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.

Bottom up self-assembly of functional materials at liquid-liquid interfaces has recently emerged as method to design and produce novel 2D nanostructured membranes and devices with tailored properties. Liquid-liquid interfaces can be seen as a "factory floor" for nanoparticle (NP) selfassembly, since NPs are driven there by a reduction of interfacial energy. Such 2D assembly can be characterised by reciprocal space techniques, namely X-ray and neutron scattering or reflectivity.These techniques have drawbacks however, as the structural information is averaged over the finite size of the radiation beam and non-periodic isolated assemblies in 3D or defects may not be easily detected. Real-space in-situ imaging methods are more appropriate in this context, but they often suffer from limited resolution and under-perform or fail when applied to challenging liquid-liquid interfaces. Here we study the surfactant-induced assembly of SiO2 nanoparticle monolayers at a water-oil interface using in-situ atomic force microscopy (AFM) achieving nanoscale resolved imaging capabilities. Hitherto, AFM imaging has been restricted to solid-liquid interfaces since

The force between two interacting particles as a function of distance is one of the most fundamental curves in science. In this regard, Atomic Force Microscopy (AFM) represents the most powerful tool in nanoscience but with severe limits when it is to probe attractive interactions with high sensitivity. The Force Feedback Microscope (FFM) described here, removes from AFM the well known jump to contact problem that precludes the complete exploration of the interaction curve and the study of associated energy exchanges. The FFM makes it possible to explore tip-surface interactions in the entire range of distances with a sensitivity better than 1 pN. FFM stands out as a radical change in AFM control paradigms. With a surprisingly simple arrangement it is possible to provide the AFM tip with the right counterforce to keep it fixed at any time. The counterforce is consequently equal to the tip-sample force. The force, force gradient and damping are simultaneously measured independently of the tip position. This permits the measurement of energy transfer in thermodynamic transformations. Here we show some FFM measurement examples of the complete interaction force curve and in particular that the FFM can follow the nucleation of a water bridge by measuring the capillary attractive force at all distances, without jump to contact despite the large attractive capillary force. Real time combination of the measured parameters will lead to new imaging modalities with chemical contrast in different environments.As stated by Feynman in his lectures [1], the force versus the distance between two interacting atoms is of the utmost importance in science being at the basis of our understanding of interactions between two objects. From the sharp repulsive regime felt at very short distances, a daily manifestation of the Pauli repulsion principle, the interaction extends for many tens or hundreds of nanometers in a (usually) long attractive regime. In this regime different sources (electrostatic, magnetic, chemical, capillary, van der Waals) at different scales intervene, and the quantitative measurement of the interaction over the entire span is essential for the understanding of the underlying mechanisms. This is well reflected by the wealth of AFM experimental activity in different environments and thermodynamical conditions covering physics, mechanics, biology, chemistry and soft condensed matter. However, despite the great successes obtained by AFM [2, 3,4,5] and Surface Force Apparatus (SFA) [6,7], up to now there is no instrument that can systematically and directly provide in real time the full force curve at nanoscale and at the pN levelas a real unambiguous experimental result. The SFA integrates over microareas, while in AFM, most of the time, an uncontrolled and irreversible dive of the nanotip onto the surface prevents direct and immediate access to the interaction in a large portion of the attractive regime. This jump to contact intervenes as soon as the force gradient overcomes the stiffness of soft AFM microlevers us...

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information.

Cell processes like endocytosis, membrane resealing, signaling and transcription involve conformational changes which depend on the chemical composition and the physicochemical properties of the lipid membrane. The better understanding of the mechanical role of lipids in cell membrane force-triggered and sensing mechanisms has recently become the focus of attention. Different membrane models and experimental methodologies are commonly explored. While general approaches involve controlled vesicle deformation using micropipettes or optical tweezers, due to the local and dynamic nature of the membrane, high spatial resolution atomic force microscopy (AFM) has been widely used to study the mechanical compression and indentation of supported lipid bilayers (SLBs). However, the substrate contribution remains unkown. Here, we demonstrate how pulling lipid tubes with an AFM out of model SLBs can be used to assess the nanomechanics of SLBs through the evaluation of the tube growing force (Ftube), allowing for very local evaluation with high spatial and force resolution of the lipid membrane tension. We first validate this approach to determine the contribution of different phospholipids, by varying the membrane composition, in both one-component and phase-segregated membranes. Finally, we successfully assess the contribution of the underlying substrate to the membrane mechanics, demonstrating that SLB models may represent an intermediate scenario between a free membrane (blebs) and a cytoskeleton supported membrane.