Understanding the Rhodopsin Worldview Through Atomic Force Microscopy (AFM): Structure, Stability, and Activity Studies.

Rhodopsin is a G protein-coupled receptor (GPCR) present in the rod outer segment (ROS) of photoreceptor cells that initiates the phototransduction cascade required for scotopic vision. Due to the remarkable advancements in technological tools, the chemistry of rhodopsin has begun to unravel especially over the past few decades, but mostly at the ensemble scale. Atomic force microscopy (AFM) is a tool capable of providing critical information from a single-molecule point of view. In this regard, to bolster our understanding of rhodopsin at the nanoscale level, AFM-based imaging, force spectroscopy, and nano-indentation techniques were employed on ROS disc membranes containing rhodopsin, isolated from vertebrate species both in normal and diseased states. These AFM studies on samples from native retinal tissue have provided fundamental insights into the structure and function of rhodopsin under normal and dysfunctional states. We review here the findings from these AFM studies that provide important insights on the supramolecular organization of rhodopsin within the membrane and factors that contribute to this organization, the molecular interactions stabilizing the structure of the receptor and factors that can modify those interactions, and the mechanism underlying constitutive activity in the receptor that can cause disease.

Exogenous extracellular matrix proteins decrease cardiac fibroblast activation in stiffening microenvironment through CAPG.

Controlling fibrosis is an essential part of regenerating the post-ischemic heart. In the post-ischemic heart, fibroblasts differentiate to myofibroblasts that produce collagen-rich matrix to physically stabilize the infarct area. Infarct models in adult mice result in permanent scarring unlike newborn animals which fully regenerate. Decellularized extracellular matrix (dECM) hydrogels derived from early-aged hearts have been shown to be a transplantable therapy that preserves heart function and stimulates cardiomyocyte proliferation and vascularization. In this study, we investigate the anti-fibrotic effects of injectable dECM hydrogels in a cardiac explant model in the context of age-associated tissue compliance. Treatments with adult and fetal dECM hydrogels were tested for molecular effects on cardiac fibroblast activation and fibrosis. Altered sensitivity of fibroblasts to the mechanosignaling of the remodeling microenvironment was evaluated by manipulating the native extracellular matrix in explants and also with elastomeric substrates in the presence of dECM hydrogels. The injectable fetal dECM hydrogel treatment decreases fibroblast activation and contractility and lowers the stiffness-mediated increases in fibroblast activation observed in stiffened explants. The anti-fibrotic effect of dECM hydrogel is most observable at highest stiffness. Experiments with primary cells on elastomeric substrates with dECM treatment support this phenomenon. Transcriptome analysis indicated that dECM hydrogels affect cytoskeleton related signaling including Macrophage capping protein (CAPG) and Leupaxin (LPXN). CAPG was down-regulated by the fetal dECM hydrogel. LPXN expression was decreased by stiffening the explants; however, this effect was reversed by dECM hydrogel treatment. Pharmacological disruption of cytoskeleton polymerization lowered fibroblast activation and CAPG levels. Knocking down CAPG expression with siRNA inhibited fibroblast activation and collagen deposition. Collectively, fibroblast activation is dependent on cooperative action of extracellular molecular signals and mechanosignaling by cytoskeletal integrity.

SIR proteins create compact heterochromatin fibers.

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins-Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2-4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to fibers with Sir3 alone, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which a dimer of Sir3 bridges and stabilizes two adjacent nucleosomes, while a Sir2-4 heterotetramer interacts with Sir3 associated with a nucleosomal trimer, driving fiber compaction.

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes.

Membrane proteins, including G protein-coupled receptors (GPCRs), present a challenge in studying their structural properties under physiological conditions. Moreover, to better understand the activity of proteins requires examination of single molecule behaviors rather than ensemble averaged behaviors. Force-distance curve-based AFM (FD-AFM) was utilized to directly probe and localize the conformational states of a GPCR within the membrane at nanoscale resolution based on the mechanical properties of the receptor. FD-AFM was applied to rhodopsin, the light receptor and a prototypical GPCR, embedded in native rod outer segment disc membranes from photoreceptor cells of the retina in mice. Both FD-AFM and computational studies on coarse-grained models of rhodopsin revealed that the active state of the receptor has a higher Young's modulus compared to the inactive state of the receptor. Thus, the inactive and active states of rhodopsin could be differentiated based on the stiffness of the receptor. Differentiating the states based on the Young's modulus allowed for the mapping of the different states within the membrane. Quantifying the active states present in the membrane containing the constitutively active G90D rhodopsin mutant or apoprotein opsin revealed that most receptors adopt an active state. Traditionally, constitutive activity of GPCRs has been described in terms of two-state models where the receptor can achieve only a single active state. FD-AFM data are inconsistent with a two-state model but instead require models that incorporate multiple active states.

