Punnonen E L, Ryhänen K, Marjomäki V S
Department of Biological and Environmental Science, University of Jyväskylä, Finland.
Eur J Cell Biol. 1998 Apr;75(4):344-52. doi: 10.1016/s0171-9335(98)80067-8.
Temperatures around 20 degrees C are known to block degradation of endocytosed material by preventing its transport to lysosomes, accordingly reduced temperature has been widely used to define endosomes. Newer studies have revealed that the low temperature block is proximal to perinuclear late endosomes, but it is not clear whether the block is already in early endosomes, or whether the traffic proceeds to multivesicular carrier endosomes which mediate transport from early to late compartments. We have now focused on this problem using rat cardiac myocytes. First, cell fractionation on Percoll gradients showed that at reduced temperatures (22 degrees C and 26 degrees C), with prolonged chase periods, endocytosed horseradish peroxidase was able to proceed from early endosomes to later compartments but not up to lysosomes. Further, microscopic experiments with fluorescent endocytic marker FITC-dextran showed that the marker did not accumulate in the perinuclear area, as was the case at 37 degrees C, but stayed in peripheral cytoplasm at reduced temperatures, even after 16-h chase. Second, electron microscopic pulse labeling showed that, at 22 degrees C, endocytosed gold particles (BSA-gold) are transported to compartments not accessible to HRP internalised later to early endosomes. Thus, these gold particles had reached a later compartment. Morphologically these vesicles were multivesicular bodies of 0.5-1 microm in diameter. Third, we used fluorescence microscopy to study the effect of reduced temperature on transferrin uptake and recycling. At 17 degrees C and 22 degrees C, transferrin was internalized normally to peripheral (sorting) and perinuclear (recycling) vesicles. If transferrin was first taken up at 37 degrees C, and the cells were then chased at various temperatures from 37 degrees C to 17 degrees C, the recycling was slowed down but not entirely blocked at the reduced temperatures. From these results we can conclude that (1) endocytic traffic is blocked in multivesicular carrier endosomes at and below 26 degrees C, and that (2) reduced temperature slows down transport in the recycling pathway, without a complete block.
已知20摄氏度左右的温度会通过阻止内吞物质向溶酶体的转运来阻断其降解，因此降低温度已被广泛用于界定内体。最新研究表明，低温阻滞发生在核周晚期内体的近端，但尚不清楚该阻滞是否已经发生在早期内体中，或者转运是否会继续至介导从早期区室到晚期区室转运的多囊泡载体内体。我们现在利用大鼠心肌细胞聚焦于这个问题。首先，在Percoll梯度上进行细胞分级分离显示，在降低温度（22摄氏度和26摄氏度）下，随着追踪时间延长，内吞的辣根过氧化物酶能够从早期内体转运至晚期区室，但无法到达溶酶体。此外，用荧光内吞标记物FITC-葡聚糖进行的显微镜实验表明，该标记物不会像在37摄氏度时那样在核周区域积累，而是在降低温度下停留在外周细胞质中，即使在追踪16小时后也是如此。其次，电子显微镜脉冲标记显示，在22摄氏度时，内吞的金颗粒（牛血清白蛋白-金）被转运至辣根过氧化物酶后来内化到早期内体时无法到达的区室。因此，这些金颗粒已到达晚期区室。从形态学上看，这些囊泡是直径为0.5 - 1微米的多囊泡体。第三，我们利用荧光显微镜研究降低温度对转铁蛋白摄取和循环的影响。在17摄氏度和22摄氏度时，转铁蛋白正常内化至外周（分选）和核周（循环）囊泡。如果转铁蛋白首先在37摄氏度时被摄取，然后将细胞在从37摄氏度到17摄氏度的不同温度下进行追踪，循环会减慢，但在降低温度下不会完全阻断。从这些结果我们可以得出结论：（1）在26摄氏度及以下，内吞转运在多囊泡载体内体中被阻断；（2）降低温度会减慢循环途径中的转运，但不会完全阻断。
