Septins regulate cytokinesis and multicellular development in the closest living relatives of animals

This study demonstrates that septins in the choanoflagellate *Salpingoeca rosetta*, the closest living relatives of animals, are essential for regulating cytokinesis, cell size, and multicellular colony development, suggesting that the evolution of animal multicellularity involved adapting cell division mechanisms to meet the mechanical demands of cell adhesion.

The Gist

Imagine a tiny, single-celled organism called Salpingoeca rosetta (let's call it "Rosie"). Rosie is special because she is the closest living cousin to all animals, including humans. She can live as a lonely swimmer, but when she gets a specific chemical signal from bacteria, she transforms into a beautiful, flower-like cluster of cells called a "rosette."

Scientists have long wondered: How did our ancient ancestors learn to stick together to form complex bodies? To answer this, they looked at a specific group of proteins inside Rosie called Septins.

Think of Septins as the construction crew and traffic police of the cell. In fungi and animals, these proteins act like a scaffolding or a rubber band that helps a cell pinch in half to divide (a process called cytokinesis). But nobody knew what they did in Rosie, our animal cousin.

Here is what the researchers discovered, explained simply:

1. The "Rubber Band" Breaks

The team used a molecular pair of scissors (CRISPR) to cut the genes that make these Septin proteins in Rosie. They found that without these proteins, the cells got confused.

• The Result: Instead of dividing cleanly into two perfect, normal-sized cells, the cells failed to finish the job. They tried to split but then snapped back together, creating giant, blob-like cells with multiple nuclei (like a monster with too many brains).
• The Analogy: Imagine trying to cut a piece of dough in half with a string. If the string is weak or missing, the dough doesn't separate; it just stretches and then snaps back together, leaving you with one giant, misshapen lump instead of two perfect rolls.

2. The "Group Hug" Gets Messy

When the scientists tried to turn these broken Septin cells into a rosette (a multicellular flower), things got even worse.

• The Result: The cells couldn't hold the shape of the flower. When they gave the culture a little shake (simulating a wave or wind), the rosettes fell apart instantly.
• The Analogy: Think of a multicellular rosette like a group of people holding hands in a circle. If the people (cells) are weak or confused, the circle breaks apart as soon as someone pushes them. The Septins are the "glue" or the "muscle" that keeps the circle tight and strong.

3. The "Double Duty" Discovery

The most exciting part of the study is why this matters for the history of life.

• The Finding: The Septin proteins worked fine when Rosie was a single swimmer, but they struggled massively when she tried to become a multicellular flower.
• The Big Idea: When the first animals evolved, they started building complex bodies with a shared "skin" (extracellular matrix) that held them together. The researchers suggest that this new "group hug" put mechanical stress on the cells.
• The Metaphor: Imagine you are a solo dancer spinning on a stage. It's easy to spin. But now, imagine you are dancing while holding hands with five other people, all spinning in a tight circle. It's much harder to spin without tripping! The "shared skin" of the animal body made cell division much harder. The Septins had to evolve to become stronger and more precise to handle this new pressure.

The Takeaway

This paper tells us that the evolution of animals wasn't just about learning to stick together; it was also about learning how to divide while stuck together.

The Septin proteins are the unsung heroes that allowed cells to master this difficult trick. By regulating how cells pinch in half, they ensured that as life became more complex and multicellular, the cells could still reproduce without falling apart. It links the story of how we stick together directly to how we split apart, showing that these two processes are deeply connected in the history of life on Earth.

Technical Summary

1. Problem Statement

Septins are conserved cytoskeletal GTPases known to regulate cytokinesis (cell division) and cell shape in fungi and bilaterian animals. However, their function in choanoflagellates—the closest living relatives of animals—remained largely unknown. While choanoflagellates possess septin genes, it was unclear whether these proteins function in cytokinesis or play a role in the transition from unicellular to multicellular life, a key step in animal evolution. Specifically, the study sought to determine if the evolution of multicellularity in the ancestor of animals imposed new mechanical or regulatory demands on cell division machinery.

2. Methodology

The authors utilized the choanoflagellate model organism Salpingoeca rosetta, which can switch between unicellular "slow swimmer" forms and multicellular "rosette" colonies in response to bacterial sulfonolipids (Rosette Inducing Factors, or RIF-OMVs).

