Chemotactic cell trapping in controlled alternating gradient fields
Chemotactic cell trapping in controlled alternating gradient fields
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DOI：
10.1073/pnas.1014853108
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发表时间：
2011-07-12
影响因子：
11.1
通讯作者：
Heinrich, Doris
中科院分区：
文献类型：
--
作者：
Meier, Born;Zielinski, Alejandro;Heinrich, Doris
定向细胞迁移到时空变化的趋化刺激需要快速的细胞骨架重组。许多研究提供证据表明，肌动蛋白重组是由信号分子的细胞内再分布控制的，如PI 4，5 P2/PI 3，4，5 P3梯度。然而，探索潜在的机制是困难的，需要仔细的时空控制外部趋化刺激。我们设计了一个微流控装置，以产生交替的趋化梯度场，同时多细胞曝光，大大方便了统计分析。对于细胞内的反应动力学的定量描述，我们应用交替的时间序列的空间均匀的浓度梯度在300 μ m，重新定向的时间尺度下降到几秒钟。Dictyosteoblastdiscoideum amoeoblasts对低于0.02 Hz的梯度转换率的反应是重新适应它们的迁移方向。对于更快的转换，细胞复极化停止，并完全停滞在0.1 Hz。在这种“化学活性捕获”的细胞状态下，外部刺激的交替比细胞内反馈更快，能够通过定向迁移的开始来响应。为了研究梯度转换过程中细胞内肌动蛋白皮质重排，我们将迁移细胞反应与肌动蛋白重聚合动力学相关联，通过GFP融合蛋白LimE Delta CC的荧光分布矩进行定量。我们发现了两种根本不同的细胞极化类型，我们可以揭示PI 3-激酶在细胞复极化中的作用。在早期聚集阶段，PI 3-Kinase增强D. discoideum细胞重新调整其极性，在空间上交替梯度场，而在聚集主管细胞的PI 3-激酶扰动的影响变得不太相关。
Directed cell migration toward spatio-temporally varying chemotactic stimuli requires rapid cytoskeletal reorganization. Numerous studies provide evidence that actin reorganization is controlled by intracellular redistribution of signaling molecules, such as the PI4,5P2/PI3,4,5P3 gradient. However, exploring underlying mechanisms is difficult and requires careful spatio-temporal control of external chemotactic stimuli. We designed a microfluidic setup to generate alternating chemotactic gradient fields for simultaneous multicell exposure, greatly facilitating statistical analysis. For a quantitative description of intracellular response dynamics, we apply alternating time sequences of spatially homogeneous concentration gradients across 300 mu m, reorienting on timescales down to a few seconds. Dictyostelium discoideum amoebae respond to gradient switching rates below 0.02 Hz by readapting their migration direction. For faster switching, cellular repolarization ceases and is completely stalled at 0.1 Hz. In this "chemotactically trapped" cell state, external stimuli alternate faster than intracellular feedback is capable to respond by onset of directed migration. To investigate intracellular actin cortex rearrangement during gradient switching, we correlate migratory cell response with actin repolymerization dynamics, quantified by a fluorescence distribution moment of the GFP fusion protein LimE Delta cc. We find two fundamentally different cell polarization types and we could reveal the role of PI3-Kinase for cellular repolarization. In the early aggregation phase, PI3-Kinase enhances the capability of D. discoideum cells to readjust their polarity in response to spatially alternating gradient fields, whereas in aggregation competent cells the effect of PI3-Kinase perturbation becomes less relevant.