Menu

Close

A robust method for transfection in choanoflagellates illuminates their cell biology and the ancestry of animal septins

David Booth, Heather Middleton, Nicole King

Preprint posted on June 09, 2018 https://www.biorxiv.org/content/early/2018/06/09/343111

The development of transfection technologies in choanoflagellates sheds light on ancestral cell scaffolding machinery.

Selected by Maya Emmons-Bell

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?

 

Posted on: 18th June 2018 , updated on: 19th June 2018

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the biochemistry category:

    Site-specific K63 ubiquitinomics reveals post-initiation regulation of ribosomes under oxidative stress

    Songhee Back, Christine Vogel, Gustavo M Silva



    Selected by Srivats Venkataramanan

    1

    Template switching causes artificial junction formation and false identification of circular RNAs

    Chong Tang, Tian Yu, Yeming Xie, et al.



    Selected by Fabio Liberante

    Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant

    Vilius Kurauskas, Audrey Hessel, François Dehez, et al.

    AND

    Major concerns with the integrity of the mitochondrial ADP/ATP carrier in dodecyl-phosphocholine used for solution NMR studies

    Martin S. King, Paul G. Crichton, Jonathan J. Ruprecht, et al.



    Selected by Reid Alderson

    1

    Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin

    Kwo-Kwang Abraham Wang, Tai L. Ng, Peng Wang, et al.



    Selected by Ellis O'Neill

    TTL proteins scaffold brassinosteroid signaling components at the plasma membrane to optimize signal transduction in plant cells

    Vitor Amorim-Silva, Alvaro Garcia-Moreno, Araceli G Castillo, et al.



    Selected by Martin Balcerowicz

    1

    OptoGranules reveal the evolution of stress granules to ALS-FTD pathology

    Peipei Zhang, Baochang Fan, Peiguo Yang, et al.



    Selected by Srivats Venkataramanan

    1

    Structural Basis of Tubulin Recruitment and Assembly by Tumor Overexpressed Gene (TOG) domain array Microtubule Polymerases

    Stanley Nithiananatham, Brian Cook, Fred Chang, et al.

    AND

    Roles for tubulin recruitment and self-organization by TOG domain arrays in Microtubule plus-end tracking and polymerase

    Brian Cook, Fred Chang, Ignacio Flor-Parra, et al.



    Selected by Ben Craske, Thibault Legal and Toni McHugh

    SWI/SNF remains localized to chromatin in the presence of SCHLAP1

    Jesse R Raab, Keriayn N Smith, Camarie C Spear, et al.



    Selected by Carmen Adriaens

    1

    A non-canonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells

    Saipraveen Srinivasan, Christoph J. Burckhardt, Madhura Bhave, et al.



    Selected by Penelope La-Borde

    HIF1-alpha expressing cells induce a hypoxic-like response in neighbouring cancer cells

    Hannah Harrison, Henry J Pegg, Jamie Thompson, et al.



    Selected by Anh Hoang Le

    Higher-Order Organization Principles of Pre-translational mRNPs

    Mihir Metkar, Hakan Ozadam, Bryan R. Lajoie, et al.



    Selected by Carmen Adriaens

    Capturing the onset of PRC2-mediated repressive domain formation

    Ozgur Oksuz, Varun Narendra, Chul-Hwan Lee, et al.



    Selected by Boyan Bonev

    RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs

    Mark A McClintock, Carly I Dix, Christopher M Johnson, et al.

    AND

    Recruitment of Two Dyneins to an mRNA-Dependent Bicaudal D Transport Complex

    Thomas E. Sladewski, Neil Billington, M. Yusuf Ali, et al.



    Selected by Dmitry Nashchekin

    Dynamic Kinetochore Size Regulation Promotes Microtubule Capture And Chromosome Biorientation In Mitosis

    Carlos Sacristan, Misbha Ahmad, Jenny Keller, et al.

    AND

    Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

    Cláudia Pereira, Rita M Reis, José B Gama, et al.



    Selected by Gautam Dey

    Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

    Tess C Branon, Justin A Bosch, Ariana D Sanchez, et al.



    Selected by Ralph Böttcher

    Nuclear envelope assembly defects link mitotic errors to chromothripsis

    Shiwei Liu, Mijung Kwon, Mark Mannino, et al.



    Selected by Gautam Dey

    Also in the cell biology category:

    SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

    Jocelyn Y. Kishi, Brian J. Beliveau, Sylvain W. Lapan, et al.



