mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

Single molecule tracking on supported membranes with arrays of optical nanoantennas.

Coupling of the localized surface plasmons between two closely apposed gold nanoparticles (nanoantenna) can cause strong enhancements of fluorescence or Raman signal intensity from molecules in the plasmonic "hot-spot". Harnessing these properties for practical applications is challenging due to the need to fabricate gold particle arrays with well-defined nanometer spacing and a means of delivering functional molecules to the hot-spot. We report fabrication of billions of plasmon-coupled nanostructures on a single substrate by a combination of colloid lithography and plasma processing. Controlled spacing of the nanoantenna gaps is achieved by taking advantage of the fact that polystyrene particles melt together at their contact point during plasma processing. The resulting polymer thread shadows a gap of well-defined spacing between each pair of gold triangles in the final array. Confocal surface-enhanced Raman spectroscopy imaging confirms the array is functionally uniform. Furthermore, a fully intact supported membrane can be formed on the intervening substrate by vesicle fusion. Trajectories of freely diffusing individual proteins are traced as they sequentially pass through, and are enhanced by, multiple gaps. The nanoantenna array thus enables enhanced observation of a fluid membrane system without static entrapment of the molecules.

The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation.

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

Micropatterning fluid lipid bilayers on solid supports.

Lithographically patterned grids of photoresist, aluminum oxide, or gold on oxidized silicon substrates were used to partition supported lipid bilayers into micrometer-scale arrays of isolated fluid membrane corrals. Fluorescently labeled lipids were observed to diffuse freely within each membrane corral but were confined by the micropatterned barriers. The concentrations of fluorescent probe molecules in individual corrals were altered by selective photobleaching to create arrays of fluid membrane patches with differing compositions. Application of an electric field parallel to the surface induced steady-state concentration gradients of charged membrane components in the corrals. In addition to producing patches of membrane with continuously varying composition, these gradients provide an intrinsically parallel means of acquiring information about molecular properties such as the diffusion coefficient in individual corrals.

Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion.

Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion.

Peroxynitrite: reactive, invasive and enigmatic.

The reactive oxygen and nitrogen species superoxide ion, nitric oxide, nitrogen dioxide and peroxynitrite ion all react with biological target molecules. Some of these interactions are carefully orchestrated segments of signal transduction cascades or part of the armamentarium of the immune system, others are pathological events and may lie at the root of many diseases. As a result of these small reactive molecules, proteins, particularly metalloproteins, can be altered with loss of function, DNA can be cleaved and lipid components can be oxidized to disrupt membranes. The interactions of these species with each other and their aftermath can be sensed by the cell, resulting in a variety of responses including gene regulation and transcription. Indeed, there is recent, tantalizing evidence that the currency of reactive oxygen and nitrogen species is central to the life and death cellular decisions in homeostasis or the initiation of apoptosis. New families of metallopharmaceuticals may serve both to probe the nature and mechanisms of these events and to effect the outcome.

Nitric oxide synthase: models and mechanisms.

The overproduction or underproduction of nitric oxide has been implicated in pathological symptoms such as endotoxic shock, diabetes, allograft rejection, and myocardial ischimia/reperfusion injury. A thorough understanding of the biosynthesis of nitric oxide is necessary to probe and manipulate these signaling events. There is also considerable pharmacological interest in developing selective inhibitors of the several isoforms of nitric oxide synthase. The recently determined crystal structures of complexes between nitric oxide synthase and substrate, the mechanisms of the enzymatic reaction that generate nitric oxide and chemical precedents and models for these reactions are now coming into focus, but there are still numerous fascinating and unanswered questions regarding nitric oxide biosynthesis.

Amphiphilic peroxynitrite decomposition catalysts in liposomal assemblies.

BACKGROUND: Peroxynitrite (ONOO-), a toxic biological oxidant, has been implicated in many pathophysiological conditions. The water-soluble porphyrins 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphinato iron(III) (FeTMPyP) and manganese(III) (MnTMPyP) have recently emerged as potential drugs for ONOO- detoxification, and FeTMPyP has demonstrated activity in models of ONOO- related disease states. We set out to develop amphiphilic analogs of FeTMPyP and MnTMPyP suitable for liposomal delivery in sterically stabilized liposomes (SLs). RESULTS: Three amphiphilic iron porphyrins (termed 1a-c.) and three manganese porphyrins (termed 2a-c.) bound to liposomes and catalyzed the decomposition of ONOO-. The polyethylene-glycol-linked metalloporphyrins 1b. and 2b. proved the most effective of these catalysts, rapidly decomposing ONOO- with second-order rate constants (kcat) of 2.9 x 10(5) M-1 s-1 and 5.0 x 10(5) M-1 s-1, respectively, in dimyristoylphosphatidylcholine liposomes. Catalysts 1b. and 2b. also bound to SLs, and these metalloporphyrin-SL constructs efficiently catalyzed ONOO- decomposition (kcat approximately 2 x 10(5) M-1 s-1). The analogous metalloporphyrins 1a. and 2a., which are not separated from the vesicle membrane surface by polyethylene glycol linkers, were significantly less effective (kcat approximately 3.5 x 10(4) M-1 s-1). CONCLUSIONS: For these amphiphilic analogs of FeTMPyP and MnTMPyP, the polarity of the environment of the metalloporphyrin headgroup is intimately related to the efficiency of the catalyst; a polar aqueous environment is essential for effective catalysis of ONOO- decomposition. Thus, catalysts 1b. and 2b. react rapidly with ONOO- and are potential therapeutic agents that, unlike their water-soluble TMPyP analogs, could be administered as liposomal formulations in SLs. These SL-bound amphiphilic metalloporphyrins may prove to be highly effective in the exploration and treatment of ONOO- related disease states.

