Cytoskeleton Methods and Protocols

Deborah J. Wessels, Daniel F. Lusche, Spencer Kuhl, Amanda Scherer, Edward Voss, David R. Soll

This chapter describes 2D quantitative methods for motion analysis as well as 3D motion analysis and reconstruction methods. Emphasis is placed on the analysis of dynamic cell shape changes that occur through extension and retraction of force generating structures such as pseudopodia and lamellipodia. Quantitative analysis of these structures is an underutilized tool in the field of cell migration. Our intent, therefore, is to present methods that we developed in an effort to elucidate mechanisms of basic cell motility, directed cell motion during chemotaxis, and metastasis. We hope to demonstrate how application of these methods can more clearly define alterations in motility that arise due to specific mutations or disease and hence, suggest mechanisms or pathways involved in normal cell crawling and treatment strategies in the case of disease. In addition, we present a 4D tumorigenesis model for high-resolution analysis of cancer cells from cell lines and human cancer tissue in a 3D matrix. Use of this model led to the discovery of the coalescence of cancer cell aggregates and unique cell behaviors not seen in normal cells or normal tissue. Graphic illustrations to visually display and quantify cell shape are presented along with algorithms and formulae for calculating select 2D and 3D motion analysis parameters.