    Selected by Yen-Chung Chen

    PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection

    Catherine M Buckley, Victoria L Heath, Aurelie Gueho, et al.



    Selected by Giuliana Clemente

    1

    Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

    Sydney M Shaffer, Benjamin L Emert, Ann E. Sizemore, et al.



    Selected by Leighton Daigh

    1

    Aurora A depletion reveals centrosome-independent polarization mechanism in C. elegans

    Kerstin Klinkert, Nicolas Levernier, Peter Gross, et al.

    AND

    Centrosome Aurora A gradient ensures a single PAR-2 polarity axis by regulating RhoGEF ECT-2 localization in C. elegans embryos

    Sachin Kotak, Sukriti Kapoor



    Selected by Giuliana Clemente

    Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling

    Amber N Stratman, Olivia M Farrelly, Constantinos M Mikelis, et al.



    Selected by Coert Margadant

    Neural crest cells regulate optic cup morphogenesis by promoting extracellular matrix assembly

    Chase Dallas Bryan, Rebecca Lynne Pfeiffer, Bryan William Jones, et al.



    Selected by Ashrifia Adomako-Ankomah

    1

    mRNA localisation in endothelial cells regulates blood vessel sprouting

    Guilherme Costa, Nawseen Tarannum, Shane Herbert



    Selected by Andreas van Impel

    Local protein synthesis in axon terminals and dendritic spines differentiates plasticity contexts

    Anne-Sophie Hafner, Paul Donlin-Asp, Beulah Leitch, et al.



    Selected by Dipen Rajgor

    The cytoskeleton as a smart composite material: A unified pathway linking microtubules, myosin-II filaments and integrin adhesions

    Nisha Mohd Rafiq, Yukako Nishimura, Sergey V. Plotnikov, et al.



    Selected by Coert Margadant

    Quantitative, real-time, single cell analysis in tissue reveals expression dynamics of neurogenesis

    Cerys S Manning, Veronica Biga, James Boyd, et al.



    Selected by Teresa Rayon

    Profiling the surface proteome identifies actionable biology for TSC1 mutant cells beyond mTORC1 signaling

    Junnian Wei, Kevin K. Leung, Charles Truillet, et al.



    Selected by Rob Hynds

    1

    Optogenetic dissection of mitotic spindle positioning in vivo

    Lars-Eric Fielmich, Ruben Schmidt, Daniel J Dickinson, et al.



    Selected by Angika Basant

    1

    Excitable RhoA dynamics drive pulsed contractions in the early C. elegans embryo.

    Jonathan B Michaux, Francois B Robin, William M McFadden, et al.



    Selected by Sundar Naganathan

    Moving beyond P values: Everyday data analysis with estimation plots

    Joses Ho, Tayfun Tumkaya, Sameer Aryal, et al.



    Selected by Gautam Dey

    1

    A limited number of double-strand DNA breaks are sufficient to delay cell cycle progression.

    Jeroen van den Berg, Anna G. Manjon, Karoline Kielbassa, et al.



    Selected by Leighton Daigh

    Optogenetic manipulation of medullary neurons in the locust optic lobe

    Hongxia Wang, Richard B. Dewell, Markus U. Ehrengruber, et al.



    Selected by Ana Patricia Ramos

    Also in the evolutionary biology category:

    millepattes micropeptides are an ancient developmental switch required for embryonic patterning

    Suparna Ray, Miriam I Rosenberg, Hélène Chanut-Delalande, et al.



    Selected by Erik Clark

    Conserved phosphorylation hotspots in eukaryotic protein domain families

    Marta J Strumillo, Michaela Oplova, Cristina Vieitez, et al.



    Selected by Gautam Dey

    Peculiar features of the plastids of the colourless alga Euglena longa and photosynthetic euglenophytes unveiled by transcriptome analyses

    Kristina Zahonova, Zoltan Fussy, Erik Bircak, et al.



    Selected by Ellis O'Neill

    1

    The Ly6/uPAR protein Bouncer is necessary and sufficient for species-specific fertilization

    Sarah Herberg, Krista R Gert, Alexander Schleiffer, et al.



    Selected by James Gagnon

    Timed collinear activation of Hox genes during gastrulation controls the avian forelimb position

    Chloe Moreau, Paolo Caldarelli, Didier Rocancourt, et al.



    Selected by Wouter Masselink

    The genomic basis of colour pattern polymorphism in the harlequin ladybird

    Mathieu Gautier, Junichi Yamaguchi, Julien Foucaud, et al.



