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Spatiotemporal characterization of the human proteome
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.ORCID iD: 0000-0003-0750-1070
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Characterizing the molecular components of the basic unit of life; the cell, is crucial for a complete understanding of human biology. The cell is divided into compartments to create a suitable environment for the resident proteins to fulfill their functions. Therefore, spatial mapping of the human proteome is essential to understand protein function in health and disease.

 

Spatial proteomics is most commonly investigated using mass spectrometry or imaging, combined with machine learning for the data analysis. Until now, studies have been limited to high abundant proteins and relied on the purification of organelle fractions from a bulk of cells. Within the scope of this thesis, we were able to systematically localize proteins in their native cellular environment using antibody-based imaging techniques, and to investigate protein subcellular localization and dynamics on a single cell level, introducing a major advance within the field of spatial proteomics.

 

Paper I of this thesis presents a subcellular map of the human proteome, where the spatial distribution of 12,003 human proteins was mapped into 30 subcellular structures, half of which were not previously localized. Besides providing a valuable dataset for cell biology, this study is the first to reveal the spatial complexity of human cells with proteins localizing to multiple compartments and pronounced single cell variations. Paper II reports on the systematic temporal dissection of these single cell variations and the identification of cell cycle correlated variations. We identified 258 novel cell cycle regulated proteins and showed that several of these proteins may be connected to proliferative diseases. A key finding of Paper II is that proteins showing non-cell cycle dependent variations are significantly enriched in mitochondria, whereas cell cycle dependent proteins are enriched in nucleoli. In Paper III and IV, we spatiotemporally characterized the proteomes of these two organelles, mitochondria and nucleoli, in greater detail.

In Paper III, we expanded the mitochondrial proteome with 560 novel proteins. As many as 20% of the mitochondrial proteome showed variations in their expression pattern at the single cell level, most often independent of the cell cycle. Paper IV provides a complete characterization of the nucleolar proteome. Nucleoli are not only important for ribosome synthesis and assembly, but are also crucial for cell cycle regulation through the recruitment of its proteins to the chromosomal periphery during cell division. Here, we presented the first proteome-wide spatiotemporal analysis of the nucleolus with its sub-compartments, and identified 69 nucleolar proteins that relocated to the chromosomes periphery during mitosis.

 

In conclusion, this thesis unravels the spatiotemporal proteome organization of the human cell over the course of a cell cycle and offers a valuable starting point for a better understanding of human cell biology in health and disease.

Place, publisher, year, edition, pages
Stockholm, Sweden: KTH Royal Institute of Technology, 2019. , p. 65
Series
TRITA-CBH-FOU ; 2019:48
Keywords [en]
Spatial proteomics, Spatiotemporal proteomics, Immunofluorescence, Human Protein Atlas, Cell compartments, Single cell proteomics, Cell cycle, Cancer
National Category
Cell and Molecular Biology Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-261245ISBN: 978-91-7873-302-6 (print)OAI: oai:DiVA.org:kth-261245DiVA, id: diva2:1357435
Public defence
2019-10-25, Atrium, Nobels väg 12B, Wargentinhuset, solna, 09:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 2019-10-04

Available from: 2019-10-04 Created: 2019-10-03 Last updated: 2019-10-04Bibliographically approved
List of papers
1. A subcellular map of the human proteome
Open this publication in new window or tab >>A subcellular map of the human proteome
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 356, no 6340, article id 820Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2017
Keywords
antibody, proteome, biology, cells and cell components, disease incidence, image analysis, physiological response, protein, proteomics, spatial distribution, Article, cell organelle, cellular distribution, human, human cell, immunofluorescence microscopy, mass spectrometry, priority journal, protein analysis, protein localization, protein protein interaction, single cell analysis, transcriptomics
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-216588 (URN)10.1126/science.aal3321 (DOI)000401957900032 ()2-s2.0-85019201137 (Scopus ID)
Note