Mechanisms of peroxynitrite decomposition catalyzed by FeTMPS, a bioactive sulfonated iron porphyrin.

Peroxynitrite is a known cytotoxic agent that plays a role in many pathological conditions. Various peroxynitrite decomposition catalysts and pathways are being explored to develop efficient therapeutic agents that can safely remove peroxynitrite from cells and tissues. Water-soluble porphyrins, such as iron(III) meso-tetra(2,4,6-trimethyl-3,5-disulfonato)porphine chloride (FeTMPS) and iron(III) meso-tetra(N-methyl4-pyridyl)porphine chloride (FeTMPyP), have been shown to react catalytically with peroxynitrite (ONOO-). However, their mechanisms are yet to be fully understood. In this study, we have explored the reactivity of FeTMPS in the catalytic decomposition of peroxynitrite. The mechanism of this complex process has been determined. According to this mechanism, Fe(III)TMPS is oxidized by peroxynitrite to produce oxoFe(lV)TMPS and NO2 (k1 = 1.3 x 10(5) M(-1)(s(-1). The porphyrin is then reduced back to Fe(III)TMPS by nitrite, but this rate (k2 = 1.4 x 10(4) M(-1)s(-1)) is not sufficient to maintain the catalytic process at the observed rate. The overall rate of peroxynitrite decomposition catalysis, kcat, was determined to be 6 x 10(4) M(-1)s(-1), under typical conditions. We have postulated that an additional reduction pathway must exist. Kinetic simulations showed that a reaction of oxoFe(IV)TMPS with NO2 (k3 = 1.7 x 10(7) M((-1)s(-1)) could explain the behavior of this system and account for the fast reduction of oxoFe(IV)TMPS to Fe(III). Using the kinetic simulation analysis, we have also shown that two other rearrangement reactions, involving FeTMPS and peroxynitrite, are plausible pathways for peroxynitrite decay. A "cage-return" reaction between the generated oxoFe(IV)TMPS and NO2 (k8 = 5.4 x 10(4) M(-1)s(-1)), affording Fe(III)TMPS and nitrate, and a reaction between oxoFe(IV)TMPS and peroxynitrite (k7 = 2.4 x 10(4) M(-1)s(-1)) that affords oxoFe(IV)TMPS and nitrate are presented. The mechanism of FeTMPS-catalyzed peroxynitrite decay differs markedly from that of FeTMPyP, providing some insight into the reactivity of metal centers with peroxynitrite and biologically important radicals such as NO2.

Deep vascular congestion in dural venous thrombosis on computed tomography.

In a 64-year-old female patient with verified dural venous thrombosis, postcontrast computed tomography showed dilatation of the transcerebral medullary veins, a feature that is probably pathognomonic.

Electric field-induced concentration gradients in planar supported bilayers.

A simple method of generating electric field-induced concentration gradients in planar supported bilayers has been developed. Gradients of charged, fluorescently labeled probes were visualized by epifluorescence microscopy and could be observed at field strengths as low as 1 V/cm. Steady-state concentration gradients can be described by a simple competition between random diffusion and electric field-induced drift. A model based on this principle has been used to determine the diffusion coefficient of the fluorescent probes. This technique achieves a degree of electrical manipulation of supported bilayers that offers a variety of possibilities for the development of new molecular architectures and the study of biological membranes.

Continuous crystalline carbonate apatite thin films. A biomimetic approach.

In contrast to extensive studies on hydroxyapatite thin films, very little has been reported on the thin films of carbonated apatite (dahllite). In this report, we describe the synthesis and characterization of a highly crystalline dahllite thin film assembled via a biomimetic pathway. A free-standing continuous precursor film of carbonated calcium phosphate in an amorphous phase was first prepared by a solution-inhibited templating method (template-inhibition) at an air-water interface. A stearic acid surface monolayer acted as the template, while a carbonate-phosphate solution composed a binary inhibition system. The precursor film formed at the air/water interface was heated at 900 degrees C and transformed into a dense crystalline film that retained the overall shape of the precursor. The crystalline phase was characterized by XRD and IR to be a single-phase carbonate apatite, with carbonate substitutions in both type A (OH-) and type B (PO4(3-)) lattice positions.