Adaptations in rod outer segment disc membranes in response to environmental lighting conditions.

The light-sensing rod photoreceptor cell exhibits several adaptations in response to the lighting environment. While adaptations to short-term changes in lighting conditions have been examined in depth, adaptations to long-term changes in lighting conditions are less understood. Atomic force microscopy was used to characterize the structure of rod outer segment disc membranes, the site of photon absorption by the pigment rhodopsin, to better understand how photoreceptor cells respond to long-term lighting changes. Structural properties of the disc membrane changed in response to housing mice in constant dark or light conditions and these adaptive changes required output from the phototransduction cascade initiated by rhodopsin. Among these were changes in the packing density of rhodopsin in the membrane, which was independent of rhodopsin synthesis and specifically affected scotopic visual function as assessed by electroretinography. Studies here support the concept of photostasis, which maintains optimal photoreceptor cell function with implications in retinal degenerations.

Effect of dietary docosahexaenoic acid on rhodopsin content and packing in photoreceptor cell membranes.

Docosahexaenoic acid (DHA) is enriched in photoreceptor cell membranes. DHA deficiency impairs vision due to photoreceptor cell dysfunction, which is caused, at least in part, by reduced activity of rhodopsin, the light receptor that initiates phototransduction. It is unclear how the depletion of membrane DHA impacts the structural properties of rhodopsin and, in turn, its activity. Atomic force microscopy (AFM) was used to assess the impact of DHA deficiency on membrane structure and rhodopsin organization. AFM revealed that signaling impairment in photoreceptor cells is independent of the oligomeric status of rhodopsin and causes adaptations in photoreceptor cells where the content and density of rhodopsin in the membrane is increased. Functional and structural changes caused by DHA deficiency were reversible.

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor-Ligand Using AFM and SPR.

The goal of this research was to develop linkage chemistry for the study of bivalent interactions between a receptor and its ligand using atomic force microscopy (AFM) and surface plasmon resonance (SPR). We conceived a three-arm structure composed of flexible chains connected to a large rigid core with orthogonal functional groups at their ends for formation and attachment (or immobilization) of bivalent ligands. To demonstrate the principle, we chose the well-known biotin-streptavidin interaction as a model system. On the basis of a crystal structure of the biotin-streptavidin complex, we designed and synthesized a bisbiotin ligand to have a Y shape with two biotin motifs on its arms for binding and a functional group on its stem for immobilization or attachment, referred to as y-bisbiotin. First, we found that the y-bisbiotin ligand stabilized the streptavidin more than its monobiotin counterpart did in solution, which indicates that the bivalent interaction was synergistic. The y-bisbiotin was attached to AFM tips through a click reaction for the force measurement experiments, which showed that unbinding the bisbiotin from streptavidin needed twice the force of unbinding a monobiotin. For the SPR study, we added a ω-thiolated alkyl chain to y-bisbiotin for its incorporation into a monolayer. The SPR data indicated that the streptavidin dissociated from a mixed monolayer bearing y-bisbiotin much slower than from the one bearing monobiotin. This work demonstrates unique chemistry for the study of bivalent interactions using AFM and SPR.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Recent Progress in Molecular Recognition Imaging Using Atomic Force Microscopy.