    Selected by Fillip Port

    Altering the temporal regulation of one transcription factor drives sensory trade-offs

    Ariane Ramaekers, Simon Weinberger, Annelies Claeys, et al.



    Selected by Mariana R.P. Alves

    A robust method for transfection in choanoflagellates illuminates their cell biology and the ancestry of animal septins

    David Booth, Heather Middleton, Nicole King



    Selected by Maya Emmons-Bell

    A SoxB gene acts as an anterior gap gene and regulates posterior segment addition in the spider Parasteatoda tepidariorum

    Christian L. B. Paese, Anna Schoenauer, Daniel J. Leite, et al.



    Selected by Erik Clark

    1

    Germ layer specific regulation of cell polarity and adhesion gives insight into the evolution of mesoderm.

    Miguel Salinas-Saavedra, Amber Q. Rock, Mark Q. Martindale



    Selected by ClaireS & SophieM

    1

    Wnt/β-catenin regulates an ancient signaling network during zebrafish scale development

    Andrew J Aman, Alexis N Fulbright, David M Parichy



    Selected by Andreas van Impel

    Bacteriophage cooperation suppresses CRISPR-Cas3 and Cas9 immunity

    Adair L Borges, Jenny Y Zhang, MaryClare Rollins, et al.

    AND

    Anti-CRISPR phages cooperate to overcome CRISPR-Cas immunity

    Mariann Landsberger, Sylvain Gandon, Sean Meaden, et al.



    Selected by Fillip Port

    Dynamic Kinetochore Size Regulation Promotes Microtubule Capture And Chromosome Biorientation In Mitosis

    Carlos Sacristan, Misbha Ahmad, Jenny Keller, et al.

    AND

    Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

    Cláudia Pereira, Rita M Reis, José B Gama, et al.



    Selected by Gautam Dey

    Individual- and population-level drivers of consistent foraging success across environments

    Lysanne Snijders, Ralf HJM Kurvers, Stefan Krause, et al.



    Selected by Rasmus Ern

    The ancestral animal genetic toolkit revealed by diverse choanoflagellate transcriptomes

    Daniel Richter, Parinaz Fozouni, Michael Eisen, et al.



    Selected by Rafael Galupa

    1

    Genome-wide selection scans integrated with association mapping reveal mechanisms of physiological adaptation across a salinity gradient in killifish

    Reid S. Brennan, Timothy M. Healy, Heather J. Bryant, et al.



    Selected by Andy Turko

    Also in the genetics category:

    millepattes micropeptides are an ancient developmental switch required for embryonic patterning

    Suparna Ray, Miriam I Rosenberg, Hélène Chanut-Delalande, et al.



    Selected by Erik Clark

    Neural crest cells regulate optic cup morphogenesis by promoting extracellular matrix assembly

    Chase Dallas Bryan, Rebecca Lynne Pfeiffer, Bryan William Jones, et al.



    Selected by Ashrifia Adomako-Ankomah

    1

    The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila



    Selected by Clarice Hong

    1

    Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages is necessary for functional spinal cord regeneration in zebrafish

    Themistoklis M. Tsarouchas, Daniel Wehner, Leonardo Cavone, et al.



    Selected by Shikha Nayar

    1

    JNK-mediated spindle reorientation in stem cells promotes dysplasia in the aging intestine

    Daniel Hu, Heinrich Jasper



    Selected by Maiko Kitaoka

    Phenotypic landscape of schizophrenia-associated genes defines candidates and their shared functions

    Summer B. Thyme, Lindsey M. Pieper, Eric H. Li, et al.



    Selected by Daniel Grimes

    Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

    Hannah A. Grunwald, Valentino M. Gantz, Gunnar Poplawski, et al.



    Selected by Rebekah Tillotson

    1

    The Ly6/uPAR protein Bouncer is necessary and sufficient for species-specific fertilization

    Sarah Herberg, Krista R Gert, Alexander Schleiffer, et al.



    Selected by James Gagnon

    Functional testing of a human PBX3 variant in zebrafish reveals a potential modifier role in congenital heart defects

    Gist H. Farr III, Kimia Imani, Darren Pouv, et al.



    Selected by Hannah Brunsdon

    A robust method for transfection in choanoflagellates illuminates their cell biology and the ancestry of animal septins

    David Booth, Heather Middleton, Nicole King



    Selected by Maya Emmons-Bell

    SWI/SNF remains localized to chromatin in the presence of SCHLAP1

    Jesse R Raab, Keriayn N Smith, Camarie C Spear, et al.



