APPLICATIONS OF CELL-DERIVED VESICLES: FROM SINGLE MOLECULE STUDIES TO DRUG DELIVERY

Single molecule studies can provide information of biological molecules which otherwise is lost in ensemble studies. A wide variety of fluorescence-based techniques are utilized for single molecule studies. While these tools have been widely applied for imaging soluble proteins, single molecule studies of transmembrane proteins are much more complicated. A primary reason for this is that, unlike membrane proteins, soluble proteins can be easily isolated from the cellular environment. One approach to isolate membrane proteins into single molecule level involves a very low label expression of the protein in cells. However, cells generate background fluorescence leading to a very low signal to noise ratio. An alternative approach involves isolating membrane proteins in artificial membrane derived vesicles. This approach is limited to proteins which can be solubilized or stabilized in detergent solution. This intermediate step endangers the structural integrity of proteins with multiple subunits. Hence, we isolated transmembrane proteins into cell-derived vesicles which maintain the proteins in their physiological membrane without compromising their functional integrity. We studied the stoichiometric assembly of α3β4 nicotinic receptors which are pentameric receptor with possible stoichiometry of (α3)2(β4)3 and (α3)3(β4)2. We found that (α3)2(β4)3 is the predominant stoichiometry, and we have verified our finding with both single and double color experiments. We have also demonstrated that cell-derived vesicles can be utilized to study ligand receptor interactions. Cell-derived vesicles generated from cellular preparations provide a method to study the overall structural and functional properties of membrane proteins. However, organelle specific information is not available in this approach. Alternatively, separating vesicles based on their original organelle could provide information on the assembly and trafficking of membrane proteins. For example, it has been hypothesized that nicotine acts as a pharmacological chaperone of α4β2 nicotinic receptors and nicotine alters the assembly of the nicotinic receptors towards the high sensitivity isoform in the ER. To validate this hypothesis, we isolated α4β2 nicotinic receptors located on vesicles derived from the ER and plasma membrane origins and utilized single molecule studies to determine the stoichiometric assembly of the receptor. The data suggested that the ER has a higher percentage of the low sensitivity isoform ((α4)3(β2)2) than the plasma membrane indicating that the high sensitivity isoform trafficked more efficiently to the cell surface. When nicotine was added, the distribution of nicotinic receptors changes in those compartments. In both the ER and plasma membrane, the percentage of high sensitivity isoform was greater than the sample without the presence of nicotine. The results suggested that nicotine altered the assembly of nicotinic receptors to form the high sensitivity isoform in the ER and the altered assembly trafficked to the plasma membrane efficiently increasing the ratio of this isoform in the plasma membrane. The cell derived vesicles we utilized to isolate single receptors are structurally similar to liposomes, an FDA approved drug delivery system, which is spherical vesicles composed of at least one lipid bilayer. Hence, cell-derived vesicles possess potential to be utilized as drug delivery vehicles. I explored the applicability of cell-derived vesicles as general delivery vehicles to cultured cells. Additionally, we implanted xenografts into immune compromised nude mice and prepared cell derived vesicles labeled with dye molecules. The vesicles were injected in a mouse containing a xenograft to monitor whether these vesicles can reach to the xenograft. Our data suggested that cell-derived vesicles can successfully reach the xenograft and thus have potential to be utilized as a drug delivery vehicle

The Nicotine Metabolite, Cotinine, Alters the Assembly and Trafficking of a Subset of Nicotinic Acetylcholine Receptors

Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs), leading to their up-regulation on the plasma membrane. Although the mechanism is not fully understood, nicotine-induced up-regulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH-sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry

Organelle-Specific Single-Molecule Imaging of \u3cem\u3eα\u3c/em\u3e4\u3cem\u3eβ\u3c/em\u3e2 Nicotinic Receptors Reveals the Effect of Nicotine on Receptor Assembly and Cell-Surface Trafficking

Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER versus those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure

Mammalian Cell-Derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly

Impact of regulatory light chain mutation K104E on the ATPase and motor properties of cardiac myosin

Mutations in the cardiac myosin regulatory light chain (RLC, MYL2 gene) are known to cause inherited cardiomyopathies with variable phenotypes. In this study, we investigated the impact of a mutation in the RLC (K104E) that is associated with hypertrophic cardiomyopathy (HCM). Previously in a mouse model of K104E, older animals were found to develop cardiac hypertrophy, fibrosis, and diastolic dysfunction, suggesting a slow development of HCM. However, variable penetrance of the mutation in human populations suggests that the impact of K104E may be subtle. Therefore, we generated human cardiac myosin subfragment-1 (M2β-S1) and exchanged on either the wild type (WT) or K104E human ventricular RLC in order to assess the impact of the mutation on the mechanochemical properties of cardiac myosin. The maximum actin-activated ATPase activity and actin sliding velocities in the in vitro motility assay were similar in M2β-S1 WT and K104E, as were the detachment kinetic parameters, including the rate of ATP-induced dissociation and the ADP release rate constant. We also examined the mechanical performance of α-cardiac myosin extracted from transgenic (Tg) mice expressing human wild type RLC (Tg WT) or mutant RLC (Tg K104E). We found that α-cardiac myosin from Tg K104E animals demonstrated enhanced actin sliding velocities in the motility assay compared with its Tg WT counterpart. Furthermore, the degree of incorporation of the mutant RLC into α-cardiac myosin in the transgenic animals was significantly reduced compared with wild type. Therefore, we conclude that the impact of the K104E mutation depends on either the length or the isoform of the myosin heavy chain backbone and that the mutation may disrupt RLC interactions with the myosin lever arm domain