Atomic force microscopy (AFM) is an extremely powerful tool in the field of bionanotechnology because of its ability to image single molecules and make measurements of molecular interaction forces with piconewton sensitivity. It works in aqueous media, enabling studies of molecular phenomenon taking place under physiological conditions. Samples can be imaged in their near-native state without any further modifications such as staining or tagging. The combination of AFM imaging with the force measurement added a new feature to the AFM technique, that is, molecular recognition imaging. Molecular recognition imaging enables mapping of specific interactions between two molecules (one attached to the AFM tip and the other to the imaging substrate) by generating simultaneous topography and recognition images (TREC). Since its discovery, the recognition imaging technique has been successfully applied to different systems such as antibody-protein, aptamer-protein, peptide-protein, chromatin, antigen-antibody, cells, and so forth. Because the technique is based on specific binding between the ligand and receptor, it has the ability to detect a particular protein in a mixture of proteins or monitor a biological phenomenon in the native physiological state. One key step for recognition imaging technique is the functionalization of the AFM tips (generally, silicon, silicon nitrides, gold, etc.). Several different functionalization methods have been reported in the literature depending on the molecules of interest and the material of the tip. Polyethylene glycol is routinely used to provide flexibility needed for proper binding as a part of the linker that carries the affinity molecule. Recently, a heterofunctional triarm linker has been synthesized and successfully attached with two different affinity molecules. This novel linker, when attached to AFM tip, helped to detect two different proteins simultaneously from a mixture of proteins using a so-called "two-color" recognition image. Biological phenomena in nature often involve multimolecular interactions, and this new linker could be ideal for studying them using AFM recognition imaging. It also has the potential to be used extensively in the diagnostics technique. This Account includes fundamentals behind AFM recognition imaging, a brief discussion on tip functionalization, recent advancements, and future directions and possibilities.

A three-arm scaffold carrying affinity molecules for multiplex recognition imaging by atomic force microscopy: the synthesis, attachment to silicon tips, and detection of proteins.

We have developed a multiplex imaging method for detection of proteins using atomic force microscopy (AFM), which we call multiplex recognition imaging (mRI). AFM has been harnessed to identify protein using a tip functionalized with an affinity molecule at a single molecule level. However, many events in biochemistry require identification of colocated factors simultaneously, and this is not possible with only one type of affinity molecule on an AFM tip. To enable AFM detection of multiple analytes, we designed a recognition head made from conjugating two different affinity molecules to a three-arm linker. When it is attached to an AFM tip, the recognition head would allow the affinity molecules to function in concert. In the present study, we synthesized two recognition heads: one was composed of two nucleic acid aptamers, and the other one composed of an aptamer and a cyclic peptide. They were attached to AFM tips through a catalyst-free click reaction. Our imaging results show that each affinity unit in the recognition head can recognize its respective cognate in an AFM scanning process independently and specifically. The AFM method was sensitive, only requiring 2 to 3 µL of protein solution with a concentration of ∼2 ng/mL for the detection with our current setup. When a mixed sample was deposited on a surface, the ratio of proteins could be determined by counting numbers of the analytes. Thus, this mRI approach has the potential to be used as a label-free system for detection of low-abundance protein biomarkers.

Aggregation of rhodopsin mutants in mouse models of autosomal dominant retinitis pigmentosa.

Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.

Application of catalyst-free click reactions in attaching affinity molecules to tips of atomic force microscopy for detection of protein biomarkers.

Atomic force microscopy (AFM) has been extensively used in studies of biological interactions. Particularly, AFM based force spectroscopy and recognition imaging can sense biomolecules on a single molecule level, having great potential to become a tool for molecular diagnostics in clinics. These techniques, however, require affinity molecules to be attached to AFM tips in order to specifically detect their targets. The attachment chemistry currently used on silicon tips involves multiple steps of reactions and moisture sensitive chemicals, such as (3-aminopropyl)triethoxysilane (APTES) and N-hydroxysuccinimide (NHS) ester, making the process difficult to operate in aqueous solutions. In the present study, we have developed a user-friendly protocol to functionalize the AFM tips with affinity molecules. A key feature of it is that all reactions are carried out in aqueous solutions. In summary, we first synthesized a molecular anchor composed of cyclooctyne and silatrane for introduction of a chemically reactive function to AFM tips and a bifunctional polyethylene glycol linker that harnesses two orthogonal click reactions, copper free alkyne-azide cycloaddition and thiol-vinylsulfone Michael addition, for attaching affinity molecules to AFM tips. The attachment chemistry was then validated by attaching antithrombin DNA aptamers and cyclo-RGD peptides to silicon nitride (SiN) tips, respectively, and measuring forces of unbinding these affinity molecules from their protein cognates human α-thrombin and human α5ß1-integrin immobilized on mica surfaces. In turn, we used the same attachment chemistry to functionalize silicon tips with the same affinity molecules for AFM based recognition imaging, showing that the disease-relevant biomarkers such as α-thrombin and α5ß1-integrin can be detected with high sensitivity and specificity by the single molecule technique. These studies demonstrate the feasibility of our attachment chemistry for the use in functionalization of AFM tips with affinity molecules.

