Pulmonary Surfactant: A Mighty Thin Film.

Pulmonary surfactant is a critical component of lung function in healthy individuals. It functions in part by lowering surface tension in the alveoli, thereby allowing for breathing with minimal effort. The prevailing thinking is that low surface tension is attained by a compression-driven squeeze-out of unsaturated phospholipids during exhalation, forming a film enriched in saturated phospholipids that achieves surface tensions close to zero. A thorough review of past and recent literature suggests that the compression-driven squeeze-out mechanism may be erroneous. Here, we posit that a surfactant film enriched in saturated lipids is formed shortly after birth by an adsorption-driven sorting process and that its composition does not change during normal breathing. We provide biophysical evidence for the rapid formation of an enriched film at high surfactant concentrations, facilitated by adsorption structures containing hydrophobic surfactant proteins. We examine biophysical evidence for and against the compression-driven squeeze-out mechanism and propose a new model for surfactant function. The proposed model is tested against existing physiological and pathophysiological evidence in neonatal and adult lungs, leading to ideas for biophysical research, that should be addressed to establish the physiological relevance of this new perspective on the function of the mighty thin film that surfactant provides.

Internalization of α-synuclein oligomers into SH-SY5Y cells.

Aggregates of misfolded α-synuclein are a distinctive feature of Parkinson's disease. Small oligomers of α-synuclein are thought to be an important neurotoxic agent, and α-synuclein aggregates exhibit prion-like behavior, propagating misfolding between cells. α-Synuclein is internalized by both passive diffusion and active uptake mechanisms, but how uptake varies with the size of the oligomer is less clear. We explored how α-synuclein internalization into live SH-SY5Y cells varied with oligomer size by comparing the uptake of fluorescently labeled monomers to that of engineered tandem dimers and tetramers. We found that these α-synuclein constructs were internalized primarily through endocytosis. Oligomer size had little effect on their internalization pathway, whether they were added individually or together. Measurements of co-localization of the α-synuclein constructs with fluorescent markers for early endosomes and lysosomes showed that most of the α-synuclein entered endocytic compartments, in which they were probably degraded. Treatment of the cells with the Pitstop inhibitor suggested that most of the oligomers were internalized by the clathrin-mediated pathway.

Amyloids of alpha-synuclein affect the structure and dynamics of supported lipid bilayers.

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71-82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.

Lipid-coated gold nanoparticles promote lamellar body formation in A549 cells.

Gold nanoparticles (GNPs) have been applied as diagnostic and therapeutic agents because they can be targeted, localized, and be heated to cause cell death. However, their use has been limited by their relatively low biocompatibility. In this work, we coated the GNPs' surface by a biocompatible phospholipid bilayer composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (SOPG). We tested their interaction with A549 cells to investigate their uptake and intracellular fate as well as the response of the cells to the presence of the GNPs. We used flow cytometry and confocal microscopy to show that the SOPG coated GNPs were readily taken up by the A549 cells. Transmission electron microscopy (TEM) images and fluorescence images further showed that the number of granular structures in the cells was increased following exposure to the lipid coated GNPs. Co-localization experiments demonstrated that SOPG coated GNPs localize in acidic compartments in a time dependent manner and that the number of these increase as the cells are exposed to the GNPs suggesting that they induce formation of lamellar bodies (LBs) which in A549 cells in turn can serve as a means of exporting the GNPs.

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant.

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir-Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.

Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein.

The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90-232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al(3+)-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al(3+), Zn(2+), and free-base). Buffer ionic strength also affected the binding, with K(d) increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~4-5 PcTS molecules per PrP molecule.

A ToF-SIMS study of the lateral organization of lipids and proteins in pulmonary surfactant systems.

