Quantifying compartment-specific protein translocation in astrocytes by object-oriented image analysis: Mitochondrial translocation of PKCδ

Abstract

Assessing the amount and subcellular distribution of protein expression is a key component in modern cell biological and medical research. We studied protein kinase Cδ (PKCδ) as a potential regulator of mitochondrial metabolism in astrocytes, and sought to evaluate mitochondrial translocation of PKCδ since this is an important determinant of its function. Apart from visualizing compartment specific localization of mobile proteins such as PKCδ, we also wanted to determine what amount of a cell’s total content of a particular protein is located to a specific compartment, or translocated comparing control and experimental condition. We develop a semiquantitative parameter that indicates the relative protein distribution to two subcellular compartments, starting from standard two-channel fluorescence microscopy images. We studied the mitochondrial translocation of PKCδ in astrocytes using double immunofluorescence microscopy and object-oriented image analysis. In one channel, the protein of interest (PKCδ) is labeled, in the other the compartment or organelle of interest (mitochondria, using cytochrome oxidase IV). Both channels were binarized, turned into object populations, and the channel specific values for total area and integrated intensity extracted. From these values, the “intensity density ratio” (IDR) is calculated, a standardized parameter to easily compare distribution patterns in different cells or ROIs. IDR is highly sensitive to changes in localization pattern, and thus easily detects protein translocation in comparison between control and experimental condition. In our study, medium application of glutamate was found to result in partial PKCδ translocation to mitochondria, a statistically highly significant result based only on a limited number of acquired images.