Analytical strategy for studying the formation and stability of multilayered films containing gold nanoparticles.

The design of layer-by-layer (LbL) polyelectrolyte films including nanoparticles is a growing field of innovation in a wide range of biomedical applications. Gold nanoparticles (AuNPs) are very attractive for further biomolecule coupling to induce a pharmacological effect. Nanostructured LbL films coupled with such metallic species show properties that depend on the conditions of construction, i.e. the polymer nature and dissolution buffer. Tripartite LbL films (polycation, AuNP, and polyanion) were evaluated using two different polycationic polymers (poly(allylamine hydrochloride) (PAH), poly(ethylene imine) (PEI)) and various medium conditions (salts, i.e. phosphate, Tris or Tris-NaCl buffers, and concentration). AuNP incorporation and film stability were analysed by visible spectrophotometry, capillary zone electrophoresis, a quartz crystal microbalance, and high-performance liquid chromatography. The ideal compromise between AuNP loading and film stability was obtained using PAH prepared in Tris-NaCl buffer (0.01-0.15 M). This condition allowed the formation of a LbL film that was more stable than the film with PEI and provided an AuNP quantity that was 4.8 times greater than that of the PAH-PBS-built film. In conclusion, this work presents an analytical strategy for the characterization of nanostructured multilayer films and optimization of LbL films enriched with AuNPs to design biomedical device coatings.

HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate.

HIV-1 transcriptional activator (Tat) enables viral transcription and is also actively released by infected cells. Extracellular Tat can enter uninfected cells and affect some cellular functions. Here, we examine the effects of Tat protein on the secretory activity of neuroendocrine cells. When added to the culture medium of chromaffin and PC12 cells, Tat was actively internalized and strongly impaired exocytosis as measured by carbon fiber amperometry and growth hormone release assay. Expression of Tat mutants that do not bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] did not affect secretion, and overexpression of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the major PtdIns(4,5)P2 synthesizing enzyme, significantly rescued the Tat-induced inhibition of neurosecretion. This suggests that the inhibition of exocytosis may be the consequence of PtdIns(4,5)P2 sequestration. Accordingly, expression of Tat in PC12 cells interfered with the secretagogue-dependent recruitment of annexin A2 to the plasma membrane, a PtdIns(4,5)P2-binding protein that promotes the formation of lipid microdomains that are required for exocytosis. In addition Tat significantly prevented the reorganization of the actin cytoskeleton necessary for the movement of secretory vesicles towards plasma membrane fusion sites. Thus, the capacity of extracellular Tat to enter neuroendocrine cells and sequester plasma membrane PtdIns(4,5)P2 perturbs several PtdIns(4,5)P2-dependent players of the exocytotic machinery, thereby affecting neurosecretion. We propose that Tat-induced inhibition of exocytosis is involved in the neuronal disorders associated with HIV-1 infection.

Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells.

Most HIV-1 Tat is unconventionally secreted by infected cells following Tat interaction with phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane. Extracellular Tat is endocytosed by uninfected cells before escaping from endosomes to reach the cytosol and bind PI(4,5)P2. It is not clear whether and how incoming Tat concentrates in uninfected cells. Here we show that, in uninfected cells, the S-acyl transferase DHHC-20 together with the prolylisomerases cyclophilin A (CypA) and FKBP12 palmitoylate Tat on Cys31 thereby increasing Tat affinity for PI(4,5)P2. In infected cells, CypA is bound by HIV-1 Gag, resulting in its encapsidation and CypA depletion from cells. Because of the lack of this essential cofactor, Tat is not palmitoylated in infected cells but strongly secreted. Hence, Tat palmitoylation specifically takes place in uninfected cells. Moreover, palmitoylation is required for Tat to accumulate at the plasma membrane and affect PI(4,5)P2-dependent membrane traffic such as phagocytosis and neurosecretion.

Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot.

HIV-1 Tat is efficiently secreted by HIV-1-infected or Tat-transfected cells. Accordingly, Tat concentrations in the nanomolar range have been measured in the sera of HIV-1-infected patients, and this protein acts as a viral toxin on bystander cells. Nevertheless, assaying Tat concentration in media or sera is not that straightforward because extracellular Tat is unstable and particularly sensitive to oxidation. Moreover, most anti-Tat antibodies display limited affinity. Here, we describe methods to quantify extracellular Tat using a sandwich ELISA or Western blotting when Tat is secreted by suspension or adherent cells, respectively. In both cases it is important to capture exported Tat using antibodies before any Tat oxidation occurs; otherwise it will become denatured and unreactive toward antibodies.

Cell guidance into quiescent state through chromatin remodeling induced by elastic modulus of substrate.

Substrate stiffness is known to strongly influence the fate of adhering cells. Yet, little is known about the influence of the substrate stiffness on chromatin. Chromatin integrates a multitude of biochemical signals interpreted by activation or gene silencing. Here we investigate for the first time the organization of chromatin of epithelial cells on substrate with various mechanical properties. On stiff substrates (100-200 kPa), where cells preferentially adhere, chromatin is mainly found in its euchromatin form. Decreasing the Young modulus to 50 kPa is correlated with a partial shift from euchromatin to heterochromatin. On very soft substrates (≪10 kPa) this is accompanied by cell lysis. On these very soft substrates, histone deacetylase inhibition by adding a drug preserves acetylated histone and thus maintains the euchromatin form, thereby keeping intact the nuclear envelope as well as a residual intermediate filament network around the nucleus. This allows cells to survive in a non-adherent state without undergoing proliferation. When transfer on a stiff substrate these cells retain their capacity to adhere, to spread and to enter a novel mitotic cycle. A similar effect is observed on soft substrates (50 kPa) without need of histone deacetylase inhibition. These new results suggest that on soft substrates cells might enter in a quiescence state. Cell quiescence may thus be triggered by the Young modulus of a substrate, a major result for strategies focusing on the design of scaffold in tissue engineering.