Microenvironment Stiffness Amplifies Post-ischemia Heart Regeneration in Response to Exogenous Extracellular Matrix Proteins in Neonatal Mice.

The cardiogenesis of the fetal heart is absent in juveniles and adults. Cross-transplantation of decellularized extracellular matrix (dECM) can stimulate regeneration in myocardial infarct (MI) models. We have previously shown that dECM and tissue stiffness have cooperative regulation of heart regeneration in transiently regenerative day 1 neonatal mice. To investigate underlying mechanisms of mechano-signaling and dECM, we pharmacologically altered heart stiffness and administered dECM hydrogels in non-regenerative mice after MI. The dECM combined with softening exhibits preserved cardiac function, LV geometry, increased cardiomyocyte mitosis and lowered fibrosis while stiffening further aggravated ischemic damage. Transcriptome analysis identified a protein in cardiomyocytes, CLCA2, confirmed to be upregulated after MI and downregulated by dECM in a mechanosensitive manner. Synthetic knock-down of CLCA2 expression induced mitosis in primary rat cardiomyocytes in the dish. Together, our results indicate that therapeutic efficacy of extracellular molecules for heart regeneration can be modulated by heart microenvironment stiffness in vivo.

Differential adaptations in rod outer segment disc membranes in different models of congenital stationary night blindness.

Rod photoreceptor cells initiate scotopic vision when the light receptor rhodopsin absorbs a photon of light to initiate phototransduction. These photoreceptor cells are exquisitely sensitive and have adaptive mechanisms in place to maintain optimal function and to overcome dysfunctional states. One adaptation rod photoreceptor cells exhibit is in the packing properties of rhodopsin within the membrane. The mechanism underlying these adaptations is unclear. Mouse models of congenital stationary night blindness with different molecular causes were investigated to determine which signals are important for adaptations in rod photoreceptor cells. Night blindness in these mice is caused by dysfunction in either rod photoreceptor cell signaling or bipolar cell signaling. Changes in the packing of rhodopsin within photoreceptor cell membranes were examined by atomic force microscopy. Mice expressing constitutively active rhodopsin did not exhibit any adaptations, even under constant dark conditions. Mice with disrupted bipolar cell signaling exhibited adaptations, however, they were distinct from those in mice with disrupted phototransduction. These differential adaptations demonstrate that although multiple molecular defects can lead to a similar primary defect causing disease (i.e., night blindness), they can cause different secondary effects (i.e., adaptations). The lighting environment or signaling defects present from birth and during early rearing can condition mice and affect the adaptations occurring in more mature animals. A comparison of effects in wild-type mice, mice with defective phototransduction, and mice with defective bipolar cell signaling, indicated that bipolar cell signaling plays a role in this conditioning but is not required for adaptations in more mature animals.

Loss of PRCD alters number and packaging density of rhodopsin in rod photoreceptor disc membranes.

Progressive rod-cone degeneration (PRCD) is a small protein localized to photoreceptor outer segment (OS) disc membranes. Several mutations in PRCD are linked to retinitis pigmentosa (RP) in canines and humans, and while recent studies have established that PRCD is required for high fidelity disc morphogenesis, its precise role in this process remains a mystery. To better understand the part which PRCD plays in disease progression as well as its contribution to photoreceptor OS disc morphogenesis, we generated a Prcd-KO animal model using CRISPR/Cas9. Loss of PRCD from the retina results in reduced visual function accompanied by slow rod photoreceptor degeneration. We observed a significant decrease in rhodopsin levels in Prcd-KO retina prior to photoreceptor degeneration. Furthermore, ultrastructural analysis demonstrates that rod photoreceptors lacking PRCD display disoriented and dysmorphic OS disc membranes. Strikingly, atomic force microscopy reveals that many disc membranes in Prcd-KO rod photoreceptor neurons are irregular, containing fewer rhodopsin molecules and decreased rhodopsin packing density compared to wild-type discs. This study strongly suggests an important role for PRCD in regulation of rhodopsin incorporation and packaging density into disc membranes, a process which, when dysregulated, likely gives rise to the visual defects observed in patients with PRCD-associated RP.

