N-Formylation modifies membrane damage associated with PSMα3 interfacial fibrillation.

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining atomic force microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains only in the fluid phase of membranes and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

Aptamer-based nanotrains and nanoflowers as quinine delivery systems.

In this study, we designed aptamer-based self-assemblies for the delivery of quinine. Two different architectures were designed by hybridizing quinine binding aptamers and aptamers targeting Plasmodium falciparum lactate dehydrogenase (PfLDH): nanotrains and nanoflowers. Nanotrains consisted in controlled assembly of quinine binding aptamers through base-pairing linkers. Nanoflowers were larger assemblies obtained by Rolling Cycle Amplification of a quinine binding aptamer template. Self-assembly was confirmed by PAGE, AFM and cryoSEM. The nanotrains preserved their affinity for quinine and exhibited a higher drug selectivity than nanoflowers. Both demonstrated serum stability, hemocompatibility, low cytotoxicity or caspase activity but nanotrains were better tolerated than nanoflowers in the presence of quinine. Flanked with locomotive aptamers, the nanotrains maintained their targeting ability to the protein PfLDH as analyzed by EMSA and SPR experiments. To summarize, nanoflowers were large assemblies with high drug loading ability, but their gelating and aggregating properties prevent from precise characterization and impaired the cell viability in the presence of quinine. On the other hand, nanotrains were assembled in a selective way. They retain their affinity and specificity for the drug quinine, and their safety profile as well as their targeting ability hold promise for their use as drug delivery systems.

Structure and mechanics of the human nuclear pore complex basket using correlative AFM-fluorescence superresolution microscopy.

Nuclear pore complexes (NPCs) are the only gateways between the nucleus and cytoplasm in eukaryotic cells. They restrict free diffusion to molecules below 5 nm while facilitating the active transport of selected cargoes, sometimes as large as the pore itself. This versatility implies an important pore plasticity. Recently, cryo-EM and AI-based protein modeling of human NPC revealed with acute precision how most constituents are arranged. But the basket, a fish trap-like structure capping the nucleoplasmic side of the pore, remains poorly resolved. Here by atomic force microscopy (AFM) coupled to single molecule localization microscopy (SMLM) we revealed that the basket is very soft and explores a large conformational landscape: apart from its canonical basket shape, it dives into the central pore channel or opens, with filaments reaching to the pore sides. Our observations highlight how this structure can adapt and let morphologically diverse cargoes shuttle through NPCs.

WITHDRAWN: Aptamer-based nanotrains and nanoflowers as quinine delivery systems.

This article has been withdrawn: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been withdrawn at the request of the author, editor and publisher. The publisher regrets that an error occurred during the publication of this paper, which was intended to be published in International Journal of Pharmaceutics: X (not International Journal of Pharmaceutics). This error bears no reflection on the scientific content of this article or its authors. The publisher apologizes to the readers for this unfortunate error.

Correlative AFM and fluorescence imaging demonstrate nanoscale membrane remodeling and ring-like and tubular structure formation by septins.

Septins are ubiquitous cytoskeletal filaments that interact with the inner plasma membrane and are essential for cell division in eukaryotes. In cellular contexts, septins are often localized at micrometric Gaussian curvatures, where they assemble onto ring-like structures. The behavior of budding yeast septins depends on their specific interaction with inositol phospholipids, enriched at the inner leaflet of the plasma membrane. Septin filaments are built from the non-polar self-assembly of short rods into filaments. However, the molecular mechanisms regulating the interplay with the inner plasma membrane and the resulting interaction with specific curvatures are not fully understood. In this report, we have imaged dynamical molecular assemblies of budding yeast septins on PIP2-containing supported lipid bilayers using a combination of high-speed AFM and correlative AFM-fluorescence microscopy. Our results clearly demonstrate that septins are able to bind to flat supported lipid bilayers and thereafter induce the remodeling of membranes. Short septin rods (octamers subunits) can indeed destabilize supported lipid bilayers and reshape the membrane to form 3D structures such as rings and tubes, demonstrating that long filaments are not necessary for septin-induced membrane buckling.

Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization.

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

Imaging Artificial Membranes Using High-Speed Atomic Force Microscopy.

Supported lipid bilayers represent a very attractive way to mimic biological membranes, especially to investigate molecular mechanisms associated with the lateral segregation of membrane components. Observation of these model membranes with high-speed atomic force microscopy (HS-AFM) allows the capture of both topography and dynamics of membrane components, with a spatial resolution in the nanometer range and image capture time of less than 1 s. In this context, we have developed new protocols adapted for HS-AFM to form supported lipid bilayers on small mica disks using the vesicle fusion or Langmuir-Blodgett methods. In this chapter we describe in detail the protocols to fabricate supported artificial bilayers as well as the main guidelines for HS-AFM imaging of such samples.

Structural basis for the oligomerization of the MADS domain transcription factor SEPALLATA3 in Arabidopsis.

In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.