Structural basis of human Nav1.5 gating mechanisms.

Voltage-gated Nav1.5 channels are central to the generation and propagation of cardiac action potentials1. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure2-5. Despite decades of progress in Nav1.5 biology6-8, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Nav1.5 in previously unanticipated open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show the IFM motif repositioned in the receptor site but not dislodged. In particular, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction results in the fast inactivation of Nav1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Nav1.5 and the mechanisms underlying CTD-related channelopathies.

Revolving ATPase motors as asymmetrical hexamers in translocating lengthy dsDNA via conformational changes and electrostatic interactions in phi29, T7, herpesvirus, mimivirus, E. coli, and Streptomyces.

Investigations of the parallel architectures of biomotors in both prokaryotic and eukaryotic systems suggest a similar revolving mechanism in the use of ATP to drive translocation of the lengthy double-stranded (ds)DNA genomes. This mechanism is exemplified by the dsDNA packaging motor of bacteriophage phi29 that operates through revolving but not rotating dsDNA to "Push through a one-way valve". This unique and novel revolving mechanism discovered in phi29 DNA packaging motor was recently reported in other systems including the dsDNA packaging motor of herpesvirus, the dsDNA ejecting motor of bacteriophage T7, the plasmid conjugation machine TraB in Streptomyces, the dsDNA translocase FtsK of gram-negative bacteria, and the genome-packaging motor in mimivirus. These motors exhibit an asymmetrical hexameric structure for transporting the genome via an inch-worm sequential action. This review intends to delineate the revolving mechanism from a perspective of conformational changes and electrostatic interactions. In phi29, the positively charged residues Arg-Lys-Arg in the N-terminus of the connector bind the negatively charged interlocking domain of pRNA. ATP binding to an ATPase subunit induces the closed conformation of the ATPase. The ATPase associates with an adjacent subunit to form a dimer facilitated by the positively charged arginine finger. The ATP-binding induces a positive charging on its DNA binding surface via an allostery mechanism and thus the higher affinity for the negatively charged dsDNA. ATP hydrolysis induces an expanded conformation of the ATPase with a lower affinity for dsDNA due to the change of the surface charge, but the (ADP+Pi)-bound subunit in the dimer undergoes a conformational change that repels dsDNA. The positively charged lysine rings of the connector attract dsDNA stepwise and periodically to keep its revolving motion along the channel wall, thus maintaining the one-way translocation of dsDNA without reversal and sliding out. The finding of the presence of the asymmetrical hexameric architectures of many ATPases that use the revolving mechanism may provide insights into the understanding of translocation of the gigantic genomes including chromosomes in complicated systems without coiling and tangling to speed up dsDNA translocation and save energy.

A simple colorimetric experiment using mammalian cell culture to study metabolism.

The goal of this laboratory exercise is to give upper-level undergraduate students an introduction to sterile technique in mammalian cell culture and metabolism. The experiment can be completed within a 3-h lab period and can be performed either in conjunction with other biochemistry/metabolism experiments or used as a stand-alone experiment. In this experiment, students are tasked with relating the acidification of cell culture medium to metabolism in order to elucidate the mechanism of action for a compound. Students can relate their experimental results to topics covered on glycolysis and oxidative phosphorylation in upper-level biochemistry classes as well as gain valuable experience relating metabolism to drug discovery.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis.

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Cryo-electron Microscopy Structures of Chimeric Hemagglutinin Displayed on a Universal Influenza Vaccine Candidate.

