Unsupervised Clustering of Subcellular Protein Expression Patterns in High-Throughput Microscopy Images Reveals Protein Complexes and Functional Relationships between Proteins

Protein subcellular localization has been systematically characterized in budding yeast using fluorescently tagged proteins. Based on the fluorescence microscopy images, subcellular localization of many proteins can be classified automatically using supervised machine learning approaches that have been trained to recognize predefined image classes based on statistical features. Here, we present an unsupervised analysis of protein expression patterns in a set of high-resolution, high-throughput microscope images. Our analysis is based on 7 biologically interpretable features which are evaluated on automatically identified cells, and whose cell-stage dependency is captured by a continuous model for cell growth. We show that it is possible to identify most previously identified localization patterns in a cluster analysis based on these features and that similarities between the inferred expression patterns contain more information about protein function than can be explained by a previous manual categorization of subcellular localization. Furthermore, the inferred cell-stage associated to each fluorescence measurement allows us to visualize large groups of proteins entering the bud at specific stages of bud growth. These correspond to proteins localized to organelles, revealing that the organelles must be entering the bud in a stereotypical order. We also identify and organize a smaller group of proteins that show subtle differences in the way they move around the bud during growth. Our results suggest that biologically interpretable features based on explicit models of cell morphology will yield unprecedented power for pattern discovery in high-resolution, high-throughput microscopy images.Author Summary: The location of a particular protein in the cell is one of the most important pieces of information that cell biologists use to understand its function. Fluorescent tags are a powerful way to determine the location of a protein in living cells. Nearly a decade ago, a collection of yeast strains was introduced, where in each strain a single protein was tagged with green fluorescent protein (GFP). Here, we show that by training a computer to accurately identify the buds of growing yeast cells, and then making simple fluorescence measurements in context of cell shape and cell stage, the computer could automatically discover most of the localization patterns (nucleus, cytoplasm, mitochondria, etc.) without any prior knowledge of what the patterns might be. Because we made the same, simple measurements for each yeast cell, we could compare and visualize the patterns of fluorescence for the entire collection of strains. This allowed us to identify large groups of proteins moving around the cell in a coordinated fashion, and to identify new, complex patterns that had previously been difficult to describe.