De-adhesion dynamics of melanoma cells from brain endothelial layer.

Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers.

Direct mapping of melanoma cell - endothelial cell interactions.

The most life-threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high-resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi-physiological conditions. Hereby, we present our latest results by using this melanoma-decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single-cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic-related processes.

Expression of pattern recognition receptors and activation of the non-canonical inflammasome pathway in brain pericytes.

Cerebral pericytes are mural cells embedded in the basement membrane of capillaries. Increasing evidence suggests that they play important role in controlling neurovascular functions, i.e. cerebral blood flow, angiogenesis and permeability of the blood-brain barrier. These cells can also influence neuroinflammation which is highly regulated by the innate immune system. Therefore, we systematically tested the pattern recognition receptor expression of brain pericytes. We detected expression of NOD1, NOD2, NLRC5, NLRP1-3, NLRP5, NLRP9, NLRP10 and NLRX mRNA in non-treated cells. Among the ten known human TLRs, TLR2, TLR4, TLR5, TLR6 and TLR10 were found to be expressed. Inflammatory mediators induced the expression of NLRA, NLRC4 and TLR9 and increased the levels of NOD2, TLR2, inflammasome-forming caspases and inflammasome-cleaved interleukins. Oxidative stress, on the other hand, upregulated expression of TLR10 and NLRP9. Activation of selected pattern recognition receptors can lead to inflammasome assembly and caspase-dependent secretion of IL-1ß. TNF-α and IFN-γ increased the levels of pro-IL-1ß and pro-caspase-1 proteins; however, no canonical activation of NLRP1, NLRP2, NLRP3 or NLRC4 inflammasomes could be observed in human brain vascular pericytes. On the other hand, we could demonstrate secretion of active IL-1ß in response to non-canonical inflammasome activation, i.e. intracellular LPS or infection with E. coli bacteria. Our in vitro results indicate that pericytes might have an important regulatory role in neuroinflammation.

DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

Antimicrobial nodule-specific cysteine-rich peptides disturb the integrity of bacterial outer and inner membranes and cause loss of membrane potential.

BACKGROUND: Certain legume plants produce a plethora of AMP-like peptides in their symbiotic cells. The cationic subgroup of the nodule-specific cysteine-rich (NCR) peptides has potent antimicrobial activity against gram-negative and gram-positive bacteria as well as unicellular and filamentous fungi. FINDINGS: It was shown by scanning and atomic force microscopies that the cationic peptides NCR335, NCR247 and Polymyxin B (PMB) affect differentially on the surfaces of Sinorhizobium meliloti bacteria. Similarly to PMB, both NCR peptides caused damages of the outer and inner membranes but at different extent and resulted in the loss of membrane potential that could be the primary reason of their antimicrobial activity. CONCLUSIONS: The primary reason for bacterial cell death upon treatment with cationic NCR peptides is the loss of membrane potential.

Interaction of cysteine-rich cationic antimicrobial peptides with intact bacteria and model membranes.

Antimicrobial peptides are small proteins that exhibit a broad spectrum of antimicrobial activity. Their chemical structure allows them to interact (attach and insert) with membranes. The fine details about this interaction and their mode of action are not fully clarified yet. In order to better understand this mechanism, we have performed in situ atomic force microscopy studies using two types of nodule specific cysteine-rich NCR peptides on Escherichia coli bacteria and on natural purple membrane. On intact bacteria, both NCR247 and NCR335 caused increase in the surface roughness, indicating the damage of the bacterial cell envelope. In case of the tightly packed purple membrane, it is clear that the peptides prefer to disrupt the border of the disks indicating a strong lipid preference of the interaction. These results verify the concept that the first target of NCR peptides is probably the bacterial cell envelope, especially the lipid matrix.

κ-Casein terminates casein micelle build-up by its "soft" secondary structure.