Pulmonary surfactant is a complex lipid-protein mixture whose main function is to reduce the surface tension at the air-liquid interface of alveoli to minimize the work of breathing. The exact mechanism by which surfactant monolayers and multilayers are formed and how they lower surface tension to very low values during lateral compression remains uncertain. We used time-of-flight secondary ion mass spectrometry to study the lateral organization of lipids and peptide in surfactant preparations ranging in complexity. We show that we can successfully determine the location of phospholipids, cholesterol and a peptide in surfactant Langmuir-Blodgett films and we can determine the effect of cholesterol and peptide addition. A thorough understanding of the lateral organization of PS interfacial films will aid in our understanding of the role of each component as well as different lipid-lipid and lipid-protein interactions. This may further our understanding of pulmonary surfactant function.

Trapping of BMP receptors in distinct membrane domains inhibits their function in pulmonary arterial hypertension.

Bone morphogenetic proteins (BMPs) are pleiotrophic growth factors that influence diverse processes such as skeletal development, hematopoiesis, and neurogenesis. They play crucial roles in diseases such as pulmonary arterial hypertension (PAH). In PAH, mutants of the BMP type II receptors (BMPR2) were detected, and their functions were impaired during BMP signaling. It is thought that expression levels of these receptors determine the fate of BMP signaling, with low levels of expression leading to decreased Smad activation in PAH. However, our studies demonstrate, for the first time, that the localization of receptors on the plasma membrane, in this case BMPR2, was misdirected. Three BMPR2 mutants, D485G, N519K, and R899X, which are known to be involved in PAH, were chosen as our model system. Our results show that all three BMPR2 mutants decreased BMP-dependent Smad phosphorylation and Smad signaling. Although the three mutants reached the cell membrane and their expression was lower than that of BMPR2, they formed smaller clusters and associated differently with membrane domains, such as caveolae and clathrin-coated pits. The disruption of these domains restored the Smad signaling of D485G and N519K to the level of wild-type BMPR2, showing that these mutants were trapped in the domains, rather than just expressed at a lower level on the surface. Therefore, new treatment options for PAH should also target receptor localization, rather than just expression level.

Photon counting histogram analysis for two-dimensional systems.

Photon counting statistics in 3D photon counting histogram analysis for one-photon excitation is a function of the number of molecules of particular brightness in the excitation-detection volume of a confocal microscope. In mathematical form that volume is approximated by a three-dimensional Gaussian function which is embedded in the PCH theoretical equations. PCH theory assumes that a molecule can be found anywhere inside the excitation-detection volume with equal probability. However, one can easily imagine systems in which this assumption is violated because molecules are constrained by the geometry of the sample. For example, molecules on a surface or in a membrane would be constrained to two dimensions. To enable the analysis of such systems by PCH, the theoretical framework requires modification. Herein, we present an extension of the PCH analysis to systems where molecules exist in thin structures that are effectively two-dimensional. The method, aptly called two-dimensional photon counting histogram (2D PCH), recovers the number of fluorescent particles per unit area and their molecular brightness. Both theoretical background and experimental results are presented. The theory was tested using computer-simulated and experimental 2D PCHs obtained from confocal experiments. We demonstrate that this modification of the theoretical framework provides a tool to extract data that reveal states of aggregation, surface photophysics, and reactivity.

The COVID-19 pandemic: a target for surfactant therapy?

INTRODUCTION: The dramatic impact of COVID-19 on humans worldwide has initiated an extraordinary search for effective treatment approaches. One of these is the administration of exogenous surfactant, which is being tested in ongoing clinical trials. AREAS COVERED: Exogenous surfactant is a life-saving treatment for premature infants with neonatal respiratory distress syndrome. This treatment has also been tested for acute respiratory distress syndrome (ARDS) with limited success possibly due to the complexity of that syndrome. The 60-year history of successes and failures associated with surfactant therapy distinguishes it from many other treatments currently being tested for COVID-19 and provides the opportunity to discuss the factors that may influence the success of this therapy. EXPERT OPINION: Clinical data provide a strong rationale for using exogenous surfactant in COVID-19 patients. Success of this therapy may be influenced by the mechanical ventilation strategy, the timing of treatment, the doses delivered, the method of delivery and the preparations utilized. In addition, future development of enhanced preparations may improve this treatment approach. Overall, results from ongoing trials may not only provide data to indicate if this therapy is effective for COVID-19 patients, but also lead to further scientific understanding and improved treatment strategies.

