Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic β cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the β cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary β cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin β1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between β1 integrins and the extracellular matrix with an anti-β1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by β1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion.

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Because of these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5 nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used green fluorescent proteins (GFPs) with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. Using a VEGF-GFP fusion protein and confocal time-lapse microscopy, we observed the intracellular routing, secretion and immobilization of VEGF in scratch-activated living astrocytes. We found VEGF to be directly transported to cell-extracellular matrix attachments where it is incorporated into fibronectin fibrils. VEGF accumulated at β1 integrin containing fibrillar adhesions and was translocated along the cell surface prior to internalization and degradation. We also found that only the astrocyte-derived, matrix-bound, and not soluble VEGF decreases β1 integrin turnover in fibrillar adhesions. We suggest that polarized VEGF release and ECM remodeling by VEGF secreting cells is key to control the local concentration and signaling of VEGF. Our findings highlight the importance of astrocytes in directing VEGF functions and identify these mechanisms as promising target for angiogenic approaches.

Heterodimeric integrin receptors control cell adhesion, migration and extracellular matrix assembly. While the α integrin subunit determines extracellular ligand specificity, the β integrin chain binds to an acidic residue of the ligand, and cytoplasmic adapter protein families such as talins, kindlins and paxillin, to form mechanosensing cell matrix adhesions. Alternative splicing of the β1 integrin cytoplasmic tail creates ubiquitously expressed β1A, and the heart and skeletal musclespecific β1D form. To study the physiological difference between these forms, we developed fluorescent β1 integrins and analyzed their dynamics, localization, and cytoplasmic adapter recruitment and effects on cell proliferation. On fibronectin, GFP-tagged β1A integrin showed dynamic exchange in peripheral focal adhesions, and long, central fibrillar adhesions. In contrast, GFP-β1D integrins exchanged slowly, forming immobile and short central adhesions. While adhesion recruitment of GFP-β1A integrin was sensitive to C-terminal tail mutagenesis, GFP-β1D integrin was recruited independently of the distal NPXY motif. In addition, a P786A mutation in the proximal, talin-binding NPXY 783 motif switched β1D to a highly dynamic integrin. In contrast, the inverse A786P mutation in β1A integrin interfered with paxillin recruitment and proliferation. Thus, differential β1 integrin splicing controls integrin-dependent adhesion signaling, to adapt to the specific physiological needs of differentiated muscle cells.

The transmembrane domain of kpOmpA possesses four long extracellular loops which exhibit substantial sequence variability throughout OmpA homologues in Enterobacteria. These loops are responsible for the immunological properties of the protein, such as cellular and humoral recognitions. Using liquid state NMR we have determined the 3D structure of kpOmpA in DHPC micelles (M. Renault et al., J. Mol. Biol. 2009). In a micellar environment, a complex dynamical behavior has been observed: a rigid barrel core, ms motion at the micellar-water interface, and sub-ns motion within the loops. Using solid state NMR relaxation and proteolysis experiments, we have demonstrated the persistence of this complex motional behavior in E. coli polar lipid bilayers (I. Iordanov et al., Biochim. Biophys. Acta, 2012). Using single molecule force spectroscopy (with D. Muller and A. Engel) we have shown that kpOmpA is able to unfold and refold reversibly its b-barrel core (P. Bosshart et al., Structure 2012). Recent advances involve: a) characterizing the structure of its C-terminal domain and its interaction with the peptido-glycane; b) analyzing ssNMR spectra of N-terminal membrane domain in liposomes using MAS at 1 GHz and 60 kHz spinning frequency (with G. Pintacuda); c) comparing the NMR spectra of the various domains and the full length protein in solution, in liposomes and in intact cell envelopes using cellular solid state NMR as established in (M. Renault et al., PNAS 2012).