Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Ulm, Germany.
Anal Chem. 2012 Jul 3;84(13):5716-22. doi: 10.1021/ac300775j. Epub 2012 Jun 22.
Hybrid atomic force microscopy (AFM)-fluorescence microscopy (FM) investigation of exocytosis in lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with adenosine-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase in cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 5), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to nonfused LBs, concluding that different "dynamic states" of LBs or locations from the PM are captured during imaging.
利用原子力显微镜-荧光显微镜（AFM-FM）联合技术研究肺上皮细胞（ATII 细胞）的胞吐作用，可以通过 FM 检测到单个胞吐事件，并通过 AFM 同时对单个细胞的结构变化进行相关分析。板层小体（LB）的胞吐作用代表了一种在许多非神经元细胞中发现的缓慢形式的胞吐作用。在受到腺苷-5'-三磷酸（ATP）和佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）刺激后，LB 与质膜（PM）融合时通过 P2X(4)受体导致阳离子内流，这应该会引起细胞高度/体积的暂时增加。在单细胞表面进行的单线扫描中进行 AFM 测量。刺激后 5 分钟，ATII 细胞的细胞高度和体积分别增加了 13.7%±4.1%和 15.9%±4.8%（N=9）。这些短暂的变化依赖于 LB-PM 融合的胞吐作用。未受刺激的细胞和缺乏 LB 融合的细胞的细胞高度/体积没有明显变化（N=8）。此外，在 UTP 和 PMA 刺激下，ATII 细胞的细胞高度也观察到下降，这些激动剂诱导 LB 与 PM 融合，但不会激活 P2X(4)受体。细胞高度和体积分别下降了-8.6%±3.6%和-11.2%±3.9%（N=5）。此外，还对刺激后细胞核周围细胞区域进行了低力接触和动态模式 AFM 成像。与未融合的 LB 相比，融合的 LB 在 AFM 形貌图像中更加明显，这表明在成像过程中捕获了 LB 的不同“动态状态”或与 PM 的不同位置。
