Ramoino P, Fronte P, Fato M, Beltrame F, Diaspro A
Department for the Study of the Territory and its Resources, University of Genoa, Corso Europa 26, I-16132 Genoa, Italy.
J Microsc. 2002 Dec;208(Pt 3):167-76. doi: 10.1046/j.1365-2818.2002.01078.x.
In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY FL C12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 microm BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10-15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4Cl reduces the lipid storage. Low temperature (4 degrees C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.
在双小核草履虫中，通过荧光共聚焦激光扫描光学显微镜和荧光类似物胆固醇 - BODIPY FL C12（BODIPY - CE）研究了胆固醇酯的摄取和细胞内流动情况。BODIPY FL荧光团具有发射绿色荧光的特性，随着探针浓缩，荧光会发生红移。在用25微摩尔BODIPY - CE孵育30秒的细胞中，在细胞后半部胞咽周围的囊泡中发现荧光。随后，脂质被食物泡内化，荧光囊泡分布于整个细胞，细胞内膜被标记。连续标记10 - 15分钟后食物泡数量最多，之后减少，直至在喂食30分钟的细胞中找不到食物泡。BODIPY - CE积聚在位于细胞前半部分的红色标记的细胞质液滴中。当用三氟拉嗪抑制食物泡形成时，在细胞膜和顶端极的绿色标记小囊泡中发现荧光。网格蛋白介导的内吞作用的抑制并不干扰双小核草履虫中BODIPY - CE的细胞内流动：细胞前部的内膜和储存液滴被染色。相反，使用固醇结合药物可防止脂质在液滴中积累，使脂质停留在细胞质膜内。此外，用莫能菌素和细胞松弛素B处理的细胞显示出细胞膜和脂质液滴的标记，而氯化铵会减少脂质储存。低温（4摄氏度）并不阻止BODIPY - CE的内化，然而，它定位于细胞质膜水平，并不积聚在储存液滴中。此外，BODIPY - CE抑制吞噬作用，通过比较仅喂食乳胶颗粒的对照细胞与同时喂食乳胶颗粒和BODIPY - CE的细胞的食物泡形成动力学可以证明这一点。总之，这项研究指出，在双小核草履虫中，胆固醇酯通过食物泡和质膜进入细胞，并且在细胞内会改变细胞功能。
