Membranes confining cells and cellular compartments are essential for life. Membrane proteins are molecular machines that equip cell membranes with highly sophisticated functionality. Examples of such functions are signaling, ion pumping, energy conversion, molecular transport, specific ligand binding, cell adhesion and protein trafficking. However, it is not well understood how most membrane proteins work and how the living cell regulates their function. We review how atomic force microscopy (AFM) can be applied for structural and functional investigations of native membrane proteins. High-resolution time-lapse AFM imaging records membrane proteins at work, their oligomeric state and their dynamic assembly. The AFM stylus resembles a multifunctional toolbox that allows the measurement of several chemical and physical parameters at the nanoscale. In the single-molecule force spectroscopy (SMFS) mode, AFM quantifies and localizes interactions in membrane proteins that stabilize their folding and modulate their functional state. Dynamic SMFS discloses fascinating insights into the free energy landscape of membrane proteins. Single-cell force spectroscopy quantifies the interactions of live cells with their environment to single-receptor resolution. In the future, technological progress in AFM-based approaches will enable us to study the physical nature of biological interactions in more detail and decipher how cells control basic processes.

Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein — MBP — and proteolipid protein — PLP — (respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation.

Gap junction channels regulate cell-cell communication by passing metabolites, ions, and signaling molecules. Gap junction channel closure in cells by acidification is well documented; however, it is unknown whether acidification affects connexins or modulating proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly inhibit connexin channel activity in an isoform-specific manner as shown in previously published studies. High-resolution atomic force microscopy of force-dissected connexin26 gap junctions revealed that in HEPES buffer, the pore was closed at pH < 6.5 and opened reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate buffers. Increasing the protonated HEPES concentration did not close the pore, indicating that a saturation of the binding sites occurs at 10 mM HEPES. Analysis of the extracellular surface topographs reveals that the pore diameter increases gradually with pH. The outer connexon diameter remains unchanged, and there is a ϳ6.5°rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca 2؉ -induced closure.Gap junction channels (GJC) 3 are dynamic macromolecular complexes capable of opening and closing the channel pore in response to a number of stimuli such as divalent cations, signaling molecules, phosphorylation, pH, and modulators of specific isoforms (1). These regulated conduits for the passage of small molecules greatly influence homeostasis, development, ionic transmission, and other cellular processes. Whereas there exist strong cell biological, biochemical, and biophysical evidence for the effects of these modulators, there is not much information at the structural level as to the conformational changes that occur in closing the pore in response to these stimuli.Each connexin (Cx) channel is composed of two hexamers (connexons) that dock at their apposed extracellular surfaces. The cyclic arrangement of the subunits within the hexamers suggests that gating can occur by a rotation and translation of the transmembrane segments within all six monomers. It has been postulated that gating occurs as a "camera iris" shutter (2). An alternate hypothesis has been proposed in which intra-connexin associations occur to produce either a particle-receptor blockage at the cytoplasmic surface (3, 4) or as a physical gate near the extracellular surface ("loop gate") (5). Whether these proposed mechanisms correlate to the closure of fast and/or slow gates that have been characterized by electrophysiological methods (see Ref. 6) remain to be determined.Gating by intracellular acidification is one way that connexin channels open and close in response to stimuli. Experimentally determined decreases in intracellular pH are known to decrease junctional electrical coupling in cardiomyocytes and in Purkinje fibers (7-10) as well as in teleost and amphibian embryos (11). Stergiopoulos et al. (12) showed that many, but not all, connexins close in a pH-sensit...

Aggregation of Tau into amyloid-like fibrils is a key process in neurodegenerative diseases such as Alzheimer. To understand how natively disordered Tau stabilizes conformations that favor pathological aggregation, we applied single-molecule force spectroscopy. Intramolecular interactions that fold polypeptide stretches of ϳ19 and ϳ42 amino acids in the functionally important repeat domain of full-length human Tau (hTau40) support aggregation. In contrast, the unstructured N terminus randomly folds long polypeptide stretches >100 amino acids that prevent aggregation. The pro-aggregant mutant hTau40⌬K280 observed in frontotemporal dementia favored the folding of short polypeptide stretches and suppressed the folding of long ones. This trend was reversed in the anti-aggregant mutant hTau40⌬K280/PP. The aggregation inducer heparin introduced strong interactions in hTau40 and hTau40⌬K280 that stabilized aggregation-prone conformations. We show that the conformation and aggregation of Tau are regulated through a complex balance of different intra-and intermolecular interactions.Amyloid forming proteins such as ␣-synuclein, the prion protein (1), and Tau (2) contain unstructured domains or belong to the family of natively unfolded or intrinsically disordered proteins (IDPs) 3 (3). The aggregation of Tau into amyloid-like fibers, known as paired helical filaments (4, 5), is a key process in human protein aggregation diseases that are summarized as tauopathies. In vivo, Tau binds and stabilizes microtubules (MTs) to regulate the cellular MT network. The dissociation of Tau from MTs is controlled by the phosphorylation of Tau at multiple sites (6, 7). The longest human Tau isoform, hTau40 (441 amino acids (aa)), contains a ϳ250-aa long N terminus of unknown function, whereas the C terminus comprises the Tau repeat domain, which encompasses four ϳ31-aa long semi-conserved repeats (R1 to R4) flanked by proline-rich stretches (Fig. 1A). Both, binding to MTs and fibril assembly are mediated through the Tau repeat domain (8, 9).As most IDPs, Tau shows a high content of charged aa residues and a low hydrophobicity, which result in an extended solution conformation with a large radius of gyration (10). In solution, Tau has no stable secondary and tertiary structure, as judged by CD and Fourier transform infrared spectroscopy (10). The Stokes radius of Tau increases upon chemical denaturation with urea or guanidine hydrochloride (11, 12) indicating some limited folding. NMR experiments revealed transient secondary structures in hTau40 that partially interact with other polypeptide regions (13). Two hexapeptide motifs, PHF6* in R2 and PHF6 in R3, can adopt ␤-strand conformation and are predominantly responsible for Tau aggregation into fibrils (9, 14). Using Förster resonance energy transfer (11), the transient "paper clip"-like folding of the C and N termini onto the repeat domain was detected in hTau40. After removing the N-and C-terminal domains, the Tau repeat domain exhibits faster aggregation than full-length Tau (15). Th...

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