Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts

Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism

Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death

Background: The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail. Methods: We use optical techniques, including fluorescence confocal microscopy and lifetime imaging microscopy (FLIM) both in model membranes built up as giant unilamellar vesicles (GUVs) and cultured cells. These experiments are complemented with computational studies to unravel the molecular mechanism that makes NAO cytotoxic. Results: We have obtained direct evidence that NAO promotes strong membrane adhesion of negatively charged vesicles. The attractive forces are derived from van der Waals interactions between anti-parallel H-dimers of NAO molecules from opposing bilayers. Semi-empirical calculations have confirmed the supramolecular scenario by which anti-parallel NAO molecules form a zipper of bonds at the contact region. The membrane remodeling effect of NAO, as well as the formation of H-dimers, was also confirmed in cultured fibroblasts, as shown by the ultrastructure alteration of the mitochondrial cristae. Conclusions: We conclude that membrane adhesion induced by NAO stacking accounts for the supramolecular basis of its cytotoxicity. General significance: Mitochondria are a potential target for cancer and gene therapies. The alteration of the mitochondrial structure by membrane remodeling agents able to form supramolecular assemblies via adhesion properties could be envisaged as a new therapeutic strategy

Co-cultivo de neuronas y microglía tratadas con neuromelanina como modelo de estudio de los procesos involucrados en la agregación de proteínas en la enfermedad del Parkinson

Parkinson's disease is a common neurodegenerative disease that affects more than 10 million people worldwide which causes motor abnormalities such as tremor, muscle stiffness, paucity of voluntary movements and postural instability. It is mainly characterized by neural degeneration in the substantia nigra of the mid brain. Misfolding and aggregation of alpha-synuclein (aSyn) is one of the major molecular hallmarks of the disease but it is not clear which are the initial molecular and cellular mechanisms leading to protein aggregation, although cellular stress caused by oxidative damage with the involvement of neuromelanin and microglia-mediated inflammatory responses appear to be involved. No experimental model has been able so far to accurately replicate the situation within the human brain. In this work, we have used co-cultures of in vitro-differentiated dopaminergic neurons (SH-SY5Y) together with an immortalized human microglia cell line (HMC3), treated with synthetic neuromelanin, to approach the initial processes leading to aSyn aggregation using 2D models. <br /

Lipid-peptide bioconjugation through pyridyl disulfide reaction chemistry and its application in cell targeting and drug delivery

BackgroundThe design of efficient drug delivery vectors requires versatile formulations able to simultaneously direct a multitude of molecular targets and to bypass the endosomal recycling pathway of cells. Liposomal-based vectors need the decoration of the lipid surface with specific peptides to fulfill the functional requirements. The unspecific binding of peptides to the lipid surface is often accompanied with uncontrolled formulations and thus preventing the molecular mechanisms of a successful therapy.ResultsWe present a simple synthesis pathway to anchor cysteine-terminal peptides to thiol-reactive lipids for adequate and quantitative liposomal formulations. As a proof of concept, we have synthesized two different lipopeptides based on (a) the truncated Fibroblast Growth Factor (tbFGF) for cell targeting and (b) the pH sensitive and fusogenic GALA peptide for endosomal scape.ConclusionsThe incorporation of these two lipopeptides in the liposomal formulation improves the fibroblast cell targeting and promotes the direct delivery of cargo molecules to the cytoplasm of the cell