Casein kinase 2 beta-subunit is a regulator of bone morphogenetic protein 2 signaling.

Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate processes as diverse as neurogenesis, skeletal formation, and hematopoesis. They signal through a hetero-oligomer complex of BMP receptors. Binding of the ligand to the receptors activates several pathways, including Smad and p38. BMP signaling is controlled in the extracellular space, the plasma membrane, and the intracellular space; however, the mechanism of receptor signaling at the plasma membrane and proteins that regulate this process still need to be identified. The experiments presented here identify the protein kinase casein kinase II (CK2) as a BMP receptor type Ia (BRIa) interacting protein. Fluorescence resonance energy transfer revealed that this interaction occurs at the plasma membrane. BMP2 stimulation of C2C12 cells leads to the release of CK2 from BRIa. Blocking this interaction with specific peptides that inhibit the binding sites for CK2 on BRIa demonstrated a redistribution of BRIa on the plasma membrane. Signaling was initiated once CK2 was released from BRIa, leading to the mineralization of C2C12 cells. These data suggest that CK2 is a negative regulator of BMP signaling and osteoblast differentiation.

Solution phase interactions controlled ordered arrangement of gold nanoparticles in dried state.

Gold (Au) nanoparticles (NPs) were synthesized in the presence of water soluble biomolecules such as DNA, chitosan, phospholipids, and BSA by using seed-mediated approach at room temperature. All reactions produced mostly spherical geometries with comparable size (< or = 20 nm). The NPs were arranged in a typical pearl-necklace type arrangement except in the presence of BSA. Different measurements such as UV-visible, TEM, XRD, and XPS were used to characterize the Au NPs. Fluorescence spectroscopy was used to identify the interactions between biomolecules and blank (uncapped) Au NPs in aqueous colloidal solutions. It was concluded that the favorable interactions between Au NPs and biomolecules in aqueous phase, in fact, drive them into pearl-necklace type arrangement in the dried state.

FRET reveals novel protein-receptor interaction of bone morphogenetic proteins receptors and adaptor protein 2 at the cell surface.

Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosis, differentiation, and proliferation. Several different pathways such as Smad, p38, and PI3/Akt are activated by BMPs. Signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. BMPRs shuttle between membrane domains such as caveolae enriched with caveolin-1 beta-isoform and caveolae of the caveolin-1 alpha/beta-isoforms. It is hypothesized that there are other membrane domains to which the receptors localize. We used immunoprecipitation, Western blots, image cross-correlation spectroscopy, and fluorescence resonance energy transfer to investigate the interaction of BMPRs with proteins in clathrin-coated pits (CCPs). Our data indicate that these domains are associated with at least two of the BMPRs: BRIa and BRII. For the first time, to our knowledge, we showed what we believe are specific interactions between BRIa and BRII with a key component of CCPs, adaptor protein 2. Further, disruption of CCPs resulted in increased BRIa aggregation at the cell surface and activation of the BMP pathway even in the absence of BMP2. Therefore, CCPs seem to function as a negative regulatory membrane domain for BMP pathway activation.

Current perspectives in pulmonary surfactant--inhibition, enhancement and evaluation.

Pulmonary surfactant (PS) is a complicated mixture of approximately 90% lipids and 10% proteins. It plays an important role in maintaining normal respiratory mechanics by reducing alveolar surface tension to near-zero values. Supplementing exogenous surfactant to newborns suffering from respiratory distress syndrome (RDS), a leading cause of perinatal mortality, has completely altered neonatal care in industrialized countries. Surfactant therapy has also been applied to the acute respiratory distress syndrome (ARDS) but with only limited success. Biophysical studies suggest that surfactant inhibition is partially responsible for this unsatisfactory performance. This paper reviews the biophysical properties of functional and dysfunctional PS. The biophysical properties of PS are further limited to surface activity, i.e., properties related to highly dynamic and very low surface tensions. Three main perspectives are reviewed. (1) How does PS permit both rapid adsorption and the ability to reach very low surface tensions? (2) How is PS inactivated by different inhibitory substances and how can this inhibition be counteracted? A recent research focus of using water-soluble polymers as additives to enhance the surface activity of clinical PS and to overcome inhibition is extensively discussed. (3) Which in vivo, in situ, and in vitro methods are available for evaluating the surface activity of PS and what are their relative merits? A better understanding of the biophysical properties of functional and dysfunctional PS is important for the further development of surfactant therapy, especially for its potential application in ARDS.

