Nanomechanics of multidomain neuronal cell adhesion protein contactin revealed by single molecule AFM and SMD

Contactin-4 (CNTN4) is a complex cell adhesion molecule (CAM) localized at neuronal membranes, playing a key role in maintaining the mechanical integrity and signaling properties of the synapse. CNTN4 consists of six immunoglobulin C2 type (IgC2) domains and four fibronectin type III (FnIII) domains that are shared with many other CAMs. Mutations in CNTN4 gene have been linked to various psychiatric disorders. Toward elucidating the response of this modular protein to mechanical stress, we studied its force-induced unfolding using single molecule atomic force microscopy (smAFM) and steered molecular dynamics (SMD) simulations. Extensive smAFM and SMD data both indicate the distinctive mechanical behavior of the two types of modules distinguished by unique force-extension signatures. The data also reveal the heterogeneity of the response of the individual FNIII and IgC2 modules, which presumably plays a role in the adaptability of CNTN4 to maintaining cell-cell communication and adhesion properties under different conditions. Results show that extensive sampling of force spectra, facilitated by robot-enhanced AFM, can help reveal the existence of weak stabilizing interactions between the domains of multidomain proteins, and provide insights into the nanomechanics of such multidomain or heteromeric proteins.

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability

Thomas Barker, Jeffrey Alan Hubbell, Mikaël Martino, Mayumi Mochizuki Martino, Dominique André Rothenfluh

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9-10) affects its integrin specificity. Through production of FN III9-10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin alpha(5)beta(1) and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of alpha(5)beta(1) integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that alpha(5)beta(1) has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for alpha(5)beta(1) versus alpha(v)beta(3) results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.

Elsevier2008

Functional Dissection of Intersubunit Interactions in the EspR Virulence Regulator of Mycobacterium tuberculosis

Benjamin Blasco, Stewart Cole, Matteo Dal Peraro, Giovanni Dietler, Aleksandre Japaridze, Florence Pojer, Marco Stenta, Basile Isidore Martin Wicky

The nucleoid-associated protein EspR, a chromosome organizer, has pleiotropic effects on expression of genes associated with cell wall function and pathogenesis in Mycobacterium tuberculosis. In particular, EspR binds to several sites upstream of the espACD locus to promote its expression, thereby ensuring full function of the ESX-1 secretion system, a major virulence determinant. The N terminus of EspR contains the helix-turn-helix DNA-binding domain, whereas the C-terminal dimerization domain harbors residues involved in intersubunit interactions. While direct binding to DNA appears to be mediated by an EspR dimer-of-dimers, where two helix-turn-helix motifs remain free for long-range interactions, the mechanism of EspR higher-order organization and its impact on chromosome structure and gene expression are not understood. To investigate these processes, we identified seven amino acid residues using molecular dynamics and replaced them with Ala in order to probe interactions at either the dimer or the dimer-of-dimers interfaces. Arg70, Lys72, and Arg101 were important for protein stability and optimal DNA-binding activity. Moreover, the Arg70 mutant showed decreased dimerization in a mycobacterial two-hybrid system. To correlate these defects with higher-order organization and transcriptional activity, we used atomic force microscopy to observe different EspR mutant proteins in complex with the espACD promoter region. In addition, complementation of an M. tuberculosis espR knockout mutant was performed to measure their impact on EspA expression. Our results pinpoint key residues required for EspR function at the dimer (Arg70) and the dimer-of-dimers (Lys72) interface and demonstrate that EspR dimerization and higher-order oligomerization modulate espACD transcriptional activity and hence pathogenesis.

Amer Soc Microbiology2014

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

Sandor Kasas, Giovanni Longo, Petar Stupar

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.