Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.

The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer1-3. 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.

Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder.

The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment: the nucleoli rim. We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that the expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.

Spatial Characterization of the Human Centrosome Proteome Opens Up New Horizons for a Small but Versatile Organelle.

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.

Targeting of the Drosophila protein CG2254/Ldsdh1 to a subset of lipid droplets.

Lipid droplets (LDs) are the principal organelles of lipid storage. They consist of a hydrophobic core of storage lipids, surrounded by a phospholipid monolayer with proteins attached. While some of these proteins are known to be essential for the regulation of cellular and organismic lipid metabolism, key questions concerning LD protein function, such as their targeting to LDs, are still unanswered. Intriguingly, some proteins are restricted to subsets of LDs by an as-yet-unknown mechanism. This finding makes LD targeting even more complex. Here, we characterize the Drosophila protein CG2254, which is targeted to subsets of LDs in cultured cells and in different larval Drosophila tissues, where the prevalence of subsets of LDs appears highly dynamic. We find that an amphipathic amino acid stretch mediates CG2254 LD localization. Additionally, we identified a juxtaposed sequence stretch limiting CG2254 localization to a subset of LDs. This sequence is sufficient to restrict a chimeric protein consisting of the subset-targeting sequence introduced to an otherwise pan-LD-localized protein sequence to a subset of LDs. Based on its subcellular localization and annotated function, we suggest that CG2254 is renamed Lipid droplet subset dehydrogenase 1 (Ldsdh1).

The evolutionarily conserved protein CG9186 is associated with lipid droplets, required for their positioning and for fat storage.

Lipid droplets (LDs) are specialized cell organelles for the storage of energy-rich lipids. Although lipid storage is a conserved feature of all cells and organisms, little is known about fundamental aspects of the cell biology of LDs, including their biogenesis, structural assembly and subcellular positioning, and the regulation of organismic energy homeostasis. We identified a novel LD-associated protein family, represented by the Drosophila protein CG9186 and its murine homolog MGI:1916082. In the absence of LDs, both proteins localize at the endoplasmic reticulum (ER). Upon lipid storage induction, they translocate to LDs using an evolutionarily conserved targeting mechanism that acts through a 60-amino-acid targeting motif in the center of the CG9186 protein. Overexpression of CG9186, and MGI:1916082, causes clustering of LDs in both tissue culture and salivary gland cells, whereas RNAi knockdown of CG9186 results in a reduction of LDs. Organismal RNAi knockdown of CG9186 results in a reduction in lipid storage levels of the fly. The results indicate that we identified the first members of a novel and evolutionarily conserved family of lipid storage regulators, which are also required to properly position LDs within cells.

The human secretome.

The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immunoassays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.

The human protein atlas: A spatial map of the human proteome.

The correct spatial distribution of proteins is vital for their function and often mis-localization or ectopic expression leads to diseases. For more than a decade, the Human Protein Atlas (HPA) has constituted a valuable tool for researchers studying protein localization and expression in human tissues and cells. The centerpiece of the HPA is its unique antibody collection for mapping the entire human proteome by immunohistochemistry and immunocytochemistry. By these approaches, more than 10 million images showing protein expression patterns at a single-cell level were generated and are publicly available at www.proteinatlas.org. The antibody-based approach is combined with transcriptomics data for an overview of global expression profiles. The present article comprehensively describes the HPA database functions and how users can utilize it for their own research as well as discusses the future path of spatial proteomics.

A subcellular map of the human proteome.

Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

Lipid droplets control the maternal histone supply of Drosophila embryos.

BACKGROUND: Histones are essential for chromatin packing, yet free histones not incorporated into chromatin are toxic. While in most cells multiple regulatory mechanisms prevent accumulation of excess histones, early Drosophila embryos contain massive extranuclear histone stores, thought to be essential for development. Excess histones H2A, H2B, and H2Av are bound to lipid droplets, ubiquitous fat storage organelles especially abundant in embryos. It has been proposed that sequestration on lipid droplets allows safe transient storage of supernumerary histones. RESULTS: Here, we critically test this sequestration hypothesis. We find that histones are anchored to lipid droplets via the previously uncharacterized protein Jabba: Jabba localizes to droplets, coimmunoprecipitates with histones, and is necessary to recruit histones to droplets. Jabba mutants lack the maternal H2A, H2B, and H2Av deposits altogether; presumably, these deposits are eliminated unless sequestered on droplets. Jabba mutant embryos compensate for this histone deficit by translating maternal histone mRNAs. However, when histone expression is mildly compromised, the maternal histone protein deposits are essential for proper early mitoses and for viability. CONCLUSIONS: A growing number of proteins from other cellular compartments have been found to transiently associate with lipid droplets. Our studies provide the first insight into mechanism and functional relevance of this sequestration. We conclude that sequestration on lipid droplets allows embryos to build up extranuclear histone stores and provides histones for chromatin assembly during times of high demand. This work reveals a novel aspect of histone metabolism and establishes lipid droplets as functional storage sites for unstable or detrimental proteins.

Lipid droplets: a dynamic organelle moves into focus.

Lipid droplets (LDs) were perceived as static storage deposits, which passively participate in the energy homeostasis of both cells and entire organisms. However, this view has changed recently after the realization of a complex and highly dynamic LD proteome. The proteome contains key components of the fat mobilization system and proteins that suggest LD interactions with a variety of cell organelles, including the endoplasmic reticulum, mitochondria and peroxisomes. The study of LD cell biology, including cross-talk with other organelles, the trafficking of LDs in the cell and regulatory events involving the LD coat proteins is now on the verge of leaving its infancy and unfolds that LDs are highly dynamic cellular organelles.