Abstract
A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division. Kinetochore perturbations lead to aneuploidy, which is a hallmark of cancer cells. Hence, understanding kinetochore homeostasis and regulation are important for a global understanding of cell division and genome integrity. The 16 budding yeast kinetochores colocalize within the nucleus to form a single focus. Here we have created a set of freely-available tools to allow high-throughput quantitation of kinetochore foci fluorescence. We use this 'FociQuant' tool to compare methods of kinetochore quantitation and we show proof of principle that FociQuant can be used to identify changes in kinetochore protein levels in a mutant that affects kinetochore function. This analysis can be applied to any protein that forms discrete foci in cells.
Highlights
The advent of genome-wide green fluorescence protein (GFP) labeling has enabled the location of most cellular proteins to be determined in vivo [1]
We find that changes www.jbmethods.org in the fluorescence intensity of foci of yellow fluorescent protein (YFP) linked to kinetochore protein Dad4 in mad1∆ cells are detected using FociQuant
FociQuant and Volocity software consistently detect a ~70% increase in fluorescence intensity of diploid Mtw1-YFP kinetochore foci, compared with haploid strains
Summary
The advent of genome-wide green fluorescence protein (GFP) labeling has enabled the location of most cellular proteins to be determined in vivo [1]. Altered levels of kinetochore proteins may disrupt normal chromosome segregation and play a role in tumorigenesis or tumor development In support of this notion, over-expression of several kinetochore and checkpoint genes has been found in tumor cells [13,14,15,16]. One of the steps in understanding kinetochore biology is to understand how the levels of the kinetochore proteins change in response to perturbations such as mutations, overexpression of endogenous genes or drug treatments This requires a high-throughput method of assessing kinetochore protein levels using fluorescence imaging. We find that changes www.jbmethods.org in the fluorescence intensity of foci of yellow fluorescent protein (YFP) linked to kinetochore protein Dad in mad1∆ cells are detected using FociQuant These data suggest that changes in kinetochore homeostasis can be used to identify mutants that lead to chromosomal instability. The software could be used to quantify other types of foci, such as those formed by centrosome proteins and we show proof of principle for simultaneously quantifying two different foci in the same images
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