Biology subjects

Lundberg, E.

Publications and source records attributed to Lundberg, E..

39 records · Page 3Linked to original sources

bioRxiv2020

A sample preparation protocol for high throughput immunofluorescence of suspension cells

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, nonadherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines and peripheral blood mononucleated cells (PBMC) and human platelets. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

cell biology↗

bioRxiv2019

Spatiotemporal dissection of the cell cycle regulated human proteome

Cellular division is a fundamental source of cell-to-cell variability, and studies of transcript and protein abundances have revealed several hundred genes that are regulated by the cell cycle1-8. However, none of these studies provide single-cell resolution of protein expression, leaving an incomplete understanding of cell-to-cell heterogeneity and the roles of cycling transcripts and proteins. Here, we present the first comprehensive map of spatiotemporal heterogeneity of the human proteome by integrating proteomics at subcellular resolution, single-cell transcriptomics, and pseudotime measurements of individual cells within the cell cycle. We identify that 17% of the human proteome displays cell-to-cell variability, of which 26% is correlated to cell cycle progression, and we present the first evidence of cell cycle association for 235 proteins. Only 15% of proteomic cell cycle regulation is due to transcriptomic cycling, which points to other means of regulation such as post-translational modifications. For proteins regulated at the transcript level, we observe a 7.7 hour delay between peak expression of transcript and protein on average. This spatially resolved proteomic map of the cell cycle has been integrated into the Human Protein Atlas and serves as a valuable resource for accelerating molecular studies of the human cell cycle and cell proliferation.

cell biology↗

bioRxiv2017

Novel ‘housekeeping’ genes and an unusually heterogeneous distribution of transporter expression profiles in human tissues and cell lines, assessed using the Gini coefficient

We analyse two comprehensive transcriptome datasets from human tissues and human-derived cell lines in terms of the expression profiles of the SLC and ABC families of membrane transporters. The Gini index (coefficient) characterises inequalities of distributions, and is used in a novel way to describe the distribution of the expression of each transporter among the different tissues and cell lines. In many cases, transporters exhibit extremely high Gini coefficients, even when their supposed substrates might be expected to be available to all tissues, indicating a much higher degree of specialisation than is usually assumed. This is consistent with divergent evolution from a more restricted set of ancestors. Similar trends hold true for the expression profiles of transporters in different cell lines, suggesting that cell lines exhibit largely similar transport behaviour to that of tissues. By contrast, the Gini coefficients for ABC transporters tend to be larger in cell lines than in tissues, implying that some kind of a selection process has taken place. In particular, with some exceptions such as olfactory receptors and genes involved in keratin production, transporter genes are significantly more heterogeneously expressed than are most non-transporter genes. The Gini index also allows us to determine those transcripts with the most stable expression; these often differ significantly from the housekeeping genes commonly used for normalisation in transcriptomics and qPCR studies. The lowest four in tissues are FAM32A, ABCB7, MRPL21 and PCBP1, while the lowest three in cell lines are SF3B2, NXF1 and RBM45. PCBP1 is both reasonably highly expressed and has a low Gini coefficient in both tissues and cell lines, and is an excellent novel housekeeping gene. Overall, our analyses provide novel opportunities for the normalisation of genome-wide expression profiling data.

systems biology↗