In our previous paper (Nagy et al. in J Biol Chem 285:38811-38817, 2010) by using a multilayered model system, we showed that, from α-casein, aggregates (similar to natural casein micelles) can be built up step by step if Ca-phosphate nanocluster incorporation is ensured between the protein adsorption steps. It remained, however, an open question whether the growth of the aggregates can be terminated, similarly to in nature with casein micelles. Here, we show that, in the presence of Ca-phosphate nanoclusters, upon adsorbing onto earlier α-casein surfaces, the secondary structure of α-casein remains practically unaffected, but κ-casein exhibits considerable changes in its secondary structure as manifested by a shift toward having more ß-structures. In the absence of Ca-phosphate, only κ-casein can still adsorb onto the underlying casein surface; this κ-casein also expresses considerable shift toward ß-structures. In addition, this κ-casein cover terminates casein aggregation; no further adsorption of either α- or κ-casein can be achieved. These results, while obtained on a model system, may show that the Ca-insensitive κ-casein can, indeed, be the outer layer of the casein micelles, not only because of its "hairy" extrusion into the water phase, but because of its "softer" secondary structure, which can "occlude" the interacting motifs serving casein aggregation. We think that the revealed nature of the molecular interactions, and the growth mechanism found here, might be useful to understand the aggregation process of casein micelles also in vivo.

Adhesion and stress relaxation forces between melanoma and cerebral endothelial cells.

Mechanical parameters play a crucial role in proper cellular functions. This article examines the process of the appearance and breaking of adhesion forces during contact between the confluent cerebral endothelial cell layer and a melanoma cell attached to a tipless cantilever. This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force measurement, if the contact was prolonged for several seconds, a decrease in the load force was observed, which corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time showed a saturation-like behavior. These stress relaxation curves could be fitted with the sum of two exponentials, suggesting that two independent processes take place simultaneously. The breakup of the adhesion during the retraction of the cantilever with the attached melanoma cell is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about 20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the elementary binding force between these two cell types. In case of fixed endothelial cells, the adhesion force was strongly decreased and the jumps disappeared, whereas the stress relaxation did not show considerable change upon fixation.

Spatial and temporal dependence of the cerebral endothelial cells elasticity.

The reliable determination of the mechanical properties of a living cell is one of the most important challenges of the atomic force microscopic measurements. In the present study the spatial and temporal dependency of the force measurements on cerebral endothelial cells was investigated. Besides imaging the cells, two different sequences of force measurements were applied: Acquisition of force curves in short time at several points across the cell surface investigating spatial dependence of the elasticity. Acquisition of force curves for long time at a previously determined place, over the cell nucleus, which provides the temporal stability/variation of the measured forces/values. Three different stages of endothelial cell cultures of the hCMEC/D3 cells were used: sub-confluent living, confluent living, and confluent fixed cells. The Young's modulus was calculated from the force curves using the Hertz model and the results were plotted against time or location correspondingly. The rational of using the three stage of culture was to clarify whether the observed effect belongs to the individual cell, to the ensemble of cells or just to some, not living cell component. In case of sub-confluent cells the results revealed a softer nuclear region compared to the periphery, while an attenuated oscillation like fluctuation in time, with a period of about 10-30 min, was observed. Confluent living cells showed similar tendencies to the sub-confluent cells, but the changes were larger and the temporal oscillations had longer period. The spatial dependency of the elasticity on confluent cells was confirmed by force-volume measurement too. In case of fixed cells neither spatial nor temporal differences were observed between the nuclear and peripheral region, however the Young's modulus and the error of the measurement was larger, compared to the sub-confluent living cells.

Effect of antimicrobial peptide-amide: indolicidin on biological membranes.

Indolicidin, a cationic antimicrobial tridecapeptide amide, is rich in proline and tryptophan residues. Its biological activity is intensively studied, but the details how indolicidin interacts with membranes are not fully understood yet. We report here an in situ atomic force microscopic study describing the effect of indolicidin on an artificial supported planar bilayer membrane of dipalmitoyl phosphatidylcholine (DPPC) and on purple membrane of Halobacterium salinarum. Concentration dependent interaction of the peptide and membranes was found in case of DPPC resulting the destruction of the membrane. Purple membrane was much more resistant against indolicidin, probably due to its high protein content. Indolicidin preferred the border of membrane disks, where the lipids are more accessible. These data suggest that the atomic force microscope is a powerful tool in the study of indolicidin-membrane interaction.

Photosynthetic Reaction-Center/Graphene Biohybrid for Optoelectronics.

