While spatial proteomics by fluorescence imaging has quickly become an essential discovery tool for researchers, fast and scalable methods to classify and embed single-cell protein distributions in such images are lacking. Here, we present the design and analysis of the results from the competition Human Protein Atlas – Single-Cell Classification hosted on the Kaggle platform. This represents a crowd-sourced competition to develop machine learning models trained on limited annotations to label single-cell protein patterns in fluorescent images. The particular challenges of this competition include class imbalance, weak labels and multi-label classification, prompting competitors to apply a wide range of approaches in their solutions. The winning models serve as the first subcellular omics tools that can annotate single-cell locations, extract single-cell features and capture cellular dynamics. 

Spatial and temporal variations among individual human cell proteomes are comprehensively mapped across the cell cycle using proteomic imaging and transcriptomics. The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer(1-3). The cell cycle is driven by precise regulation of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.

After a century of research, the human centrosome continues to fascinate. Based on immunofluorescence and confocal microscopy, an extensive inventory of the protein components of the human centrosome, and the centriolar satellites, with the important contribution of over 300 novel proteins localizing to these compartments is presented. A network of candidate centrosome proteins involved in ubiquitination, including six interaction partners of the Kelch-like protein 21, and an additional network of protein phosphatases, together supporting the suggested role of the centrosome as an interactive hub for cell signaling, is identified. Analysis of multi-localization across cellular organelles analyzed within the Human Protein Atlas (HPA) project shows how multi-localizing proteins are particularly overrepresented in centriolar satellites, supporting the dynamic nature and wide range of functions for this compartment. In summary, the spatial dissection of the human centrosome and centriolar satellites described here provides a comprehensive knowledgebase for further exploration of their proteomes.

Micronuclei are small nuclei-like structures, commonly associated with cancer, that have long been used as biomarkers of DNA damage and chromosomal instability. They are caused by lagging chromosomes or chromosome fragments that fail to be incorporated into primary nuclei during mitosis. Until recently, they were thought to be inert but recent studies have shown that micronuclei may be playing an active role in cellular biology, particularly in the context of cancer and chromothripsis. However, the precise ways that micronuclei perform these functions remain unknown.

In this study, we perform the first comprehensive mapping and functional analysis of the micronuclear proteome. We demonstrate that micronuclei are involved in several known cellular processes, showing high enrichment for proteins involved in gene expression, cell cycle checkpoints, and DNA damage repair among other things. Furthermore, we show that several poorly categorized proteins also localize to micronuclei, hinting towards unknown functionality beyond the already known. Our results indicate that micronuclei are more involved in cellular activity than previously thought and that .

We anticipate this resource to be a starting point for further investigation into the functions of micronuclei, particularly for their role in DNA damage and the chromothripsis phenomenon. Additionally, we believe that it can be used for investigation into errors in mitosis and new ways of treating diseases.

Mitochondria are involved in a wide range of cellular functions beyond their role in energy metabolism. Defining the human mitochondrial proteome is crucial to understand the mitochondria’s diverse functions and role in disease. Here, we present an image-based map of the human mitochondrial proteome containing 1,121 proteins with subcellular resolution. Our analysis shows that 48.3% (n=542) of the proteins localize to additional cellular compartments, further contributing to the diverse cellular functions of mitochondria and connectivity to other organelles. Furthermore, the mitochondrial proteome reveals tissue specific clustering, suggesting tissue specific functions and physiology. Strikingly, the single cell resolution of our dataset revealed extensive heterogeneity for as much as 33.5% (n=376) of the mitochondrial proteome which could not be explained by cell cycle progression. By performing a high throughput immunofluorescence screen, we conclude that heterogeneity in mitochondria protein expression can establish metabolic states in cell populations. This map of the mitochondrial proteome, part of the Human Protein Atlas database (www.proteinatlas.org), provides a valuable knowledge resource for studies of mitochondria function, dysfunction and disease.