Preprint highlights, selected by the biological community

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

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?

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

Read preprint

(No Ratings Yet)

Issue: *

Your Name: *

Your Email: *

Details: *

Have your say Cancel reply

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

Also in the biochemistry category:

Drosophila kinesin-8 stabilises kinetochore-microtubule interaction

Tomoya Edzuka, Gohta Goshima

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

Share this:

Have your say Cancel reply

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

Also in the biochemistry category:

Drosophila kinesin-8 stabilises kinetochore-microtubule interaction

Cryo-EM structure of cardiac amyloid fibrils from an immunoglobulin light chain (AL) amyloidosis patient

PUMILIO hyperactivity drives premature aging of Norad-deficient mice

Budding yeast complete DNA replication after chromosome segregation begins

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

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

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

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

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

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

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

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

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

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

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

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

Higher-Order Organization Principles of Pre-translational mRNPs

Capturing the onset of PRC2-mediated repressive domain formation

Also in the cell biology category:

Drosophila kinesin-8 stabilises kinetochore-microtubule interaction

Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma.

Tension on kinetochore substrates is insufficient to prevent Aurora-triggered detachment

Single-cell RNA sequencing reveals novel cell differentiation dynamics during human airway epithelium regeneration

Actomyosin-driven tension at compartmental boundaries orients cell division independently of cell geometry in vivo

Molecular organization of integrin-based adhesion complexes in mouse Embryonic Stem Cells

Superresolution architecture of pluripotency guarding adhesions

Transcriptional initiation and mechanically driven self-propagation of a tissue contractile wave during axis elongation

Bridging the divide: bacteria synthesizing archaeal membrane lipids

Extensive transfer of membrane lipid biosynthetic genes between Archaea and Bacteria

Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

Signaling dynamics control cell fate in the early Drosophila embryo

Lysosome exocytosis is required for mitosis

Three-dimensional tissue stiffness mapping in the mouse embryo supports durotaxis during early limb bud morphogenesis

PUMILIO hyperactivity drives premature aging of Norad-deficient mice

3D Tissue elongation via ECM stiffness-cued junctional remodeling

Transient intracellular acidification regulates the core transcriptional heat shock response

Budding yeast complete DNA replication after chromosome segregation begins

Also in the evolutionary biology category:

Bridging the divide: bacteria synthesizing archaeal membrane lipids

Extensive transfer of membrane lipid biosynthetic genes between Archaea and Bacteria

PUMILIO hyperactivity drives premature aging of Norad-deficient mice

Eukaryotic acquisition of a bacterial operon

millepattes micropeptides are an ancient developmental switch required for embryonic patterning

Conserved phosphorylation hotspots in eukaryotic protein domain families

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

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

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

The genomic basis of colour pattern polymorphism in the harlequin ladybird

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