利用三維追蹤顯微技術追蹤自噬體及含鋅離子囊泡和溶小體在鋅離子螯合後的型態變化

Abstract

細胞內有各式各樣的膜狀胞器或囊泡，以便分工執行各種生理活動，同時也作為離子儲存的場所，以維持胞內離子恆定。多數膜狀構造多在1 um以下，並藉由分裂與融合機制，維持其動態平衡。然而一般螢光顯微鏡無法提供適當解析度，以觀察這些膜狀構造的動態變化。在本實驗中，我們在顯微鏡相機成像前，加裝一組圓柱狀透鏡，改變螢光亮點在不同聚焦面的成像形狀，再以函數計算，還原此亮點在空間中正確的位置，其精確度可達0.1 um。我們先以H2O2刺激大鼠腎上腺髓質嗜鉻細胞瘤細胞 (PC12 cell)，提高胞內鋅離子濃度，以觸發自噬體的產生，並以綠螢光蛋白修飾的微管相關蛋白1A/1B輕鏈3 (Microtubule-associated protein 1A/1B-light chain 3, LC3) 標記自噬體。結果顯示我們能成功追蹤到自噬體的形成與增大，且觀測到的自噬體平均大小成長為41.0 ± 8.5％。由於鋅離子恆定的重要性，以鋅離子螢光染劑，標記初級培養的大鼠胚胎皮質神經細胞中的鋅離子分布，發現在一些細胞中有高濃度的鋅離子聚集，以鋅離子螯合劑處理後，這些聚集亮點的型態迅速萎縮且螢光強度逐漸消散，其形態大小平均下降47.3 ± 8.3％，而螢光強度平均下降了32.0 ± 3.5％。顯示鋅離子對於維持含鋅顆粒的完整性很重要。有些研究報導溶小體 (lysosome) 也會聚集鋅離子，且溶小體大小與前述鋅離子聚集點相當，因此我們以溶小體染劑處理神經細胞，再加上鋅離子螯合劑，結果顯示溶小體的型態以及螢光強度也會快速萎縮，大小平均下降59.0 ± 6.9％，而螢光強度平均下降44.3 ± 7.0％。顯示鋅離子對於維持溶小體的完整性更為重要。為進一步了解鋅離子螯合劑所造成的溶小體形態變化，與其分裂融合機制的關係，我們先以管化 (tubulation) 抑制劑處理細胞以抑制其分裂，發現溶小體在鋅離子螯合劑處理後，其形態以及螢光消散的程度明顯地降低，大小平均下降16.0 ± 4.6％，而螢光強度平均下降29.3 ± 4.6％。顯示了鋅離子螯合所導致的萎縮和溶小體管化有很大的關係，抑制管化可以阻止鋅離子螯合所導致的萎縮。這些結果顯示鋅離子對維持溶小體的動態平衡扮演重要的角色，缺乏鋅離子，會加速溶小體的分裂，導致其變小。因此我們所用的技術，可以穩定追蹤胞內相關膜狀構造的動態變化，以探討相關生理功能。

Cells utilize various membrane-enclosed vesicles to execute different physiological activities. These vesicles are intracellular stores of ions to maintain the ionic homeostasis in the cells. Most membrane-enclosed structures are smaller than 1 um and their dynamic equilibrium is maintained by fission and fusion. However, an ordinary fluorescence microscope doesn’t have enough resolution to monitor the dynamic changes of these membrane-enclosed structures. In this study, we introduced a 3D tracking microscopic technique with a cylindrical lens before the camera to produce astigmatism. With this technique, the shape of a fluorescent particle varies in different focal planes. Recording the shape and orientation of the fluorescent particle and calculating the correlating mathematical functions, we can determine the location of the fluorescent particle with the accuracy of ~100 nm. First, we stimulated PC12 cell with H2O2 to escalate the concentration of zinc ions in the cell and accelerate the formation of autophagosomes. Meanwhile, we also tagged a well-known autophagosome marker of the microtubule-associated protein 1A/1B-light chain 3 (LC3-Ⅱ) with green fluorescent proteins (GFP). The experimental results showed that we could successfully monitor the formation and enlargement of autophagosomes. The average enlargement of the autophagosomes is 41.0 ± 8.5％. Moreover, we monitored the distribution of zinc ions in cultured neuron cells with the aid of zinc fluorescent dye. We found intracellular zinc aggregation in some cells. However, after the zinc chelator treatment, these fluorescent aggregation points dispersed dramatically with the reduction of morphological size by 47.3 ± 8.3％ and the fluorescence intensity by 32.0 ± 3.5％, indicating the importance of zinc for maintaining the completeness of zinc-containing puncta. It has been reported that zinc aggregates in lysosomes with the size of the lysosomes similar to the zinc aggregation points. Therefore, we labeld the lysosomes with lysotracker and monitored their morphological changes after zinc chelation. The result observed in lysosomes is similar to the zinc aggregation points with the dramatic reduction of morphological size by 59.0 ± 6.9％and fluorescence intensity by 44.3 ± 7.0％, indicating that zinc plays crucial roles for maintaining the completeness of lysosomes. To further understand the morphological changes of lysosomes caused by zinc chelation and their relationships between fission and fusion, we treated the cells with a tubulation inhibitor to inhibit the lysosomal fission. After the zinc chelation, the measured shrinkage of lysosomes was insignificant with the decreases of morphological size only by 16.0 ± 4.6％ and fluorescence intensity by 29.3 ± 4.6％, revealing the relation of lysosomal tubulation to its morphological changes after zinc chelation. The inhibition of tubulation can prevent the shrinkage of lysosomes caused by zinc chelation. These results showed that zinc ions play a vital role in maintaining the dynamic equilibrium of lysosomes, in which zinc deficiency will accelerate the fission of lysosome and lead to the shrinkage of lysosomes. Finally, our 3D tracking microscopic technique allowed us to monitor the dynamic changes of intracellular membrane-enclosed structures and explore the related physiological activities