QC 20171208

Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2019-10-03Bibliographically approved
2. Spatiotemporal dissection of the cell cycle regulated human proteome
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Here we present a spatiotemporal dissection of proteome single cell heterogeneity in human cells, performed with subcellular resolution over the course of a cell cycle. We identify 17% of the human proteome to display cell-to-cell variability, of which we could attribute 25% as correlated to cell cycle progression, and present the first evidence of cell cycle association for 258 proteins. A key finding is that the variance, of many of the cell cycle associated proteins, is only partially explained by the cell cycle, which hints at cross-talk between the cell cycle and other signaling pathways. We also demonstrate that several of the identified cell cycle regulated proteins may be clinically significant in proliferative disorders. This spatially resolved proteome map of the cell cycle, integrated into the Human Protein Atlas, serves as a valuable resource to accelerate the molecular knowledge of the cell cycle and opens up novel avenues for the understanding of cell proliferation.

Keywords
Proteomics, Single cell variation, Immunofluorescence, Human Protein Atlas, Cell cycle, Cell cycle dependent proteome
National Category
Natural Sciences Medical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-261234 (URN)10.1101/54323 (DOI)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20191007

Available from: 2019-10-03 Created: 2019-10-03 Last updated: 2019-10-07Bibliographically approved
3. An image-based map of the human mitochondrial proteome and its heterogeneity
Open this publication in new window or tab >>An image-based map of the human mitochondrial proteome and its heterogeneity
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mitochondria is involved in a numerous variety of cellular functions beyond its 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 1,098 proteins. The single cell resolution revealed extensive heterogeneity for as much as 20% (n=226) of the mitochondrial proteome.  These variations are independent of cell cycle position and likely represent metabolic fluctuations in the cell. Our analysis shows that 48% (n=524) of the proteins localize to additional cellular compartments, further contributing to the diverse cellular functions of mitochondria. This map of the mitochondrial proteome, part of the Cell Atlas of the Human Protein Atlas database (www.proteinatlas.org), provides a valuable knowledge resource for studies of mitochondria function, dysfunction and disease.

Keywords
Mitochondria, Human Protein Atlas, Human Proteome, Immunofluorescence, Multilocalizing protein
National Category
Natural Sciences Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-261236 (URN)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20191007

Available from: 2019-10-03 Created: 2019-10-03 Last updated: 2019-10-07Bibliographically approved
4. Spatial organization of the nucleolar proteome during mitosis
Open this publication in new window or tab >>Spatial organization of the nucleolar proteome during mitosis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In the interphase cell, the membrane-less nucleoli are the sites of ribosome biogenesis. As part of the Human Protein Atlas we created an image catalogue comprising 1,314 nucleolar proteins using antibody-based proteomics. We show experimental evidence for 1,027 proteins localizing to the whole nucleoli and 287 to the fibrillar center or dense fibrillar component. We also propose a new sub-compartment located in the nucleoplasmic border denoted as nucleoli rim, comprising at least 131 proteins. As a step toward better understanding of nucleolar protein function during cell division, we additionally generated confocal images of 68 nucleolar proteins being recruited to the chromosomal periphery in mitosis. Thanks to the single cell resolution we were able to define three expression phenotypes among the mitotic chromosome proteins; early, intermediate and late recruitment suggesting phase specific functions. We also for the first time provide a proteome-wide confirmation that the nucleoli in general, but mitotic chromosome proteins in particular have a higher predicted intrinsic disorder level compared to cytoplasmic proteins, indicating that the perichromosomal layer indeed is a liquid-like layer.

Keywords
Spatial proteomics, Human Protein Atlas, Immunofluorescence, Nucleolar proteome, Intrinsic disorder, chromosomal periphery
National Category
Cell and Molecular Biology Natural Sciences
Identifiers
urn:nbn:se:kth:diva-261238 (URN)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20191007

Available from: 2019-10-03 Created: 2019-10-03 Last updated: 2019-10-07Bibliographically approved

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