Microenvironment stiffness requires decellularized cardiac extracellular matrix to promote heart regeneration in the neonatal mouse heart.

The transient period of regeneration potential in the postnatal heart suggests molecular changes with maturation influence the cardiac response to damage. We have previously demonstrated that injury and exercise can stimulate cardiomyocyte proliferation in the adult heart suggesting a sensitivity to exogenous signals. Here, we consider whether exogenous fetal ECM and mechanically unloading interstitial matrix can drive regeneration after myocardial infarction (MI) surgery in low-regenerative hearts of day5 mice. Compared to controls, exogenous fetal ECM increases cardiac function and lowers fibrosis at 3 weeks post-injury and this effect can be augmented by softening heart tissue. In vitro experiments support a mechano-sensitivity to exogenous ECM signaling. We tested potential mechanisms and observed that fetal ECM increases nuclear YAP localization which could be enhanced by pharmacological stabilization of the cytoskeleton. Blocking YAP expression lowered fetal ECM effects though not completely. Lastly we observed mechanically unloading heart interstitial matrix increased agrin expression, an extracellular node in the YAP signaling pathway. Collectively, these data support a combined effect of exogenous factors and mechanical activity in altering agrin expression, cytoskeletal remodeling, and YAP signaling in driving cardiomyocyte cell cycle activity and regeneration in postnatal non-regenerative mice. STATEMENT OF SIGNIFICANCE: With the purpose of developing regenerative strategies, we investigate the influence of the local niche on the cardiac injury response. We conclude tissue stiffness, as anticipated in aging or disease, impairs regenerative therapeutics. Most novel, mechanical unloading facilitates enhanced cardiac regeneration only after cells are pushed into a permissive state by fetal biomolecules. Specifically, mechanical unloading appears to increase extracellular agrin expression that amplifies fetal-stimulation of nuclear YAP signaling which correlates with observed increases of cell cycle activity in cardiomyocytes. The results further suggest the cytoskeleton is critical to this interaction between mechanical unloading and independently actived YAP signaling. Using animal models, tissue explants, and cells, this work indicates that local mechanical stimuli can augment proliferating-permissive cardiomyocytes in the natural cardiac niche.

Investigating the Nanodomain Organization of Rhodopsin in Native Membranes by Atomic Force Microscopy.

Membrane proteins play an integral role in cellular communication. They are often organized within the crowded cell membrane into nanoscale domains (i.e., nanodomains), which facilitates their function in cell signaling processes. The visualization of membrane proteins and nanodomains within biological membranes under physiological conditions presents a challenge and is not possible using conventional microscopy methods. Atomic force microscopy (AFM) provides an opportunity to study the organization of membrane proteins within biological membranes with sub-nanometer resolution. An example of a membrane protein organized into nanodomains is rhodopsin. Rhodopsin is expressed in photoreceptor cells of the retina and upon photoactivation initiates a series of biochemical reactions called phototransduction, which represents the first steps of vision. AFM has provided an opportunity to directly visualize the packing of rhodopsin in native retinal membranes and the quantitative analysis of AFM images is beginning to reveal insights about the nanodomain organization of rhodopsin in the membrane. In this report, we outline procedures for imaging rhodopsin nanodomains by AFM and the quantitative analysis of those AFM images.

Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins.

BACKGROUND: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. METHODS AND RESULTS: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 µg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. CONCLUSIONS: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis.

Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.

Solution-state conformation and stoichiometry of yeast Sir3 heterochromatin fibres.

Heterochromatin is a repressive chromatin compartment essential for maintaining genomic integrity. A hallmark of heterochromatin is the presence of specialized nonhistone proteins that alter chromatin structure to inhibit transcription and recombination. It is generally assumed that heterochromatin is highly condensed. However, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, formation of heterochromatin at telomeres and the homothallic silent mating type loci require the Sir3 protein. Here, we use a combination of sedimentation velocity, atomic force microscopy and nucleosomal array capture to characterize the stoichiometry and conformation of Sir3 nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome. We also find that Sir3 fibres are less compact than canonical magnesium-induced 30 nm fibres. We suggest that heterochromatin proteins promote silencing by 'coating' nucleosomal arrays, stabilizing interactions between nucleosomal histones and DNA.