A self-assembling surface layer flattens the cytokinetic furrow to aid cell division in an archaeon

Bioengineering Biophysics Microbiology Cell Biology Biochemistry Life Sciences
self-assembly surface layer cell division archaea S-layer membrane deformation cell mechanics

1. Academic Background

Since the origin of life, living organisms have needed to construct effective barriers to protect their cells from external physical and chemical damage. In the realms of bacteria and archaea, the surface layer (S-layer) is a universally present and exquisitely structured two-dimensional protein lattice that substitutes for the cell wall or capsular polysaccharides, playing crucial roles in defense and structural support. These S-layers can protect the cell membrane, offering unique defense against harsh environments, predation, osmotic pressure, and toxin exposure. However, such regular lattices are also thought to physically restrict rapid changes in cell morphology (such as cell division and cytoplasmic partitioning). Thus, how cells achieve a balance between maintaining mechanical strength and enabling rapid division has become a core scientific question in cell biology.

The archaeon Sulfolobus acidocaldarius, a representative of the thermoacidophilic family, is an ideal model for studying archaeal cell biology, environmental adaptation, and the relevance to the origin of eukaryotic cells. The S-layer structure of this species has been revealed by high-resolution technologies, and its protein components are found to be highly glycosylated and tightly embedded in the cell membrane. However, the interaction between cell division mechanisms and the S-layer in Sulfolobus acidocaldarius remains far from being fully elucidated. In particular, during division, Sulfolobus acidocaldarius remodels its cell membrane via ESCRT-III (endosomal sorting complexes required for transport-III) proteins and the Vps4 AAA-type ATPase, a molecular mechanism highly similar to that of eukaryotic cells. On one hand, this division relies on intense membrane remodeling, while on the other, it demands the integrity of the external S-layer for protection. How these two factors are coordinated is a key focus of this study.

Previous studies have found significant differences in how S-layer proteins are inserted and expanded in different species—some being concentrated at the cell midzone, while others are distributed uniformly across the surface. There has not yet been a systematic study on how the S-layer of Sulfolobus acidocaldarius self-assembles, how gaps are filled during cell growth, or the specific contribution of this protein lattice to the division machinery.

2. Source of the Paper

This article, titled “A self-assembling surface layer flattens the cytokinetic furrow to aid cell division in an archaeon,” is authored by Sherman Foo, Ido Caspy, Alice Cezanne, Tanmay A. M. Bharat, and Buzz Baum, from the Cell Biology Division and the Structural Studies Division, Medical Research Council Laboratory of Molecular Biology (MRC LMB), Cambridge, UK. The paper was published in the leading academic journal Proceedings of the National Academy of Sciences (PNAS), Vol. 122, Issue 25, 2025 (DOI: 10.1073/pnas.2501044122), and is open access.

3. Detailed Description of Research Procedures

This study systematically investigates the self-assembly properties of the Sulfolobus acidocaldarius S-layer, its molecular anchoring mechanism, and its impact on cell division. The experimental design and implementation include the following key steps:

1. Construction and Phenotypic Analysis of S-layer Related Mutants

Using molecular genetics techniques, the researchers knocked out the major S-layer structural protein SlaA (Saci2355, Δslaa), the membrane anchor protein SlaB (Saci2354, Δslab), as well as a double knockout of both (Δslaab), creating various mutants. All mutants were confirmed by genotyping and whole-genome sequencing. Furthermore, by employing fluorescein-labelled concanavalin A (ConA) (a lectin that specifically binds glycosylated proteins), the glycosylation level of the cell surface S-layer was assessed. The results showed that any single or double knockout significantly reduced surface glycosylation, although residual labelling indicated that other surface proteins or glycolipids also participate in glycosylation.

