Clathrin-coated pits on the plasma membrane invaginate into coated vesicles to internalize receptors and membrane. The clathrin adaptor epsin contains an aminoterminal ENTH domain that binds PI(4,5)P₂ and a carboxy-terminal domain that binds clathrin, and accessory proteins such as AP2. Here, we assessed how inter- and intramolecular factors affect the contribution of epsin to coated-pit function in living cells. We found Dictyostelium epsin was not required for global clathrin function, but plays an essential role in spore development. We demonstrated that clathrin, but not AP2, was critical for epsin to associate with clathrin-coated pits. We found that the carboxy-terminal region of epsin was essential, but not sufficient, for targeting epsin within clathrin-coated pits on the plasma membrane. In addition to targeting epsin to the membrane, the amino-terminal ENTH domain regulates the interaction between epsin and clathrin, an essential property that cannot be replaced by an alternate PI(4,5)P₂ binding domain. Moreover, the ENTH domain facilitates the functional interaction between clathrin and actin during late stages of endocytosis, possibly by regulating the activity of the adaptor Hip1r. Both the ability to bind PI(4,5)P₂ and another function mediated by residue T107 are critical for the activity of the ENTH domain. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits. Furthermore, we propose that the ENTH domain of epsin facilitates the membrane recruitment and phosphorylation of Hip1r, which in turn mediates the productive interaction of clathrin with the actin cytoskeleton at the plasma membrane. / text

Mercury exposure has been related to neurological diseases and poisoning. Quantification of mercury in biological fluids, such as serum or urine is an important diagnostic method for mercury exposure. We have developed an aptamer-encapsulated porous phospholipid nanoshell (PPN) sensor for sensing mercury in urine using a modified 15-mer single strand DNA.1 The probe is protected from DNAse and other biofouling species by encapsulation within the porous liposomes composed of mixed phospholipids, allowing direct application of the aptamer in biological fluids containing DNAse and other biofouling materials. The encapsulated sensor was directly tested in urine samples at physiological pH. We were able to detect below 100 ppb (500 nM) Hg2+ in urine (urine mercury threshold set by Biologischer Arbeitstoff Toleranz Wert or BAT)1 with no sample preparation other than pH adjustment. These results suggest that porous phospholipid nanoshells (PPNs) can serve as a general-purpose protection scaffold for biological sensing.

No description available.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (MMedVet (Gyn.))-University of Pretoria, 1998.

No description available.

Human Endogenous Retroviruses (HERV) are derived from exogenous retroviruses that infected inheritable germline tissues millions of years ago and account for 8% of the human genome. Like other retroviruses HERVs encode Gag, Pol and sometimes Env proteins. During a retroviral infection, retroviral Gag recruits the hosts Endosomal Sorting Complex Required for Transport (ESCRT) and associated proteins (ALIX and TSG101) to produce precisely sized viruses from endosomes or the plasma membrane. The ESCRT machinery is also involved in cytokinesis and control growth factor receptor signalling. HERV-K is the most recent HERV family to insert into the genome and is still able to produce mostly intact transcripts, including Gag. When expressed, Gag causes cells to release Virus-Like Particles (VLP) that lack HERV genomes. These retroviral VLP are remarkably similar to a sub-category of extracellular vesicles (EVs) called exosomes. Exosomes require ALIX, TSG101 and the ESCRT machinery for their production. It is possible that HERV-K Gag is required for exosome production or that HERV VLPs are a major contaminant of exosome preparations that account for many of the functions attributed to exosomes. Our data shows that HERV-K Gag over-expression or knockdown did not change the number of EVs released per cell in two cell lines. As well there was no difference in the amount of ALIX and TSG101 in the EVs in these conditions. The most intriguing observation made was the increase of cell number with expression of HERV-K Gag and decrease when HERV-K Gag was knocked down in HEK293T. We are currently unable to conclude the role of HERV-K Gag on EV production and content. We speculate that HERV-K Gag might affect cells through controlling cell proliferation or death, for example by competing with ESCRT machinery to impact signalling through growth factor receptors. This study begins to outline the potential effects HERV-K Gag might have on EV release and cell proliferation.

The stability of growing uni-lamellar vesicles is investigated using the formalism of nonequilibrium thermodynamics. The vesicles, which are assumed to be in an otherwise aqueous solution, are growing due to the accretion of lipids to the bilayer which forms the vesicle membrane. The thermodynamic description is based on the hydrodynamics of a water-lipid mixture together with a model of the vesicle as a discontinuous system in the sense of linear nonequilibrium thermodynamics. The approach assumes that the energy of the bilayer membrane is given by the spontaneous curvature model attributed to Helfrich. Furthermore, the rate at which lipids incorporate into the membrane is taken to be proportional to the surface area of the membrane. In this way, the relevant forces and fluxes of the system are identified in the context of a stability analysis. The resulting constitutive equation for the flux of water across the membrane is used to analyse the stability of spherical vesicles that are subject to different perturbations. First, a simplified approach is presented which restricts perturbations to axisymmetric ellipsoids. In that case, the analysis is carried out using an explicit Cartesian parametrisation. A perturbation theory which describes more general deformations is then developed and applied to the case of arbitrary axisymmetric perturbations. It is found that there are generically two critical radii at which changes of stability occur. For the case where the perturbation takes the form of a single zonal harmonic, only one of these radii is physical and is given by the ratio $2L_p / L_\gamma$, where $L_p$ is the hydraulic conductivity and $L_\gamma$ is the Onsager coefficient related to changes in membrane area due to lipid accretion. The stability of such perturbations is related to the value of l corresponding to the particular zonal harmonic: those with lower l are more unstable than those with higher l. The conditions under which general axisymmetric perturbations reduce to explicit ellipsoidal calculations are also found. A heuristic explanation for the results is proposed whilst possible extensions of the current work and the need for experimental input are also discussed.

acase@tulane.edu / 1 / Adedoyin Johnson

No description available.