Photon counting histogram analysis as a tool for studying the nature of intermolecular interactions.

Photon counting histogram (PCH) analysis is a statistical method that provides independent information about the relative brightness and the number of molecular entities in the system. This suggests that it could be very advantageous in the analysis of intermolecular interactions in multicomponent systems. In this paper we employ PCH to study the interaction of Rhodamine 6G (Rh-6G) with sodium dodecyl sulfate (SDS) micelles and show how the method can unveil the mechanism of quenching of Rh-6G by methyl viologen (MV).

Distribution and Co-localization of endosome markers in cells.

Clathrin mediated endocytosis is one pathway for internalization of extracellular nano materials into cells [1, 2]. In this pathway, proteins attached to receptors and the internalized materials are encapsulated in clathrin coated membrane vesicles that subsequently fuse with or transform into intracellular compartments (early and late endosomes) as their contents are being directed to the lysosomes for degradation. The following proteins are commonly used to mark the pathway at various stages: Rab5 (early endosome), Rab7 (late endosome), and LAMP-1 (lysosome). In this work, we studied the distribution and co-localization of these marker proteins in two cell lines (C2C12 and A549) to determine whether these markers are unique for specific endosome types or whether they can co-exist with other markers. We estimate the densities and sizes of the endosomes containing the three markers, as well as the number of marker antibodies attached to each endosome. We determine that the markers are not unique to one endosome type but that the extent of co-localization is different for the two cell types. In fact, we find endosomes that contain all three markers simultaneously. Our results suggest that the use of these proteins as specific markers for specific endosome types should be reevaluated. This was the first successful use of triple image cross correlation spectroscopy to qualitatively and quantitatively study the extent of interaction among three different species in cells and also the first experimental study of three-way interactions of clathrin mediated endocytic markers.

Characterizing the inhibition of α-synuclein oligomerization by a pharmacological chaperone that prevents prion formation by the protein PrP.

Aggregation of the disordered protein α-synuclein into amyloid fibrils is a central feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease. Small, pre-fibrillar oligomers of misfolded α-synuclein are thought to be the key toxic entities, and α-synuclein misfolding can propagate in a prion-like way. We explored whether a compound with anti-prion activity that can bind to unfolded parts of the protein PrP, the cyclic tetrapyrrole Fe-TMPyP, was also active against α-synuclein aggregation. Observing the initial stages of aggregation via fluorescence cross-correlation spectroscopy, we found that Fe-TMPyP inhibited small oligomer formation in a dose-dependent manner. Fe-TMPyP also inhibited the formation of mature amyloid fibrils in vitro, as detected by thioflavin T fluorescence. Isothermal titration calorimetry indicated Fe-TMPyP bound to monomeric α-synuclein with a stoichiometry of 2, and two-dimensional heteronuclear single quantum coherence NMR spectra revealed significant interactions between Fe-TMPyP and the C-terminus of the protein. These results suggest commonalities among aggregation mechanisms for α-synuclein and the prion protein may exist that can be exploited as therapeutic targets.

Early stages of aggregation of engineered α-synuclein monomers and oligomers in solution.