UNLABELLED: Influenza viruses expressing chimeric hemagglutinins (HAs) are important tools in the quest for a universal vaccine. Using cryo-electron tomography, we have determined the structures of a chimeric HA variant that comprises an H1 stalk and an H5 globular head domain (cH5/1 HA) in native and antibody-bound states. We show that cH5/1 HA is structurally different from native HA, displaying a 60° rotation between the stalk and head groups, leading to a novel and unexpected "open" arrangement of HA trimers. cH5/1N1 viruses also display higher glycoprotein density than pH1N1 or H5N1 viruses, but despite these differences, antibodies that target either the stalk or head domains of hemagglutinins still bind to cH5/1 HA with the same consequences as those observed with native H1 or H5 HA. Our results show that a large range of structural plasticity can be tolerated in the chimeric spike scaffold without disrupting structural and geometric aspects of antibody binding. IMPORTANCE: Chimeric hemagglutinin proteins are set to undergo human clinical trials as a universal influenza vaccine candidate, yet no structural information for these proteins is available. Using cryo-electron tomography, we report the first three-dimensional (3D) visualization of chimeric hemagglutinin proteins displayed on the surface of the influenza virus. We show that, unexpectedly, the chimeric hemagglutinin structure differs from those of naturally occurring hemagglutinins by displaying a more open head domain and a dramatically twisted head/stalk arrangement. Despite this unusual spatial relationship between head and stalk regions, virus preparations expressing the chimeric hemagglutinin are fully infectious and display a high glycoprotein density, which likely helps induction of a broadly protective immune response.

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications.

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd(2+) ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

Three-dimensional imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission.

UNLABELLED: HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4(+) T cells and either uninfected human CD4(+) T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread. IMPORTANCE: Our findings lead to new insights into unique aspects of HIV transmission in the brain and at T cell-T cell synapses, which are thought to be a predominant mode of rapid HIV transmission early in the infection process.

Three-Dimensional Imaging of Viral Infections.

Three-dimensional (3D) imaging technologies are beginning to have significant impact in the field of virology, as they are helping us understand how viruses take control of cells. In this article we review several methodologies for 3D imaging of cells and show how these technologies are contributing to the study of viral infections and the characterization of specialized structures formed in virus-infected cells. We include 3D reconstruction by transmission electron microscopy (TEM) using serial sections, electron tomography, and focused ion beam scanning electron microscopy (FIB-SEM). We summarize from these methods selected contributions to our understanding of viral entry, replication, morphogenesis, egress and propagation, and changes in the spatial architecture of virus-infected cells. In combination with live-cell imaging, correlative microscopy, and new techniques for molecular mapping in situ, the availability of these methods for 3D imaging is expected to provide deeper insights into understanding the structural and dynamic aspects of viral infection.

Exploring the mechanism of plasmid DNA nuclear internalization with polymer-based vehicles.

Cationic polymers are commonly used to transfect mammalian cells, but their mechanisms of DNA delivery are unknown. This study seeks to decipher the mechanism by which plasmid DNA delivered by a class of cationic polymers traffics to and enters the nucleus. While studies have been performed to elucidate the mechanism of naked plasmid DNA (pDNA) import into the nuclei of mammalian cells, our objectives were to determine the effects of polymer complexation on pDNA nuclear import and the impact of polymer structure on that import. We have performed studies in whole cells and in isolated nuclei using flow cytometry and confocal microscopy to characterize how polymer-DNA complexes (polyplexes) are able to deliver their pDNA cargo to the nuclei of their target cells. The polymers tested herein include (i) linear poly(ethylenimine) (JetPEI), a polyamine, and (ii) two poly(glycoamidoamine)s (PGAAs), polyamines that contain carbohydrate moieties (meso-galactarate, Glycofect (G4), and L-tartarate, T4) within their repeat units. Our results indicate that, when complexed with the PGAAs, pDNA association with the nuclei was severely hampered in isolated nuclei compared to whole cells. When the pDNA was complexed with JetPEI, there was slight inhibition of pDNA-nuclear interaction in isolated nuclei compared to whole cells. However, even in the case of PEI, the amount of pDNA imported into the nucleus increases in the presence of cytosolic extract, thus indicating that intracellular components also play a role in pDNA nuclear import for all polymers tested. Interestingly, PEI and G4 exhibit the highest reporter gene expression as well as inducing higher envelope permeability compared to T4, suggesting that the ability to directly permeabilize the nuclear envelope may play a role in increasing expression efficiency. In addition, both free T4 and G4 polymers are able to cross the nuclear membrane without their pDNA cargo in isolated nuclei, indicating the possibility of different modes of nuclear association for free polymers vs polyplexes. These results yield insight to how the incorporation of carbohydrate moieties influences intracellular mechanisms and will prove useful in the rational design of safe and effective polymer-based gene delivery vehicles for clinical use.

Cationic glycopolymers for the delivery of pDNA to human dermal fibroblasts and rat mesenchymal stem cells.