    Selected by Carmen Adriaens

    1

    Genetic compensation is triggered by mutant mRNA degradation

    Mohamed El-Brolosy, Andrea Rossi, Zacharias Kontarakis, et al.



    Selected by Andreas van Impel

    1

    Cellular Crowding Influences Extrusion and Proliferation to Facilitate Epithelial Tissue Repair

    Jovany Jeomar Franco, Youmna Maryline Atieh, Chase Dallas Bryan, et al.



    Selected by Helen Weavers

    Limb- and tendon-specific Adamtsl2 deletion identifies a soft tissue mechanism modulating bone length

    Dirk Hubmacher, Stetson Thacker, Sheila M Adams, et al.



    Selected by Alberto Rosello-Diez

    Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)

    Scott J Callahan, Stephanie Tepan, Yan M Zhang, et al.



    Selected by Hannah Brunsdon

    1

    PDX Finder: A Portal for Patient-Derived tumor Xenograft Model Discovery

    Nathalie Conte, Jeremy Mason, Csaba Halmagyi, et al.



    Selected by Carmen Adriaens

    Also in the molecular biology category:

    Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

    Sydney M Shaffer, Benjamin L Emert, Ann E. Sizemore, et al.



    Selected by Leighton Daigh

    1

    Site-specific K63 ubiquitinomics reveals post-initiation regulation of ribosomes under oxidative stress

    Songhee Back, Christine Vogel, Gustavo M Silva



    Selected by Srivats Venkataramanan

    1

    Quantitative, real-time, single cell analysis in tissue reveals expression dynamics of neurogenesis

    Cerys S Manning, Veronica Biga, James Boyd, et al.



    Selected by Teresa Rayon

    The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila



    Selected by Clarice Hong

    1

    Moving beyond P values: Everyday data analysis with estimation plots

    Joses Ho, Tayfun Tumkaya, Sameer Aryal, et al.



    Selected by Gautam Dey

    1

    A limited number of double-strand DNA breaks are sufficient to delay cell cycle progression.

    Jeroen van den Berg, Anna G. Manjon, Karoline Kielbassa, et al.



    Selected by Leighton Daigh

    Template switching causes artificial junction formation and false identification of circular RNAs

    Chong Tang, Tian Yu, Yeming Xie, et al.



    Selected by Fabio Liberante

    Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant

    Vilius Kurauskas, Audrey Hessel, François Dehez, et al.

    AND

    Major concerns with the integrity of the mitochondrial ADP/ATP carrier in dodecyl-phosphocholine used for solution NMR studies

    Martin S. King, Paul G. Crichton, Jonathan J. Ruprecht, et al.



    Selected by Reid Alderson

    1

    Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon

    Jessica Messier, Hongmei Chen, Zhao-Lin Cai, et al.

    AND

    High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins

    Mathias Mahn, Lihi Gibor, Katayun Cohen-Kashi Malina, et al.



    Selected by Mahesh Karnani

    2

    Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin

    Kwo-Kwang Abraham Wang, Tai L. Ng, Peng Wang, et al.



    Selected by Ellis O'Neill

    The Histone H3-H4 Tetramer is a Copper Reductase Enzyme

    Narsis Attar, Oscar A Campos, Maria Vogelauer, et al.



    Selected by Lauren Neves

    Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

    Hannah A. Grunwald, Valentino M. Gantz, Gunnar Poplawski, et al.



    Selected by Rebekah Tillotson

    1

    EFFECTORS OF THE SPINDLE ASSEMBLY CHECKPOINT BUT NOT THE MITOTIC EXIT NETWORK ARE CONFINED WITHIN THE NUCLEUS OF SACCHAROMYCES CEREVISIAE

    Lydia R Heasley, Jennifer G DeLuca, Steven M Markus



    Selected by Hiral Shah

    TTL proteins scaffold brassinosteroid signaling components at the plasma membrane to optimize signal transduction in plant cells

    Vitor Amorim-Silva, Alvaro Garcia-Moreno, Araceli G Castillo, et al.



    Selected by Martin Balcerowicz

    1

    Peculiar features of the plastids of the colourless alga Euglena longa and photosynthetic euglenophytes unveiled by transcriptome analyses

    Kristina Zahonova, Zoltan Fussy, Erik Bircak, et al.



    Selected by Ellis O'Neill

    1

    OptoGranules reveal the evolution of stress granules to ALS-FTD pathology

    Peipei Zhang, Baochang Fan, Peiguo Yang, et al.



    Selected by Srivats Venkataramanan

    1

    Close