Electric field-induced reorganization of two-component supported bilayer membranes.

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.

Paramagnetic 1H-NMR relaxation probes of stereoselectivity in metalloporphyrin catalyzed olefin epoxidation.

Enantioselective catalytic epoxidation of olefins is an important problem from both practical and mechanistic points of view. The origins of chiral induction by asymmetric porphyrin and salen complexes were investigated by FT-NMR T1 relaxation techniques. A new chiral vaulted porphyrin (1) that carries (S)-binaphthyl-L-alanine straps across both faces of the porphyrin macrocycle was synthesized and characterized. (R)-styrene oxide was obtained in > 90% ee in the initial stages of styrene epoxidation with F5PhIO catalyzed by 1-Fe(III)Cl. The transition state for olefin epoxidation with high-valent metal-oxo species was modeled by coordinating epoxides to paramagnetic copper complexes of the corresponding ligands. The epoxide enantiomer that better fit the chiral cavity of the catalyst, as revealed by T1 relaxation measurements, was also the major product of catalytic olefin epoxidation. These results are consistent with the "lock-and-key" mechanism of asymmetric catalysis by metalloporphyrins. The copper complex of a chiral salen ligand showed no differentiation in terms of T1 relaxation rates between the enantiomers of cis-beta-methylstyrene oxide in contrast to the high enantioselectivity observed for catalytic epoxidation.

Electric field-induced critical demixing in lipid bilayer membranes.

Electric fields can induce lateral reorganization of lipids in fluid bilayer membranes. The resulting concentration profiles readily are observed in planar-supported bilayers by epifluorescence microscopy. When a fluorescently labeled lipid was used to probe the field-induced separation of cardiolipin from egg-phosphatidylcholine, an enhanced sensitivity to the electric field was observed that is attributed to a critical demixing effect. A thermodynamic model of the system was used to analyze the results. The observed concentration profiles can be understood if the lipid mixture has a critical temperature equal to 75 degrees K. The steady-state distribution of lipids under the influence of an electric field is very sensitive to demixing effects, even at temperatures well above the critical temperature for spontaneous phase separation, and this may have significant consequences for organization and structural changes in natural cell membranes.

Electronic and steric factors in regioselective hydroxylation catalyzed by purified cytochrome P-450.

A reconstituted hydroxylation system consisting of electrophoretically homogeneous phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450 LM2), NADPH-cytochrome P-450 reductase, phospholipid, buffer, NADPH, and O2 was used to oxidize four cyclohexane derivatives: cyclohexene, methylcyclohexane, norcarane and norbornane. Cyclohexene gave only cyclohexene oxide and allylic cyclohexenol, while methylcyclohexane yielded all possible monohydric alcohols, but with 1 degrees:2 degrees:3 degrees ratios of 0.072:1:1.25. Norcarane yielded 2-norcaranol. While oxidation of norbornane produced exo-2- and endo-2-norborneols in a ratio of 3.4:1, replacement of all four exo-hydrogens by deuterium led to a reversal of the exo:endo ratio to 0.76:1. These and other observations are interpreted as evidence for a selective, hydrogen-abstracting enzyme-bound oxidant exhibiting a large intramolecular deuterium isotope effect. A transient substrate carbon radical is a probable intermediate in the hydroxylation process.

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.

Architecture and function of membrane proteins in planar supported bilayers: a study with photosynthetic reaction centers.

We present a simple and convenient method for creating fluid supported bilayers which contain oriented and functional photosynthetic reaction centers (RCs). The supported bilayers are prepared by fusion of proteoliposomes with a glass surface. The proteoliposomes are prepared by spontaneous insertion of RCs into preformed small, unilamellar vesicles. The RCs in these vesicles are shown to be oriented with the cytochrome c binding surface on the outside and the H-subunit facing inside. Upon fusion to glass surfaces, the RCs remain functional and highly oriented, with the cytochrome c binding surface exposed to the bulk solution. The RCs in the supported bilayers are at a surface density of order 10(11) RCs/cm2. The quality of the supported lipid bilayer is characterized by epifluorescence microscopy and the long-range lateral mobility of the lipids by fluorescence recovery after photobleaching. We demonstrate that homogeneous, fluid bilayers can be prepared over large areas (e.g., 1 cm2) of clean glass surfaces. The lipids in these supported bilayers are laterally mobile, and their diffusion coefficient agrees with values obtained in other fluid bilayer systems. This fluidity is unaffected by the presence of RCs; however, the RCs bearing a site-specific fluorescent label are immobile, despite retaining their charge separation and cytochrome c binding properties. We speculate that this results from interactions between the globular domain of the H-subunit and the glass substrate. Because of the unique spectroscopic and functional signatures associated with intact RCs, this system is one of the best characterized examples of a transmembrane protein in a supported bilayer at a nonbiological interface.