2. Identification of S-layer Anchor Proteins and Redundant Systems

Cryo-electron microscopy analysis of the cell surface ultrastructure revealed that deleting SlaA resulted in complete loss of the S-layer, while deleting SlaB left patchy remnants, suggesting the presence of a redundant anchoring system. Through homology analysis, structural prediction, and further gene knockouts, it was discovered that Saci1846 (a thermopsin family protein) is highly related to SlaB in both domains and function. Knocking out Saci1846 alone did not lead to a complete loss of the S-layer, whereas a double knockout of SlaB/Saci1846 showed a complete S-layer breakdown and shedding, further confirming their redundant anchoring roles.

3. S-layer Biogenesis Dynamics and Protein Insertion Patterns

To elucidate the mechanism of S-layer insertion, the researchers optimized a dual-channel pulse-chase labelling experiment using imido NHS ester dyes (Alexa Fluor NHS Ester). The old layer was first labeled with 488, and after two hours, the new inserted layer was labeled with 647; confocal microscopy tracked the spatial distribution of the proteins. The results demonstrated that new S-layer protein insertion occurs randomly over the cell surface and is not confined to the division midzone, contrasting with certain other bacteria/archaea that use focal insertion (like Caulobacter, Clostridium, etc.).

4. S-layer Self-Assembly and In Vitro Reconstruction Experiments

Furthermore, the researchers engineered a system to express C-terminally HA-tagged SlaA. Upon induced overexpression, the HA-tagged SlaA could be uniformly incorporated into the S-layer, repairing regions lost in cells. When induced in a Δslaa background, only protein patches appeared, gradually spreading to cover the surface, mimicking the dynamic of “spontaneous expansion of growth islands.” When exogenous purified SlaA proteins, labelled with NHS dye, were incubated with Δslaa mutants, these proteins assembled into fragmentary island-like structures that gradually merged. These results clearly demonstrated that S-layer formation depends on the capacity of molecular self-assembly—when there are available membrane anchor proteins, the protein restores complete structure via lattice expansion and gap filling.

5. Functional Verification of S-layer in Cell Division

The researchers employed flow cytometry (Hoechst-stained DNA) to quantify the chromosomal content of various mutants during exponential growth. Δslaa/Δslab/Δslaab mutants exhibited increased abnormal >2N DNA proportions, indicating division defects. To precisely analyze interactions between the S-layer and the division machinery, and the mechanical feedback during division, the team used a dominant-negative mutant of the Vps4 Walker B locus (Vps4E209Q), which blocks ESCRT-III ring disassembly, thereby “freezing” division events in time. Combined with cryo-electron microscopy and live-cell imaging, they found that in wild-type cells with a complete S-layer, even the extreme division bridge regions remained fully covered with almost no gaps, making it difficult for exogenous S-layer proteins to localize there. In contrast, absence of the S-layer resulted in exogenous proteins preferentially binding to the division bridge region.

The experiments further revealed that the S-layer lattice tends to cover regions of low curvature, showing mild exclusion at sites of high curvature (such as the center of the division bridge). Moreover, live-cell imaging of S-layer-deficient mutants showed that in cells with a complete S-layer, the division furrow became notably flatter during division, with accelerated division speed and slicing-like, rapid and even separation. Mutants lacking the S-layer exhibited highly curved division bridges, slow constriction, increased likelihood of division failure, and even cell body asymmetry. Under intensified physical stress (such as hyperosmotic 4% sucrose), S-layer-deficient cells showed a significantly increased rate of division failures, further supporting the S-layer’s mechanical protection function.

4. Interpretation of Major Findings

1. S-layer Protein Self-Assembly Mechanism Rigorously Confirmed

Whether by endogenous overexpression or exogenous addition, SlaA protein can spontaneously expand into a continuous lattice supported by membrane anchor proteins (SlaB, Saci1846, etc.). This self-assembly does not depend on real-time local protein synthesis, but occurs mainly through intrinsic protein aggregation and lattice extension.

2. Dual System of S-layer Anchoring Ensures Structural Integrity

SlaB and the newly identified Saci1846 are the primary membrane anchor proteins. When either is singly deleted, the S-layer relies on the other for partial anchoring; double deletion results in complete loss of the S-layer. This finding reveals a highly redundant and robust anchoring mechanism for the S-layer.