• Phylogenetic & Structural Analysis:
  • Constructed an updated phylogeny of septins using protein sequences from diverse opisthokonts (animals, fungi, and choanoflagellates).
  • Used AlphaFold-3 to predict the oligomeric assembly of S. rosetta septins, comparing them to known human and yeast hexameric structures.
• Genome Editing (CRISPR/Cas9):
  • Generated loss-of-function mutants for all four S. rosetta septin genes (Sros_septA, Sros_sept6, Sros_septB, Sros_sept9) by introducing premature stop codons near the 5' end of each gene.
  • Developed a novel endogenous C-terminal tagging strategy using CRISPR/Cas9 to fuse fluorescent proteins (mStayGold) and epitope tags (ALFA) to Sros_SeptA without disrupting its native regulation.
• Phenotypic Assays:
  • Cell Size & Morphology: Quantified cell area and volume in single cells and chains using flow cytometry (Attune CytPix) and confocal microscopy.
  • Rosette Assembly & Integrity: Induced rosette formation with RIF-OMVs and applied mechanical shear (vortexing) to test structural integrity.
  • Cytokinesis Analysis: Performed time-lapse DIC microscopy to observe cell division events, specifically looking for furrow ingression and abscission failures.
  • Localization Studies: Visualized the dynamic localization of tagged Sros_SeptA during interphase and cytokinesis using spinning-disk confocal microscopy.

3. Key Contributions

• Functional Characterization: This is the first study to functionally characterize septins in a choanoflagellate using CRISPR/Cas9, moving beyond transient expression to stable, endogenous mutants.
• Mechanistic Link to Multicellularity: The study provides evidence that the regulation of cell division (cytokinesis) is intrinsically linked to the evolution of multicellular organization, as septin requirements are heightened in multicellular contexts.
• Methodological Advance: The authors successfully adapted CRISPR/Cas9 for endogenous fluorescent tagging in S. rosetta, a technique that will facilitate future live-cell imaging studies in this model organism.

4. Key Results

A. Phylogeny and Structure

• S. rosetta encodes four septins (Sros_septA, Sros_sept6, Sros_septB, Sros_sept9) that cluster with animal and fungal septin families.
• AlphaFold-3 predictions suggest that Sros_septA, Sros_sept6, and Sros_septB assemble into a hexamer structurally similar to the human septin hexamer, with Sros_SeptA occupying the position of animal SEPT7 and Sros_SeptB occupying the SEPT2 position.

B. Regulation of Cell Size

• Disruption of specific septins alters cell size:
  • Loss of Sros_septA and Sros_sept6: Results in oversized cells (1.13–1.25 fold increase in mean cell size).
  • Loss of Sros_sept9: Results in smaller cells.
  • Loss of Sros_septB: No significant change in cell size.

C. Rosette Development and Integrity

• Assembly: Mutants in Sros_septA, Sros_sept6, and Sros_sept9 formed smaller, disorganized rosettes compared to wild-type.
• Integrity: Upon mechanical shear (vortexing), rosettes lacking Sros_septA or Sros_sept6 disassembled significantly more than wild-type or Sros_septB mutants.
• ECM: Despite integrity defects, staining for extracellular matrix (ECM) components (Jacalin and Rosetteless) showed no obvious reduction in ECM secretion, suggesting the defects are not due to a lack of matrix production but potentially to mechanical coupling or bridge stability.

D. Cytokinesis Failure (The Primary Mechanism)

• Late-Stage Failure: Sros_septA mutants exhibited a specific defect in late-stage cytokinesis. While furrow ingression occurred, the cells frequently fused back together after apparent separation, leading to multinucleated, enlarged cells.
• Context Dependence: The rate of cytokinesis failure in Sros_septA mutants was elevated in uninduced (unicellular) cultures but more than doubled upon rosette induction. This indicates that the multicellular context (likely involving shared ECM and cell-cell adhesion) places heightened mechanical demands on the septin cytoskeleton.
• Localization Dynamics: Endogenously tagged Sros_SeptA localized to the basal pole in interphase cells but dynamically redistributed to the cleavage furrow and nascent intercellular bridge during cytokinesis, mirroring the behavior of animal septins.

5. Significance

• Evolutionary Insight: The findings suggest that the evolution of animal multicellularity may have required the co-option or elaboration of existing cytokinetic machinery (septins) to handle the mechanical stresses of dividing cells within a shared extracellular matrix.
• Mechanical Coupling: The study proposes a model where the elaboration of the ECM in early animal ancestors imposed new physical constraints on dividing cells, linking the evolution of cell adhesion directly to the evolution of cytokinetic regulation.
• Foundational Framework: By establishing septins as critical regulators of both cell size and multicellular integrity in choanoflagellates, this work provides a framework for understanding how cell division mechanisms contributed to the emergence of animal multicellularity.