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Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

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Published on: December 22, 2011

Light microscopy to image and quantify cell movement.

Deborah J Wessels¹, Spencer Kuhl, David R Soll

¹Department of Biology, The University of Iowa, Iowa City, IA, USA.

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

|September 19, 2009

Summary

This summary is machine-generated.

This study introduces advanced 2D and 3D imaging techniques with Dynamic Image Analysis System (DIAS) software to precisely analyze basic cell motility in Dictyostelium discoideum. These methods help identify defects in cytoskeletal and signaling mutants.

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Area of Science:

Cell Biology
Biophysics
Microscopy

Background:

Dictyostelium discoideum is a model organism for studying cell motility due to its easily mutable haploid genome and distinct motile phases.
Basic motile behavior, including pseudopod extension and retraction, occurs independently of chemoattractants in cells like Dictyostelium and polymorphonuclear neutrophils (PMNs).
Understanding basic motility is crucial before attributing roles in chemotaxis to specific molecules identified through mutant analysis.

Purpose of the Study:

To present novel 2D and 3D imaging technologies integrated with Dynamic Image Analysis System (DIAS) software.
To enable detailed analysis of cell motility, shape dynamics, pseudopod formation, and molecular localization during basic motile behavior.
To provide a reproducible methodology for analyzing the 3D trajectories of surface-attached microspheres on crawling cells.

Main Methods:

Utilizing advanced two-dimensional (2D) and three-dimensional (3D) microscopy and image acquisition techniques.
Employing 2D and 3D Dynamic Image Analysis System (DIAS) software for quantitative motion analysis.
Describing methods for tracking microsphere trajectories on the cell surface and analyzing cytoskeletal and signaling mutants.

Main Results:

The described technologies and DIAS software facilitate precise measurement of cell motility parameters.
The system allows for the detailed analysis of shape changes, pseudopod dynamics, and the localization of tagged molecules.
The methodology effectively identifies defects in basic motile behavior in various mutant strains.

Conclusions:

The integrated 2D/3D DIAS approach offers a robust platform for dissecting basic cell motility.
This methodology is essential for accurately characterizing the functions of cytoskeletal and signaling proteins in cell movement.
The described techniques are adaptable for typical laboratory settings, advancing the study of cell motility mechanisms.