The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers

Understanding the molecular mechanisms governing nanoparticle-membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse-grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di-stearoyl-phosphatidylcholine (DSPC) and anionic di-stearoyl-phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 degrees C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC-DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP-membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse-grained MD simulations as a lipid-crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.Peer reviewe

Molecular mechanisms involved in spermatogonial germ cells proliferation and differentiation

Lo scopo del seguente lavoro, è comprendere alcuni dei meccanismi molecolari coinvolti nella regolazione della proliferazione e del differenziamento degli spermatogoni nel testicolo prepuberale di topo. Lo studio ha riguardato la caratterizzazione della RNA-binding-protein Nanos 3 e il suo coinvolgimento nella progressione del ciclo cellulare degli spermatogoni e l’analisi dell’espressione genica negli spermatogoni in seguito a trattamento con i fattori Kit ligand e acido retinico, coinvolti rispettivamente nella proliferazione e nel differenziamento in questo tipo cellulare. Nel topo sono stati identificati tre omologhi di Nanos , Nanos1, Nanos2 and Nanos3. L’ortologo Nanos3 è espresso nella gonade embrionale maschile e femminile e dopo la nascita è presente esclusivamente nel testicolo. E’ noto che l’eliminazione di questo gene comporta la perdita completa della cellule germinali in entrambi i sessi, ma la funzione di Nanos3 nel testicolo dopo la nascita, non è stata ancora analizzata. Nel nostro studio dimostriamo che Nanos3 è espresso negli spermatogoni indifferenziati del testicolo prepuberale di topo e che l’over espressione di questo gene comporta un aumento della percentuale di cellule nella fase G1, suggerendone il coinvolgimento nel rallentamento della progressione del ciclo cellulare degli spermatogoni. Dimostriamo inoltre, che il meccanismo di azione di Nanos 3, quale potenziale repressore della traduzione, è conservato nel topo e prevede l’interazione con una seconda RNA-binding protein murina, Pumilio2. In accordo con il possibile coinvolgimento di Nanos 3 nell’inibizione del differenziamento degli spermatogoni, il trattamento con il fattore differenziativo acido retinoico all-trans (ATRA), induce una notevole down-regolazione della sua espressione. Questi risultati permettono di concludere che nel testicolo di topo dopo la nascita, Nanos3 è un fattore importante nel mantenimento dello stato indifferenziato degli spermatogoni attraverso la regolazione del loro ciclo. Nella seconda parte del lavoro, è stata realizzata un’ analisi, mediante macroarray, dei geni regolati dal trattamento con KL o ATRA negli spermatogoni ottenuti da topi di 7 giorni di età. L’analisi ha evidenziato che il pattern di espressione dei geni che risultano indotti da KL è in accordo con i cambiamenti che intervengono nel ciclo cellulare durante le divisioni cellulari successive degli spermatogoni di tipo A e B. KL induce, infatti, l’espressione dei geni correlati alle fasi iniziali della meiosi negli spermatogoni in differenziamento e regola negativamente l’espressione dei geni tipicamente presenti negli spermatogoni durante la fase mitotica. Inoltre, l’analisi dei dati ottenuti da spermatogoni trattati con acido retinoico, ha confermato che l’ ATRA induce un pattern di espressione genica compatibile con la progressione verso il programma differenziativo della meiosi, suggerendo che l’ATRA e KL, indipendentemente, sono in grado di promuovere l’entrata nella meiosi negli spermatogoni.The aim of this work is to understand some of the molecular mechanisms which regulate spermatogonial germ cells proliferation and differentiation in the prepuberal mouse testis. In this study we characterized the RNA-binding protein Nanos3 and its role in the regulation of spermatogonial cell-cycle progression. Furthermore we performed a transcriptome analysis of spermatogonia stimulated with KL and retinoic acid, which have been previously shown to regulate their proliferaton and differentiaton, respectively. In the mouse, three Nanos homologs have been identified, Nanos1, Nanos2 and Nanos3. The Nanos3 ortholog is expressed in both male and female gonads of early embryo and, after birth, it is found only in the testis. Nanos3 targeted disruption results in the complete loss of germ cells in both sexes during embryonic development, however the role of Nanos3 in the testis during the postnatal period has not been explored yet. We found that Nanos3 is expressed in undifferentiated spermatogonia of the prepuberal testis and that its up-regulation causes accumulation of cells in the G1 phase, suggesting that this protein is able to delay the cell cycle progression of spermatogonial cells. We also demonstrate a conserved mechanism of action of Nanos3 as potential translational repressor, involving the interaction with the murine RNA-binding protein Pumilio2. According to the possible role of Nanos3 in inhibiting spermatogonia cell differentiation, treatment with the differentiating factor all-trans retinoic acid (ATRA) induces a dramatic down-regulation of its expression. These results allowed us to conclude that, in the prepuberal testis, Nanos3 is important to maintain undifferentiated spermatogonia via the regulation of their cell cycle. In the second part of the work we performed a wide genome analysis of gene expression regulated by treatment with KL or ATRA of spermatogonia from 7-day-old mice. The analysis revealed that the pattern of RNA expression induced by KL is compatible with the qualitative changes of the cell cycle that occur during the subsequent cell divisions in type A and B spermatogonia. Moreover, KL up-regulates in differentiating spermatogonia the expression of early meiotic genes whereas it down-regulates typical spermatogonial markers. Since KL modifies the expression of several genes known to be up-regulated or down-regulated in spermatogonia during the transition from the mitotic to the meiotic cell cycle, these results are consistent with a role of the KL/Kit interaction in the induction of their meiotic differentiation. Microarray analysis on stimulated spermatogonia showed that ATRA induces a pattern of gene expression which is compatible with their ongoing differentiating program toward meiosis, suggesting that ATRA and KL, independently, are able to promote meiotic entry of postnatal spermatogonia

