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Proteomic Dissection of a Giant Cell

Athena Lin, Paul D. Piehowski, Chia-Feng Tsai, Tatyana Makushok, Lian Yi, Ulises Diaz, Connie Yan, Diana Summers, Pranidhi Sood, Richard D. Smith, Tao Liu, Wallace F. Marshall
doi: https://doi.org/10.1101/2022.01.06.475287
Athena Lin
1Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, 94158 USA
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Paul D. Piehowski
2Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA
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Chia-Feng Tsai
2Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA
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Tatyana Makushok
1Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, 94158 USA
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Lian Yi
2Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA
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Ulises Diaz
1Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, 94158 USA
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Connie Yan
1Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, 94158 USA
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Diana Summers
1Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, 94158 USA
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Pranidhi Sood
1Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, 94158 USA
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Richard D. Smith
2Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA
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Tao Liu
2Biological Science Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA
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Wallace F. Marshall
1Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, 94158 USA
3Chan Zuckerberg Biohub, San Francisco, CA 94158
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  • For correspondence: wallace.marshall@ucsf.edu
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Summary

Many individual proteins have been identified as having defined positions relative to cell polarity axes, raising the question of what fraction of all proteins may have polarized localizations. We took advantage of the giant ciliate Stentor coeruleus to quantify the extent of polarized localization proteome-wide. This trumpet-shaped unicellular organism shows a clear morphological anterior-posterior axis defined by a circular array of cilia known as a membranellar band at one end, and a holdfast at the other end. Because individual Stentor cells are over a millimeter in length, we were able to cut the cells into three pieces along the anterior-posterior axis, followed by proteomic analysis of proteins enriched in each piece. We find that approximately 30% of all detected proteins show a polarized location relative to the anterior-posterior cell axis. Proteins with polarized enrichment include centrin-like proteins, calcium-regulated kinases, orthologs of SFI1 and GAS2, and proteases. At the organelle level, nuclear and mitochondrial proteins are enriched in the anterior half of the cell body, but not in the membranellar band itself, while ribosome related proteins are apparently uniformly distributed. RNAi of signaling proteins enriched in the membranellar band, which is the anterior-most structure in the cell, revealed a protein phosphatase 2 subunit b ortholog required for closure of the membranellar band into the ring shape characteristic of Stentor. These results suggest that a large fraction of the Stentor proteome has a polarized localization, and provide a protein-level framework for future analysis of pattern formation and regeneration in Stentor as well as defining a general strategy for subcellular spatial proteomics based on physical dissection of cells.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted January 06, 2022.
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Proteomic Dissection of a Giant Cell
Athena Lin, Paul D. Piehowski, Chia-Feng Tsai, Tatyana Makushok, Lian Yi, Ulises Diaz, Connie Yan, Diana Summers, Pranidhi Sood, Richard D. Smith, Tao Liu, Wallace F. Marshall
bioRxiv 2022.01.06.475287; doi: https://doi.org/10.1101/2022.01.06.475287
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Proteomic Dissection of a Giant Cell
Athena Lin, Paul D. Piehowski, Chia-Feng Tsai, Tatyana Makushok, Lian Yi, Ulises Diaz, Connie Yan, Diana Summers, Pranidhi Sood, Richard D. Smith, Tao Liu, Wallace F. Marshall
bioRxiv 2022.01.06.475287; doi: https://doi.org/10.1101/2022.01.06.475287

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