3. S-layer Dynamically Fills Gaps During Cell Growth, Insertion Is Not Focal

Insertion of new S-layer proteins mainly occurs by random gap filling, and is not focused at the division midzone. Unlike some prokaryotes that drive division through midzone S-layer insertion, Sulfolobus acidocaldarius distributes new insertions over its entire surface.

4. S-layer Aids Division, Maintaining Efficiency Especially Under Stress

Mutant data show conclusively that in the absence of the S-layer, the division process is significantly slowed, failure rates increase, the division furrow becomes abnormally curved during live-cell division, cell morphology is aberrant, and cells are more susceptible to environmental stresses. The S-layer mechanically “flattens” the division furrow, accelerating division and enhancing physical robustness.

5. Conclusion and Significance

This research, from multiple angles—molecular genetics, ultrastructure, biochemistry, cell dynamics—systematically reveals the self-assembly properties of the S-layer in Sulfolobus acidocaldarius, its redundant membrane anchoring mechanism, and its unique functions during division. It is particularly notable for showing that the S-layer is not merely a passive mechanical protector, but also actively facilitates rapid division, ensures division symmetry, and maintains division bridge stability—overturning traditional concepts about the mechanical rigidity limiting cell division.

The scientific significance of this study includes:

  • Revealing how two-dimensional surface protein lattices, through self-assembly and redundant anchoring, both protect the cell and support rapid division—a compatible mechanism that provides a new model for understanding the collaborative mechanical principles between prokaryotic cell membranes and external lattices;
  • Thoroughly comparing different S-layer insertion behaviors, enriching theoretical understanding of prokaryotic cell surface dynamics and maintenance mechanisms;
  • Elucidating the organic coupling between ESCRT-III division mechanisms and passive mechanical support, which may provide molecular evidence for interpreting early eukaryotic cell origins and division mechanism evolution.

In terms of practical applications, the relevant theories and experimental foundation are expected to inspire advances in nanomaterial self-assembly, protein engineering, and biological surface protection materials. Furthermore, this system also theoretically supports the development of highly ordered nanostructure templates and stress-resistant engineered microorganisms in synthetic biology.

6. Research Highlights

  1. First systematic analysis of the self-assembly mechanism of the Sulfolobus acidocaldarius S-layer and its physical role in division, revealing a balanced model of “coexistence of mechanical constraint and division flexibility.”
  2. Discovery that new S-layer insertion occurs via random gap filling, not focal insertion, breaking the limitations of previous reference models.
  3. Identification and functional verification of redundant membrane anchor proteins working cooperatively to ensure structural robustness under extreme conditions.
  4. Integration of multiple high-precision experimental methods (high-temperature live-cell imaging, cryo-EM, protein chemical labeling, etc.) to innovatively reconstruct the division dynamics process.
  5. This work provides a new paradigm for the association of archaeal cell division mechanisms and external protein layer dynamics, representing a milestone for understanding evolutionary strategies combining mechanical and molecular mechanisms in early life.

7. Other Valuable Information

The research was jointly conducted by a multidisciplinary team from institutions such as the UK MRC LMB and funded by multiple agencies, including the Wellcome Trust, EMBO, and Human Frontiers Science Program. The Supplementary Information (SI Appendix) contains details on molecular cloning, flow data, raw cryo-EM images, data analysis algorithms, etc., providing a strong foundation for peers in the field to reproduce or expand the research. All data and materials are completely open, reflecting the scientific integrity and spirit of collaboration of a world-class team.

This project deepens our understanding of the relationship between microbial surface self-assembled structures and cell dynamics, clarifying the dual role of the archaeal S-layer as an “ancient nanoscopic exoskeleton” in cell physiology, ecological adaptation, and evolutionary innovation. It is sure to attract further attention and in-depth study in structural biology, microbiology, and molecular mechanics.