Generation of artificial channels by multimerization of beta-strands from natural porin

General diffusion porins are passive transmembrane channels. We have explored the possibility to create artificial nanopores starting from natural beta-barrel structures. Structural elements of bacterial porins were used to build a series of artificial nanopores. The basic module was selected by multi-alignment of general diffusion porins. The sequence corresponded to a highly conserved motif containing two beta-strands, which was obtained from Escherichia coli OmpF. Dimeric to octameric repeats were obtained through cDNA recombinant technology. The hexameric repeat was used to test its properties. This protein was expressed, purified and reconstituted in the planar bilayer membranes. It was able to form channels in membranes with a conductance of 300 pS in 150 mM KCl and did not show any relevant voltage-dependence

Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are 'decapacitation factors', an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed

Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are 'decapacitation factors', an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed

Deletion of beta-strands 9 and 10 converts VDAC1 voltage-dependence in an asymmetrical process

Voltage-dependent anion selective channel isoform1 maintains the permeability of the outer mitochondrial membrane. Its voltage-gating properties are relevant in bioenergetic metabolism and apoptosis. The N-terminal domain is suspected to be involved in voltage-gating, due to its peculiar localization. However this issue is still controversial. In this work we exchanged or deleted the beta-strands that take contact with the N-terminal domain. The exchange of the whole hVDAC1 beta-barrel with the homologous hVDAC3 beta-barrel produces a chimeric protein that, in reconstituted systems, loses completely voltage-dependence. hVDAC3 beta-barrel has most residues in common with hVDAC1, including V143 and L150 considered anchor points for the N-terminus. hVDAC1 mutants completely lacking either the beta-strand 9 or both beta-strands 9 and 10 were expressed, refolded and reconstituted in artificial bilayers. The mutants formed smaller pores. Molecular dynamics simulations of the mutant structure supported its ability to form smaller pores. The mutant lacking both beta-strands 9 and 10 showed a new voltage-dependence feature resulting in a fully asymmetric behavior. These data indicate that a network of beta-strands in the pore-walls, and not single residues, are required for voltage-gating in addition to the N-terminus. (C) 2013 Elsevier B.V. All rights reserved

The Cumulus Cell Layer Protects Bovine Maturing Oocyte Against Fatty Acid-Induced Lipotoxicity

Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate; unsaturated oleate) on oocyte maturation and quality. In the current study the effects of elevated fatty acid levels on cumulus cells were investigated. The three fatty acids dose-dependently induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3-6 fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of ceramide C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed

Symmetry-breaking transitions in the early steps of protein self-assembly

International audienceProtein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer’s disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly β-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aβ(1–40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations