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Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

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Measuring secretory membrane traffic: a quantitative fluorescence microscopy approach.

Vytaute Starkuviene¹, Arne Seitz, Holger Erfle

¹University of Heidelberg, Heidelberg, Germany.

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

|December 11, 2008

Summary

This chapter details automated imaging assays for quantifying cargo transport and Golgi complex integrity. These methods support gene silencing and compound screening in biological research.

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Area of Science:

Cell Biology
Biophysics

Background:

Accurate quantification of intracellular transport and organelle integrity is crucial for understanding cellular function.
Existing methods may lack the throughput or precision required for comprehensive analysis.

Purpose of the Study:

To describe automated imaging methods for quantifying transmembrane and soluble cargo transport rates.
To present methods for evaluating Golgi complex integrity using automated imaging.
To demonstrate the applicability of these assays for various experimental scales and perturbations.

Main Methods:

Development of fluorescence intensity-based assays for cargo transport quantification.
Implementation of morphology-based assays for Golgi complex integrity assessment.
Adaptation of protocols for sample preparation, automated image acquisition, image analysis, and data quantification.

Main Results:

Established automated imaging assays capable of quantifying cargo transport rates.
Developed methods to evaluate Golgi complex integrity.
Demonstrated the utility of these assays for analyzing effects of RNAi, cDNA overexpression, and chemical compounds.

Conclusions:

Automated imaging provides robust quantitative assays for cargo transport and Golgi integrity.
These assays are adaptable for single experiments and high-throughput screening.
The described methods facilitate the study of cellular responses to genetic and chemical perturbations.