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RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-6898-6377
2018 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 28, no 8, p. 1281-1288.e3Article in journal (Refereed) Published
Abstract [en]

While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 upregulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement, and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many upregulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes, such as dual-specific tyrosine-(Y)-phosphorylation-regulated kinases and MORN domain-containing proteins that seemingly act as key regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies [1, 2], our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.

Place, publisher, year, edition, pages
2018. Vol. 28, no 8, p. 1281-1288.e3
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-354956DOI: 10.1016/j.cub.2018.02.055ISI: 000430694900049PubMedID: 29628369OAI: oai:DiVA.org:uu-354956DiVA, id: diva2:1223556
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish Research Council, 621-2009-4813EU, European Research Council, 310039-PUZZLE_CELLSwedish Foundation for Strategic Research , SSF-FFL5Available from: 2018-06-25 Created: 2018-06-25 Last updated: 2019-09-07Bibliographically approved
In thesis
1. Novel insights into protist diversity and niche adaptation using single cell transcriptomics
Open this publication in new window or tab >>Novel insights into protist diversity and niche adaptation using single cell transcriptomics
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protists are a polyphyletic group of microbes that represents the vast majority of eukaryotic diversity. Despite this, most sequencing efforts targeting eukaryotes have been focused on animals, fungi and plants. The sequencing bias towards multicellular organisms can partially be explained by the difficulty in cultivating protists, which is needed in traditional sequencing workflows. In this thesis, single-cell RNA sequencing has been used to generate transcriptome data from environmental protists, without being dependent on establishing a culture. These transcriptome data have been used to discover novel protist diversity, as well as exploring the cell biology of two ciliates.

In the first chapter, transcriptomes of cell fragments were generated for the ciliate Stentor. This ciliate is well-known for its ability to repair drastic cellular wounds, and the transcriptomes uncovered genes involved in processes such as cell cycle, signaling and microtubule-based movement to be activated during Stentor regeneration.

Spirostomum semivirescens is another ciliate, whose transcriptome was generated using single-cell RNA sequencing. The transcriptome data suggest that S. semivirescens is using rhodoquinol-dependent fumarate reduction for respiration in environments with low levels of oxygen.

Single-cell RNA sequencing was further used to target cells smaller than Stentor and Spirostomum. By generating 124 transcriptomes of environmental protists, a high number of novel lineages could be identified. The generated transcriptome data included free-living prokinetoplastids, non-photosynthetic euglenids, metamonads and katablepharids.

A few modifications to the single-cell RNA sequencing protocol Smart-seq2 were necessary to generate the 124 transcriptomes of small protists cells. The impact of these modifications to Smart-seq2 was benchmarked using Giardia intestinalis. The generated single-cell transcriptomes revealed that addition of freeze-thaw cycles to Smart-seq2 improved transcript recovery. Finally, we propose a protocol that allows identification of failed cDNA reactions, based only on measuring DNA concentration, without compromising on transcript recovery. Reducing the dependency on quality control will be important if single-cell RNA sequencing would be done in a high-throughput workflow.

In conclusion, single-cell RNA sequencing can be a powerful tool for studying protist diversity and biology. In particular, it has the potential to efficiently uncover protist diversity, provided that a robust and efficient method to isolate single cells from the environment is established.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 52
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1853
Keywords
Protists, microbial eukaryotes, cultivation-independent methods, single-cell RNA sequencing, phylogenomics
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-392618 (URN)978-91-513-0747-3 (ISBN)
Public defence
2019-10-25, B22, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-10-01 Created: 2019-09-07 Last updated: 2019-10-15

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Onsbring, HenningJamy, MahwashEttema, Thijs J. G.

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