α-Synuclein is a protein that aggregates as amyloid fibrils in the brains of patients with Parkinson's disease and dementia with Lewy bodies. Small oligomers of α-synuclein are neurotoxic and are thought to be closely associated with disease. Whereas α-synuclein fibrillization and fibril morphologies have been studied extensively with various methods, the earliest stages of aggregation and the properties of oligomeric intermediates are less well understood because few methods are able to detect and characterize early-stage aggregates. We used fluorescence spectroscopy to investigate the early stages of aggregation by studying pairwise interactions between α-synuclein monomers, as well as between engineered tandem oligomers of various sizes (dimers, tetramers, and octamers). The hydrodynamic radii of these engineered α-synuclein species were first determined by fluorescence correlation spectroscopy and dynamic light scattering. The rate of pairwise aggregation between different species was then monitored using dual-color fluorescence cross-correlation spectroscopy, measuring the extent of association between species labelled with different dyes at various time points during the early aggregation process. The aggregation rate and extent increased with tandem oligomer size. Self-association of the tandem oligomers was found to be the preferred pathway to form larger aggregates: interactions between oligomers occurred faster and to a greater extent than interactions between oligomers and monomers, indicating that the oligomers were not as efficient in seeding further aggregation by addition of monomers. These results suggest that oligomer-oligomer interactions may play an important role in driving aggregation during its early stages.

Metal nanoparticle pollutants interfere with pulmonary surfactant function in vitro.

Reported associations between air pollution and pulmonary and cardiovascular diseases prompted studies on the effects of gold nanoparticles (Au NP) on pulmonary surfactant function. Low levels (3.7 mol % Au/lipid, 0.98% wt/wt) markedly inhibited adsorption of a semisynthetic pulmonary surfactant (dipalmitoyl-phosphatidylcholine (DPPC)/palmitoyl-oleoyl-phosphatidylglycerol/surfactant protein B (SP-B); 70:30:1 wt %). Au NP also impeded the surfactant's ability to reduce surface tension (gamma) to low levels during film compression and to respread during film expansion. Transmission electron microscopy showed that Au NP generated by a seed-growth method were spherical with diameters of approximately 15 nm. Including palmitoyl-oleoyl-phosphatidylglycerol appeared to coat the NP with at least one lipid bilayer but did not affect NP shape or size. Similar overall observations occurred with dimyristoyl phosphatidylglycerol. Dipalmitoyl-phosphatidylglycerol was less effective in NP capping, although similar sized NP were formed. Including SP-B (1% wt/wt) appears to induce the formation of elongated strands of interacting threads with the fluid phosphatidylglycerols (PG). Including DPPC resulted in formation of aggregated, less spherical NP with a larger size distribution. With DPPC, strand formation due to SP-B was not observed. Agarose gel electrophoresis studies demonstrated that the aggregation induced by SP-B blocked migration of PG-coated NP. Migration was also influenced by the fluidity of the PGs. It is concluded that Au NP can interact with and sequester pulmonary surfactant phospholipids and, if inhaled from the atmosphere, could impede pulmonary surfactant function in the lung.

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films. I. Micro- and nanostructures of functional pulmonary surfactant films and the effect of SP-A.

Monolayers of a functional pulmonary surfactant (PS) can reach very low surface tensions well below their equilibrium value. The mechanism by which PS monolayers reach such low surface tensions and maintain film stability remains unknown. As shown previously by fluorescence microscopy, phospholipid phase transition and separation seem to be important for the normal biophysical properties of PS. This work studied phospholipid phase transitions and separations in monolayers of bovine lipid extract surfactant using atomic force microscopy. Atomic force microscopy showed phospholipid phase separation on film compression and a monolayer-to-multilayer transition at surface pressure 40-50 mN/m. The tilted-condensed phase consisted of domains not only on the micrometer scale, as detected previously by fluorescence microscopy, but also on the nanometer scale, which is below the resolution limits of conventional optical methods. The nanodomains were embedded uniformly within the liquid-expanded phase. On compression, the microdomains broke up into nanodomains, thereby appearing to contribute to tilted-condensed and liquid-expanded phase remixing. Addition of surfactant protein A altered primarily the nanodomains and promoted the formation of multilayers. We conclude that the nanodomains play a predominant role in affecting the biophysical properties of PS monolayers and the monolayer-to-multilayer transition.