Progenitor and pluripotent cell types offer promise as regenerative therapies but transfecting these sensitive cells has proven difficult. Herein, a series of linear trehalose-oligoethyleneamine "click" copolymers were synthesized and examined for their ability to deliver plasmid DNA (pDNA) to two progenitor cell types, human dermal fibroblasts (HDFn) and rat mesenchymal stem cells (RMSC). Seven polymer vehicle analogs were synthesized in which three parameters were systematically varied: the number of secondary amines (4-6) within the polymer repeat unit (Tr4(33), Tr5(30), and Tr6(32)), the end group functionalities [PEG (Tr4(128)PEG-a, Tr4(118)PEG-b), triphenyl (Tr4(107)-c), or azido (Tr4(99)-d)], and the molecular weight (degree of polymerization of about 30 or about 100) and the biological efficacy of these vehicles was compared to three controls: Lipofectamine 2000, JetPEI, and Glycofect. The trehalose polymers were all able to bind and compact pDNA polyplexes, and promote pDNA uptake and gene expression [luciferase and enhanced green fluorescent protein (EGFP)] with these primary cell types and the results varied significantly depending on the polymer structure. Interestingly, in both cell types, Tr4(33) and Tr5(30) yielded the highest luciferase gene expression. However, when comparing the number of cells transfected with a reporter plasmid encoding enhanced green fluorescent protein, Tr4(33) and Tr4(107)-c yielded the highest number of HDFn cells positive for EGFP. Interestingly, with RMSCs, all of the higher molecular weight analogs (Tr4(128)PEG-a, Tr4(118)PEG-b, Tr4(107)-c, Tr4(99)-d) yielded high percentages of cells positive for EGFP (30-40%).

Membrane and nuclear permeabilization by polymeric pDNA vehicles: efficient method for gene delivery or mechanism of cytotoxicity?

The aim of this study is to compare the cytotoxicity mechanisms of linear PEI to two analogous polymers synthesized by our group: a hydroxyl-containing poly(l-tartaramidoamine) (T4) and a version containing an alkyl chain spacer poly(adipamidopentaethylenetetramine) (A4) by studying the cellular responses to polymer transfection. We have also synthesized analogues of T4 with different molecular weights (degrees of polymerization of 6, 12, and 43) to examine the role of molecular weight on the cytotoxicity mechanisms. Several mechanisms of polymer-induced cytotoxicity are investigated, including plasma membrane permeabilization, the formation of potentially harmful polymer degradation products during transfection including reactive oxygen species, and nuclear membrane permeabilization. We hypothesized that since cationic polymers are capable of disrupting the plasma membrane, they may also be capable of disrupting the nuclear envelope, which could be a potential mechanism of how the pDNA is delivered into the nucleus (other than nuclear envelope breakdown during mitosis). Using flow cytometry and confocal microscopy, we show that the polycations with the highest amount of protein expression and toxicity, PEI and T4(43), are capable of inducing nuclear membrane permeability. This finding is important for the field of nucleic acid delivery in that direct nucleus permeabilization could be not only a mechanism for pDNA nuclear import but also a potential mechanism of cytotoxicity and cell death. We also show that the production of reactive oxygen species is not a main mechanism of cytotoxicity, and that the presence or absence of hydroxyl groups and polymer length play a role in polyplex size and charge in addition to protein expression efficiency and toxicity.

Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions.

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing N-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000.

Interaction of poly(ethylenimine)-DNA polyplexes with mitochondria: implications for a mechanism of cytotoxicity.

Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure-function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F(0)F(1)-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F(0)F(1)-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

A polycation scaffold presenting tunable "click" sites: conjugation to carbohydrate ligands and examination of hepatocyte-targeted pDNA delivery.

A versatile polycation scaffold that can easily be modified with targeting ligands has been designed, synthesized, and characterized. A series of galactose-containing polymers has been produced to demonstrate the ease of modification of this polynucleotide delivery vehicle motif via the click reaction and to study how various structural modifications affect recognition by ASGPr on hepatocytes. A small library of structures was created where DCS and alkyl spacer length between the targeting group and the polymer backbone was varied. The novel polymer scaffold described proves to be a valuable tool for understanding structure/activity relationships of complexes made with receptor-targeted polymers.