Institute of General Physiology, University of Ulm, Ulm, Germany.
Institute of General Physiology, University of Ulm, Ulm, Germany ; Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria.
PLoS One. 2014 Jan 21;9(1):e84926. doi: 10.1371/journal.pone.0084926. eCollection 2014.
Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca(2+) release upon ATP treatment and significant triggering of LB exocytosis. These findings are in line with the strong Ca(2+)-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca(2+) signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca(2+) signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP.
富含亮氨酸重复激酶 2（LRRK2）已知在包括帕金森病、克罗恩病、麻风病和癌症在内的各种疾病的发病机制中发挥作用。LRRK2 被认为参与许多细胞生物学过程，如囊泡运输、转录、自噬和溶酶体途径。最近对 LRRK2 敲除（LRRK2 -/-）小鼠肺部的组织学研究表明，肺泡 II 型（ATII）上皮细胞中的板层小体（LB）明显增大。LB 是大型、与溶酶体相关的肺表面活性剂储存细胞器，当 LB 出胞时被释放到肺泡腔中。在这项研究中，我们使用高分辨率、亚细胞活细胞成像测定来研究 LRRK2 -/- 大鼠的原代 ATII 细胞是否可以观察到类似的形态变化，以及这种变化是否导致 LB 出胞的改变。与小鼠的报告类似，LRRK2 -/- 大鼠的 ATII 细胞含有明显增大的 LB，导致 LB 体积增加超过 50%。用 ATP 刺激 ATII 细胞，会导致来自 LRRK2 -/- 动物的细胞中 LB 出胞的比例显著增加。LRRK2 -/- 细胞在用 ATP 处理时还显示出细胞内 Ca2+释放的增加，以及 LB 出胞的显著触发。这些发现与 LB 融合活性的强烈 Ca2+依赖性一致，并表明 LRRK2 -/- 通过调节细胞内 Ca2+信号转导来影响 ATII 细胞的出胞反应。表面活性剂分泌的融合后调节保持不变。融合囊泡的肌动蛋白涂层和随后促进表面活性剂排出的囊泡压缩在 LRRK2 -/- 和 wt 细胞中是可比的。令人惊讶的是，刺激 LB 出胞后，LRRK2 -/- 细胞的表面活性剂（磷脂）释放减少，可能是由于 LB 成熟受损和 LB 对表面活性剂的装载减少。总之，我们的结果表明，LRRK2 -/- 影响 LB 大小，调节细胞内 Ca2+信号转导，并在刺激 ATII 细胞用 ATP 时促进 LB 出胞。