Photosynthetic reaction center proteins (RC) purified from purple bacterial strains were deposited on graphene layer prepared by liquid phase exfoliation and light-induced resistance change was measured. By measuring the temperature dependence of the resistance change of the bare and RC covered graphene and comparing with the one inactivated by protein unfolding, two effects were possible to separate. One of them is the resistance change due to temperature effect. The other one clearly indicates a possible electric/electronic interaction between the charge flow in the graphene and the light-induced charge pair within the protein, which is, essentially, different in the open (dark, PBPheo) and closed (light, P+BPheo-) states. These results provide useful information for designing hybrid bio-photonic devices which are able to absorb and convert light energy.

Atypical transcriptional regulation and role of a new toxin-antitoxin-like module and its effect on the lipid composition of Bradyrhizobium japonicum.

A toxin-antitoxin (TA)-like system (designated as bat/bto genes) was identified in Bradyrhizobium japonicum, based on sequence homology and similarities in organization and size to known TA systems. Deletion of the bat/bto module resulted in pleiotropic alterations in cell morphology and metabolism. The generation time of the mutant was considerably decreased in rich media. Atomic force microscopy revealed the modified shape (shorter and wider) and softness of mutant cells. The synthesis of phosphatidylcholine was completely blocked in the mutant bacteria, and vaccenic acid, the predominant fatty acid of membranes of the wild-type cell, was replaced by palmitic acid in the mutant membranes. The mutant bacteria synthesized incomplete lipopolysaccharide molecules. Remarkable changes in the membrane lipid composition may explain the observed morphological alterations and growth properties of the mutant bacteria. The overlapping promoter region of bat/bto and glpD (coding for the aerobic sn-glycerol-3-phosphate dehydrogenase) genes suggests a complex regulation and the involvement of bat/bto in the control of main metabolic pathways and an important role in the maintenance of a normal physiological state of B. japonicum. These data reveal new aspects of the role of TA systems in bacteria.

Regulation of cerebral endothelial cell morphology by extracellular calcium.

Cerebral endothelial cells interconnected by tight and adherens junctions constitute the structural basis of the blood-brain barrier. Extracellular calcium ions have been reported to play an important role in the formation and maintenance of the junctional complex. However, little is known about the action of calcium depletion on the structural characteristics of cerebral endothelial cells. Using atomic force microscopy we analyzed the effect of calcium depletion and readdition on the shape and size of living brain endothelial cells. It was found that the removal of extracellular calcium from confluent cell cultures induced the dissociation of the cells from each other accompanied by an increase in their height. After readdition of calcium a gradual recovery was observed until total confluency was regained. We have also demonstrated that Rho-kinase plays an important role in the calcium-depletion-induced disassembly of endothelial tight and adherens junctions. The Rho-kinase inhibitor Y27632 could prevent the morphological changes induced by a lack of calcium as well. Our results suggest that calcium depletion induces Rho-kinase-dependent cytoskeletal changes that may be partly responsible for the disassembly of the junctional complex.

Enthalpy/entropy driven activation of the first interquinone electron transfer in bacterial photosynthetic reaction centers embedded in vesicles of physiologically important phospholipids.

The thermodynamics and kinetics of light-induced electron transfer in bacterial photosynthetic RCs are sensitive to physiologically important lipids (phosphatidylcholine, cardiolipin and phosphatidylglycerol) in the environment. The analysis of the temperature-dependence of the rate of the P(+)Q(A)(-)Q(B)-->P(+)Q(A)Q(B)(-) interquinone electron transfer revealed high enthalpy change of activation in zwitterionic or neutral micelles and vesicles and low enthalpy change of activation in vesicles constituted of negatively charged phospholipids. The entropy change of activation was compensated by the changes of enthalpy, thus the free energy change of activation ( approximately 500 meV) did not show large variation in vesicles of different lipids.

Stabilization effect of single-walled carbon nanotubes on the functioning of photosynthetic reaction centers.

The interaction between single-walled carbon nanotubes and photosynthetic reaction centers purified from purple bacterium Rhodobacter sphaeroides R-26 has been investigated. Atomic force microscopy studies provide evidence that reaction center protein can be attached effectively to the nanotubes. The typical diameter of the nanotube is 1-4 nm and 15 +/- 2 nm without and with the reaction centers, respectively. Light-induced absorption change measurements indicate the stabilization of the P+(Q(A)Q(B))- charge pair, which is formed after single saturating light excitation after the attachment to nanotubes. The separation of light-induced charges is followed by slow reorganization of the protein structure. The stabilization effect of light-initiated charges by the carbon nanotubes opens a possible direction of several applications, the most promising being in energy conversion and storage devices.

