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Analyzing Intracellular Gradients in Pollen Tubes.

Daniel S C Damineli1, Maria Teresa Portes2, José A Feijó2

  • 1Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA. danieldamineli@gmail.com.

Methods in Molecular Biology (Clifton, N.J.)
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Summary

Quantifying intracellular gradients in pollen tubes is challenging. This study introduces a computational protocol using R to analyze these gradients, enabling high-throughput spatiotemporal phenotyping of cellular processes.

Keywords:
Apical growthCa2+Computational analysisHigh-throughput phenotypingIntracellular gradientIon gradientsRatiometric probesSpatiotemporal analysisTip detection

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Area of Science:

  • Cell Biology
  • Plant Science
  • Biophysics

Background:

  • Intracellular gradients are crucial for cell polarity, particularly in pollen tube growth.
  • Quantifying these dynamic gradients presents significant technical hurdles.
  • Existing methods lack the efficiency for high-throughput analysis.

Purpose of the Study:

  • To develop a robust computational protocol for analyzing intracellular gradients in pollen tubes.
  • To enable quantitative phenotyping of gradient characteristics like steepness and intensity.
  • To filter non-representative time points for more accurate gradient analysis.

Main Methods:

  • Utilizing imaging data from pollen tubes expressing a calcium indicator (Yellow CaMeleon 3.6).
  • Employing the CHUKNORRIS method for pollen tube tip detection and kymograph reconstruction.
  • Applying local polynomial regression for mean spatial profile estimation and filtering oscillatory data points.
  • Estimating gradient slope from the linear decay in mean fluorescence.

Main Results:

  • A computational protocol for analyzing intracellular gradients in pollen tubes was successfully developed.
  • The method allows for the quantitative assessment of gradient steepness, location, intensity, and variability.
  • Non-representative time points, such as those during growth arrest, were effectively filtered out.
  • The protocol facilitates efficient, high-throughput spatiotemporal phenotyping.

Conclusions:

  • The developed R-based protocol provides a simple and efficient method for quantifying intracellular gradients in apically growing cells.
  • This approach overcomes previous technical challenges in gradient analysis.
  • Enables detailed spatiotemporal phenotyping of cellular processes driven by intracellular gradients.