A robust method for transfection in choanoflagellates illuminates their cell biology and the ancestry of animal septins
Posted on: 18 June 2018 , updated on: 19 June 2018
Preprint posted on 9 June 2018
Article now published in Molecular Biology of the Cell at http://dx.doi.org/10.1091/mbc.e18-08-0514
The development of transfection technologies in choanoflagellates sheds light on ancestral cell scaffolding machinery.
Selected by Maya Emmons-Bell
Categories: biochemistry, cell biology, evolutionary biology, genetics, molecular biology
Choanoflagellates are closely related to animals, making them a fantastic comparative model to reconstruct the cell biology of the first animals. However, until now, research in choanoflagellates has been limited by the absence of methods for efficient transgene expression.
Here, Booth et al. present the rigorous and comprehensive development of a method for efficient DNA delivery and expression in the choanoflagellate Salpingoeca rosetta. The multi-step method is the first report of efficient transgenic transformation in the system. The authors carefully dissect the contribution of each component of the protocol in producing efficient transformation, positing particular technical procedures which could assist in development of transgenics in other emerging cellular model systems. For example, choanoflagellates are encased in a thick mesh of extracellular matrix, which has historically hampered effective delivery of DNA. The authors develop a method for removing much of the ECM mesh without damaging cells, as well as novel recovery conditions, allowing for successful nucleofection and subsequent cell survival.
The group goes on to endogenously tag gene products in order to highlight cellular structures in S. rosetta. They present markers of organelles and cytoskeletal elements, uncovering novel actin and tubulin structures, and allowing for visualization of flagellar beating in vivo.
Ultimately, choanoflagellates are a powerful experimental system to interrogate the function of gene products known to be important to animal cell biology, in the hopes of reconstructing what the ancestors of animals may have looked like. To this end, Booth et al. endogenously tagged the septin SrSeptin2. Septins play central roles in cell structure, division, and polarity in animal cells, and SrSeptin2 transcript abundance is markedly increased in the multicellular life stage of the choanoflagellate S. rosetta. Incredibly, the authors found that choanoflagellate septins are localized basolaterally at cell contacts, in similar patterns to polarized epithelial cells, suggesting that spatially restricted septin expression may have evolved before the divergence of choanoflagellates and animals.
The paper reports a massive amount of careful and laborious protocol optimization, leading to a major technical advance in the field. But, it goes beyond tool development to unveil novel choanoflagellate structures and the conservation of core polarity and adhesion molecules between animals and their closest relatives.
In the future, scientists may tag any number of genes to compare the expression and localization of the products between choanoflagellates and animal cells.
Fluorescent markers expressed from reporter plasmids in the choanoflagellate S. rosetta.
Reproduced from Figure 3. of the preprint.
Questions for the authors
What general features of non-model cells might impact transfection protocol optimization? How might researchers modify your protocol to apply it to new species?
What gene families would be most informative to tag and visualize in choanoflagellates in order to draw meaningful conclusions about animal evolution?
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A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) Biochemistry/metabolism 2) Cancer cell Biology 3) Cell adhesion, migration and cytoskeleton 4) Cell organelles and organisation 5) Cell signalling and 6) Genetics
| List by | Barbora Knotkova et al. |
Keystone Symposium – Metabolic and Nutritional Control of Development and Cell Fate
This preList contains preprints discussed during the Metabolic and Nutritional Control of Development and Cell Fate Keystone Symposia. This conference was organized by Lydia Finley and Ralph J. DeBerardinis and held in the Wylie Center and Tupper Manor at Endicott College, Beverly, MA, United States from May 7th to 9th 2025. This meeting marked the first in-person gathering of leading researchers exploring how metabolism influences development, including processes like cell fate, tissue patterning, and organ function, through nutrient availability and metabolic regulation. By integrating modern metabolic tools with genetic and epidemiological insights across model organisms, this event highlighted key mechanisms and identified open questions to advance the emerging field of developmental metabolism.
| List by | Virginia Savy, Martin Estermann |
April in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) biochemistry/metabolism 2) cell cycle and division 3) cell organelles and organisation 4) cell signalling and mechanosensing 5) (epi)genetics
| List by | Vibha SINGH et al. |
Biologists @ 100 conference preList
This preList aims to capture all preprints being discussed at the Biologists @100 conference in Liverpool, UK, either as part of the poster sessions or the (flash/short/full-length) talks.
| List by | Reinier Prosee, Jonathan Townson |
February in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) biochemistry and cell metabolism 2) cell organelles and organisation 3) cell signalling, migration and mechanosensing
| List by | Barbora Knotkova et al. |
Community-driven preList – Immunology
In this community-driven preList, a group of preLighters, with expertise in different areas of immunology have worked together to create this preprint reading list.
| List by | Felipe Del Valle Batalla et al. |
January in preprints – the CellBio edition
A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. This month, categories include: 1) biochemistry/metabolism 2) cell migration 3) cell organelles and organisation 4) cell signalling and mechanosensing 5) genetics/gene expression
| List by | Barbora Knotkova et al. |
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
| List by | Nathalie Krauth |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |