Analyzing Intracellular Gradients in Pollen Tubes.
Analyzing Intracellular Gradients in Pollen Tubes.
复制标题
分析花粉管中的细胞内梯度。
DOI：
10.1007/978-1-0716-0672-8_14
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Feijó,JoséA
中科院分区：
文献类型：
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作者：
Damineli,DanielSC;Portes,MariaTeresa;Feijó,JoséA
明显的细胞内梯度显示和/或驱动细胞内极性在花粉管中。然而，量化这些梯度提出了多个技术挑战。在这里，我们提出了一个明智的计算协议，分析梯度生长的花粉管和过滤非代表性的时间点。作为一个例子，我们使用的成像数据从花粉管表达的遗传编码的比率Ca 2+探针，黄色CaMeleon 3.6，从其中提取的记波图。用CHUKNORRIS（我们以前发表的方法）检测花粉管尖端，从而重建细胞内梯度。过滤掉统计学混淆时间点，如生长停滞（梯度高度振荡），并采用局部多项式回归方法估计平均空间分布。最后，我们估计的梯度斜率的平均荧光衰减的线性部分，提供了一个定量的方法来检测梯度陡度，位置，强度和变异性的表型。所提出的数据操作协议可以使用统计编程语言R以简单有效的方式实现，从而打开路径以在顶部生长的细胞中进行细胞内梯度的高通量时空表型分析。
Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.