Gilberto Weissmüller

Membrane Recognition by Vesicular Stomatitis Virus Involves Enthalpy-Driven Protein-Lipid Interactions

ABSTRACT Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process.

Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .

Glial cells with differential neurite growth-modulating properties probed by atomic force microscopy.

Lateral (L) and medial (M) midbrain astrocytes differ in their ability to support neuritic growth (L, permissive; M, non-permissive) with properties of M glia depending on heparan sulfate (HS). Here we show by atomic force microscopy that the surfaces of formaldehyde-fixed astrocytes differ by conspicuous 250 nm protrusions in L and by a HS-dependent fibrillar network in M glia, thus, demonstrating correlations between cell surface morphology and functional properties.

Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi

Background Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility. Methodology/Principal Findings Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea. Conclusions/Significance Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating.

Artificial Laminin Polymers Assembled in Acidic pH Mimic Basement Membrane Organization

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 ± 5 and 86 ± 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1′, which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin α1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.

Psd1 binding affinity toward fungal membrane components as assessed by SPR: The role of glucosylceramide in fungal recognition and entry

Psd1 is a plant defensin that has antifungal activity against several pathogenic and nonpathogenic fungi. Previous analysis of Psd1 chemical shift perturbations by nuclear magnetic resonance (NMR) spectroscopy demonstrated that this defensin interacts with phospholipids and the sphingolipid glucosylceramide isolated from Fusarium solani (GlcCerFusarium solani). In this study, these interactions were evaluated by real‐time surface plasmon resonance (SPR) analysis. The data obtained demonstrated that Psd1 could bind more strongly to small unilamellar vesicles (SUV)‐containing GlcCerFusarium solani than to SUV that was composed of phosphatidylcholine (PC) alone or was enriched with GlcCer that had been isolated from soybeans. An increase in the SPR response after cholesterol or ergosterol incorporation in PC‐SUV was detected; however, SUV composed of PC:Erg (7:3; molar:molar) became unstable in the presence of Psd1, suggesting membrane destabilization. We also observed a lack of Psd1 internalization in Candida albicans strains that were deficient in the glucosyl ceramide synthase gene. Together, these data indicate that GlcCer is essential for Psd1 anchoring in the fungal plasma membrane as well as internalization.

A Fluorescent Mutant of the NM Domain of the Yeast Prion Sup35 Provides Insight into Fibril Formation and Stability

The Sup35 protein of Saccharomyces cerevisiae forms a prion that generates the [PSI(+)] phenotype. Its NM region governs prion status, forming self-seeding amyloid fibers in vivo and in vitro. A tryptophan mutant of Sup35 (NM(F117W)) was used to probe its aggregation. Four indicators of aggregation, Trp 117 maximum emission, Trp polarization, thio-T binding, and light scattering increase, revealed faster aggregation at 4 degrees C than at 25 degrees C, and all indicators changed in a concerted fashion at the former temperature. Curiously, at 25 degrees C the changes were not synchronized; the first two indicators, which reflect nucleation, changed more quickly than the last two, which reflect fibril formation. These results suggest that nucleation is insensitive to temperature, whereas fibril extension is temperature dependent. As expected, aggregation is accelerated when a small fraction (5%) of the nuclei produced at 4 or 25 degrees C are added to a suspension containing the soluble NM domain, although these nuclei do not seem to propagate any structural information to the growing fibrils. Fibrils grown at 4 degrees C were less stable in GdmCl than those grown at higher temperature. However, they were both resistant to high pressure; in fact, both sets of fibrils responded to high pressure by adopting an altered conformation with a higher capacity for thio-T binding. From these data, we calculated the change in volume and free energy associated with this conformational change. AFM revealed that the fibrils grown at 4 degrees C were statistically smaller than those grown at 25 degrees C. In conclusion, the introduction of Trp 117 allowed us to more carefully dissect the effects of temperature on the aggregation of the Sup35 NM domain.

Modulators of axonal growth and guidance at the brain midline with special reference to glial heparan sulfate proteoglycans

Bilaterally symmetric organisms need to exchange information between the left and right sides of their bodies to integrate sensory input and to coordinate motor control. Thus, an important choice point for developing axons is the Central Nervous System (CNS) midline. Crossing of this choice point is influenced by highly conserved, soluble or membrane-bound molecules such as the L1 subfamily, laminin, netrins, slits, semaphorins, Eph-receptors and ephrins, etc. Furthermore, there is much circumstantial evidence for a role of proteoglycans (PGs) or their glycosaminoglycan (GAG) moieties on axonal growth and guidance, most of which was derived from simplified models. A model of intermediate complexity is that of cocultures of young neurons and astroglial carpets (confluent cultures) obtained from medial and lateral sectors of the embryonic rodent midbrain soon after formation of its commissures. Neurite production in these cocultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exerted an inhibitory or non-permissive effect on neuritic growth that was correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment with GAG lyases shows minor effects of CS and discloses a major inhibitory or non-permissive role for HS. The results are discussed in terms of available knowledge on the binding of HSPGs to interative proteins and underscore the importance of understanding glial polysaccharide arrays in addition to its protein complement for a better understanding of neuron-glial interactions.

Envelope lipid-packing as a critical factor for the biological activity and stability of alphavirus particles isolated from mammalian and mosquito cells

Alphaviruses are enveloped arboviruses. The viral envelope is derived from the host cell and is positioned between two icosahedral protein shells (T = 4). Because the viral envelope contains glycoproteins involved in cell recognition and entry, the integrity of the envelope is critical for the success of the early events of infection. Differing levels of cholesterol in different hosts leads to the production of alphaviruses with distinct levels of this sterol loaded in the envelope. Using Mayaro virus, a New World alphavirus, we investigated the role of cholesterol on the envelope of alphavirus particles assembled in either mammalian or mosquito cells. Our results show that although quite different in their cholesterol content, Mayaro virus particles obtained from both cells share a similar high level of lateral organization in their envelopes. This organization, as well as viral stability and infectivity, is severely compromised when cholesterol is depleted from the envelope of virus particles isolated from mammalian cells, but virus particles isolated from mosquito cells are relatively unaffected by cholesterol depletion. We suggest that it is not cholesterol itself, but rather the organization of the viral envelope, that is critical for the biological activity of alphaviruses.

Interaction between chondroitin-6-sulfate and Entamoeba histolytica as revealed by force spectroscopy

We have observed by atomic force microscopy (AFM) the amoeba surface and probed the interaction force between Entamoeba histolytica and chondroitin-6-sulphate (C6S). We have used several substrates to adhere trophozoites. The best reproducibility in sample preparation was obtained with fibronectin-coated coverslips and when the cells were fixed with paraformaldehyde. The images obtained with the AFM showed that the trophozoite exhibits an irregular surface. Pseudopods and waving adhesion plaques could be observed. Force spectroscopy analysis showed that the trophozoite surface strongly interacts with C6S-functionalized tips. During cantilever retraction, attractive force peaks were observed at distances up to 1.3 microm above the trophozoite surface. Statistical analysis of the force distributions collected for five samples shown a reproducible 2.2 nN mean adhesion force. We observed a reduction of the adhesion force and of the interaction distance after addition of galactose to the buffer solution suggesting that the observed interaction is also Gal/GalNAc-lectin-mediated.