抗体进行标记：脂肪细胞分化相关蛋白（ADRP）、TIP47和Rab18。通过图像分析对荧光显微镜下的标记强度进行定量。还检测了突变型TIP47的定位。对小鼠RPE中的ADRP进行免疫电子显微镜检查。通过RNA干扰（RNAi）敲低ARPE - 19细胞中TIP47的表达，并通过高效液相色谱法测量其对视黄酯储存的影响。
小鼠眼睛上的强光闪烁和ARPE - 19细胞上的全反式视黄醇均导致TIP47从细胞质快速转运至LDs，而ADRP则持续分布于LDs中。任何处理均未使LDs的密度出现明显变化。当分子的氨基末端或羧基末端的一半被缺失时，TIP47在LDs上的定位被消除，但羧基末端的短缺失则增强了这种定位。通过RNAi或cDNA转染对TIP47表达进行操作，并未显著影响ARPE - 19细胞中的视黄酯含量。
视网膜中光漂白产生的全反式视黄醇诱导TIP47快速转运至RPE中的LDs。
