Analyzing GFP-tagged cytoskeletal protein colocalization in human carcinoma cells

Abstract

Cytoskeletal proteins function as dynamic, complex components involved in cellular structure and signaling. Characterizing the roles of such proteins would greatly benefit many research areas, including the study of cancer and protein-related diseases. There is currently no accurate, high throughput method of image analysis that clearly describes protein behavior within the cell. In addressing this problem, we chose to characterize proteins based on the colocalization parameter-the amount of overlap between two objects or signals. We aimed to create a single parameter that quantitatively defined colocalization yet complemented biological intuition about a complicated system. Cell culture techniques were used to transfect HeLa cells with four "marker" GFP-tagged protein constructs. Cells were fluorescently labeled in three channels-Hoechst for nucleus, Texas Red phalloidin for actin, and GFP for protein-and images were captured using Cellomics scanning microscopy. After collecting data and testing software applications, we analyzed our data with Definiens software and developed a flexible, comprehensible method of quantifying colocalization using minimal parameters.