Laboratory of Neuroendocrinology-Molecular Cell Physiology, Medical Faculty, Institute of Pathophysiology, University of Ljubljana, Slovenia.
Acta Physiol (Oxf). 2011 Dec;203(4):457-71. doi: 10.1111/j.1748-1716.2011.02337.x. Epub 2011 Aug 12.
We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker-stained vesicles in cultured rat astrocytes.
Time-lapse confocal images were acquired, and vesicle mobility was analysed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles.
At rest, when mobility was monitored for 2 min in bath with Ca(2+), two vesicle populations were discovered: (1) non-mobile vesicles (6.1%) with total track length (TL) < 1 μm, averaging at 0.33 ± 0.01 μm (n = 1305) and (2) mobile vesicles (93.9%) with TL > 1 μm, averaging at 3.03 ± 0.01 μm (n = 20,200). ALS IgG (0.1 mg mL(-1)) from 12 of 13 patients increased the TL of mobile vesicles by approx. 24% and maximal displacement (MD) by approx. 26% within 4 min, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca(2+), indicating that ALS IgG vesicle mobility enhancement involves changes in Ca(2+) homeostasis. To examine whether enhanced mobility relates to elevated Ca(2+) activity, cells were stimulated by 1 mm ATP, a cytosolic Ca(2+) increasing agent, in the presence (2 mm) and in the absence of extracellular Ca(2+). ATP stimulation triggered an increase in TL by approx. 7% and 12% and a decrease in MD by approx. 11% and 1%, within 4 min respectively. Interestingly, none of the stimuli triggered the release of vesicle cargo.
Amyotrophic lateral sclerosis-IgG-enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.
我们研究了肌萎缩侧索硬化症（ALS）患者的纯化免疫球蛋白 G（IgG）对培养的大鼠星形胶质细胞中 Lysotracker 染色囊泡的迁移和胞吐释放的影响。
在应用 ALS IgG 前后，通过时程共聚焦图像采集分析囊泡的迁移。通过对染色细胞器的囊泡计数来评估货物胞吐。
在静息状态下，当在 Ca(2+)浴中监测 2 分钟的迁移时，发现了两种囊泡群体：（1）非迁移囊泡（6.1%），总轨迹长度（TL）<1μm，平均为 0.33±0.01μm（n=1305）和（2）迁移囊泡（93.9%），TL>1μm，平均为 3.03±0.01μm（n=20200）。来自 13 例患者中的 12 例的 ALS IgG（0.1mg/mL）在 4 分钟内将迁移囊泡的 TL 增加了约 24%，最大位移（MD）增加了约 26%，而对照组的 IgG 没有改变囊泡的迁移。在缺乏 Ca(2+)的细胞外溶液中，ALS IgG 对囊泡迁移的增强作用降低，表明 ALS IgG 囊泡迁移的增强作用涉及 Ca(2+)内稳态的变化。为了研究增强的迁移是否与升高的 Ca(2+)活性有关，在存在（2mm）和不存在细胞外 Ca(2+)的情况下，用 1mm ATP（一种增加细胞质 Ca(2+)的试剂）刺激细胞。ATP 刺激在 4 分钟内分别引起 TL 增加约 7%和 12%，MD 减少约 11%和 1%。有趣的是，没有一种刺激触发囊泡货物的释放。
肌萎缩侧索硬化症 IgG 增强星形胶质细胞中的囊泡迁移涉及钙稳态的变化。
