Development of label-free biophysical markers in osteogenic maturation.

The spatial and temporal changes of morphological and mechanical properties of living cells reflect complex functionally-associated processes. Monitoring these modifications could provide a direct information on the cellular functional state. Here we present an integrated biophysical approach to the quantification of the morphological and mechanical phenotype of single cells along a maturation pathway. Specifically, quantitative phase microscopy and single cell biomechanical testing were applied to the characterization of the maturation of human foetal osteoblasts, demonstrating the ability to identify effective label-free biomarkers along this fundamental biological process.

A single point mutation reveals gating of the human ClC-5 Cl-/H+ antiporter.

ClC-5 is a 2Cl(-)/1H(+) antiporter highly expressed in endosomes of proximal tubule cells. It is essential for endocytosis and mutations in ClC-5 cause Dent's disease, potentially leading to renal failure. However, the physiological role of ClC-5 is still unclear. One of the main issues is whether the strong rectification of ClC-5 currents observed in heterologous systems, with currents elicited only at positive voltages, is preserved in vivo and what is the origin of this rectification. In this work we identified a ClC-5 mutation, D76H, which, besides the typical outward currents of the wild-type (WT), shows inward tail currents at negative potentials that allow the estimation of the reversal of ClC-5 currents for the first time. A detailed analysis of the dependence of these inward tail currents on internal and external pH and [Cl(-)] shows that they are generated by a coupled transport of Cl(-) and H(+) with a 2 : 1 stoichiometry. From this result we conclude that the inward tail currents are caused by a gating mechanism that regulates ClC-5 transport activity and not by a major alteration of the transport mechanism itself. This implies that the strong rectification of the currents of WT ClC-5 is at least in part caused by a gating mechanism that activates the transporter at positive potentials. These results elucidate the biophysical properties of ClC-5 and contribute to the understanding of its physiological role.

On the mechanism of gating charge movement of ClC-5, a human Cl(-)/H(+) antiporter.

ClC-5 is a Cl(-)/H(+) antiporter that functions in endosomes and is important for endocytosis in the proximal tubule. The mechanism of transport coupling and voltage dependence in ClC-5 is unclear. Recently, a transport-deficient ClC-5 mutant (E268A) was shown to exhibit transient capacitive currents. Here, we studied the external and internal Cl(-) and pH dependence of the currents of E268A. Transient currents were almost completely independent of the intracellular pH. Even though the transient currents are modulated by extracellular pH, we could exclude that they are generated by proton-binding/unbinding reactions. In contrast, the charge movement showed a nontrivial dependence on external chloride, strongly supporting a model in which the movement of an intrinsic gating charge is followed by the voltage-dependent low-affinity binding of extracellular chloride ions. Mutation of the external Glu-211 (a residue implicated in the coupling of Cl(-) and proton transport) to aspartate abolished steady-state transport, but revealed transient currents that were shifted by ~150 mV to negative voltages compared to E268A. This identifies Glu(ext) as a major component of the gating charge underlying the transient currents of the electrogenic ClC-5 transporter. The molecular events underlying the transient currents of ClC-5 emerging from these results can be explained by an inward movement of the side chain of Glu(ext), followed by the binding of extracellular Cl(-) ions.

Extracellular determinants of anion discrimination of the Cl-/H+ antiporter protein CLC-5.

Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.

Heparin strongly enhances the formation of beta2-microglobulin amyloid fibrils in the presence of type I collagen.

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of beta2-microglobulin (beta2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of beta2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes beta2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of beta2-m (DeltaN6beta2-m) that is a ubiquitous constituent of the natural beta2-m fibrils. The formation of this beta2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.

Collagen plays an active role in the aggregation of beta2-microglobulin under physiopathological conditions of dialysis-related amyloidosis.

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of beta(2)-microglobulin (beta(2)-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, beta(2)-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of beta(2)-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in beta(2)-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role in beta(2)-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-L-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-L-lysine is dissolved in solution with beta(2)-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as beta(2)-m, that decay on moving away from the fiber surface.