The influence of the halide ions on the photochemical reaction cycle of pharaonis halorhodopsin.

Sodium salt of chloride, bromide and iodide were used to elucidate the effect of the size of the anion on the binding to pharaonis halorhodopsin and its transport during the photocycle of this retinal protein. Spectroscopic titration revealed an apparent strong binding constant of 2 mM for chloride, 0.23 mM for bromide and 5 mM for iodide. In the case of iodide a second, week binding constant of about 10 M could be estimated. This second binding constant was similar to that observed earlier for nitrate. By changing the halide ions, only the transitions in the second half of the photocycle were affected, which contained intermediates N, O, and HR'. The O to HR' transition becomes faster with increasing ion volume, meaning that the ion uptake is accelerated. This effect shows a direct correlation with the ion radius. With increasing ion concentration the N-O-HR' equilibrium changed in such a way that the accumulated O tended to decrease. This tendency was overruled in iodide, by the appearance of the second binding constant. The increasing iodide concentration, up to 100mM decreases the accumulation of the intermediate O, due to kinetic reasons, but at higher ion concentration the amount of O increases, although its decay becomes faster. This effect correlates with the appearance of the second iodide bound to the protein.

The photochemical reaction cycle of retinal reconstituted bacteriorhodopsin.

The function of three types of bacteriorhodopsins was compared: the wild-type, the bleached and retinal reconstituted and retinal deficient bacteriorhodopsin after retinal addition. The apparent pK(a) of the proton acceptor group for the bleached BR and retinal deficient BR shifted toward higher pH values compared to the wild-type BR. Fitting the photocycle model to the absorption kinetic signals for all three proteins showed the existence of the same intermediates, but the time-dependent concentration of the intermediates was different. Although measurements were made at pH 7, the absorption kinetics and photoelectric signals in both retinal reconstituted samples acted as wild-type bacteriorhodopsin at significantly higher pH. Below pH 3 the retinal deficient and reconstituted sample bleached. These results suggested that the added retinal was not able to rebind in the same position in the protein as in native bacteriorhodopsin. This points out that care should be taken, when bleached bacteriorhodopsin is reconstituted with different retinal analogs.

Elasto-mechanical properties of living cells.

The possibility to directly measure the elasticity of living cell has emerged only in the last few decades. In the present study the elastic properties of two cell lines were followed. Both types are widely used as cell barrier models (e.g. blood-brain barrier). During time resolved measurement of the living cell elasticity a continuous quasi-periodic oscillation of the elastic modulus was observed. Fast Fourier transformation of the signals revealed that a very limited number of three to five Fourier terms fitted the signal in the case of human cerebral endothelial cells. In the case of canine kidney epithelial cells more than 8 Fourier terms did not result a good fit. Calculating the correlation between nucleus and periphery of the signals revealed a higher correlation factor for the endothelial cells compared to the epithelial cells.

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K.

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.

Kinetic isotope effects in the photochemical reaction cycle of ion transporting retinal proteins.

The kinetics of the photochemical reaction cycle of the bacteriorhodopsin, pharaonis halorhodopsin and proteorhodopsin were determined in H2O and D2O at low and high pH, to get insight in the proton dependent steps of the transport process. While all the steps of the bacteriorhodopsin photocycle at normal pH exhibited a strong isotope effect, the proton uptake step of the photocycle, measured at high pH, became independent of deuterium exchange, making plausible that this step, at low proton concentration, becomes concentration dependent, not mobility dependent. The proton transporting photocycle of the proteorhodopsin at its normal pH (9.5) shows a marked deuterium effect, while at high pH (12.2) this effect almost totally disappears. It was shown earlier that the proton uptake step of the proteorhodopsin is at the rise of the N form. As the proton concentration decreases with rising pH this step becomes the rate limiting, proton concentration dependent step, hiding all the other isotope dependent components. In the case of halorhodopsin in all the chloride, nitrate and proton transporting conditions the photocycle was not strongly affected by the deuterium exchange. While in the cases of the first two ions this seems normal, the absence of the deuterium effect in the case of the proton transporting photocycle was a puzzle. The only plausible explanation is that in the presence of azide the halorhodopsin transports not the proton, but a negatively charged ion the OH-, the mass and mobility of which is only slightly influenced by the deuterium exchange.