DEVELOPMENT OF A MICROSCOPE FLUORESCENCE SPECTRUM AND ITS APPLICATION TO CHARGED PHOSPHOLIPID VESICLE FUSION
A fluorescence resonance energy transfer (FRET) imaging microscope was developed to simultaneously study the changes of fluorescence spectrum and direct florescence image of pair-wise charged giant unilamellar lipid vesicle (GUV) fusion. The negatively charged GUV were comprised of DOPG and DOPC and were labeled with Rh-PE (red) as an acceptor, and the positively charge GUV comprised of EDOPC+ and DOPC were labeled with fluorescent probe DOC (green) as a donor. An electric control chamber was used to control individual charged vesicle movement, contact and fusion. A high-speed digital camera captured the spectrum change at a frame rate of 3ms (Princeton instrument, PentaMax). At the same time an analog color camera (Samsung SHC710) was used to record the fusion event at a frame rate of 33ms. By comparing the spectrum and fluorescence image, we found that most charged vesicle fusion events exhibit hemifusion as an intermediate stage. The time required for pair-wise vesicles to reach hemifusion after initial vesicle contact is less than 6ms.

Researcher: Guo Hua Lei

A novel development has allowed for the direct observation of single, pairwise interactions of anionic and cationic vesicles, linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, 1,2-doleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes. Anionic vesicles were composed of the anionic lipid DOPG, neutral lipids DOPC and/or DOPE and in some cases imparted with cholesterol. For the experiments involving DNA, the cationic vesicles were electrophoretically maneuvered into contact with individual pieces of DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of DOPG (100%), DOPG/DOPE (1:1) or DOPG/DOPC (1:1).

To see clips of recorded interactions click here:

Other Research:

o Cationic liposomes - application for DNA delivery / gene transfection
o Interaction of cationic lipids with membrane lipids
o Kinetics of lipoplex formation and DNA release

Researchers: Rumiana Koynova, Yury Tarahovsky, Spiro Pantazatos

     This project is currently being carried out in conjunction with the Northwestern Medical School, Division of Cardiovascular Research. Below is a summary of the project:

     Traditional means to identify the physiological severity of arterial disease are hampered by their inability to identify atheroma extent and composition. New techniques that identify atheroma in-vivo are being developed, however, accurate methodologies for atheroma are hampered, due to the heterogeneous nature of the disease process. Novel acoustic targeting and highlighting agents, such as liposomes, may overcome these problems.
     Liposomes are phospholipid vesicles enclosing an aqueous space. We have developed a unique methodology that, by process and composition, provides acoustic characteristics of liposomes. This formulation allows modification for antibody conjugation and therapeutic drug incorporation. Previous work by this group has been centered on the optimization of formulation, optimization of conjugation, and development of in-vitro and in-vivo quantitation techniques.
     This proposal describes a series of protocols to optimize highlighting and enhancing characteristics of these agents in-vitro and in-vivo. We plan to determine in-vitro and in-vivo transfection and drug delivery potential of the agents. We plan to determine if the agents and can be utilized with other imaging modalities as atheroma enhancement agents. Our long term goals are to determine, quantitate, and characterize the stage, extent, and physiologic severity of atherosclerosis and allow directed therapy to improve physiologic flow following intervention.

Researchers: Shao-ling Huang, Li Wang

To squeeze through capillaries, red blood cells must undergo a reversible shape change. The membrane skeletal network responsible for the elasticity necessry in this vital process is composed largely of one isoform of the ubiquitous protein, spectrin. Until recently, evidence for molecular mechanism (s) accounting for spectrin's elasticity has been lacking.

Spectrin consists of two, elongated monomers, the polypeptide chain of each which is folded into 17 and 20 triple helical bundles, respectively, of about 106 amino acids. Although it had been speculated that the linker regions between the triple helical bundles or repeating units are disordered, the X-ray crystal structures of 4 cloned fragments of two, connected repeats, which we obtained recently (Grum et al., 1999), showed the linker region to be alpha-helical. More importantly, our structures suggested two molecular models of spectrin flexibility--one in which conformational rearrangement leads to shortening of the spectrin chain and another in which bending of repeats at the linker regions also leads to a shortening of spectrin chain.

We are continuing structural characterization of other regions of spectrin in order to test our models and obtain evidence for other models by biophysical methods including X-ray crystallography and NMR spectroscopy. To indentify structural features correlated with stability or instability of folding of different spectrin fragments, we are also determining the free energies of folding different spectin fragments by urea and by thermal denaturation (Pantazatos and MacDonald, 1997; MacDonald and Pozharski, 2001).

In addition to spectrin flexibility, we are investigating the roles of different spectrin repeats and of nonrepeating unit domains in specific binding and localization of important regularory proteins in the cell. Recent evidence for nonerythroid spectrin (McMahon et al., 2001) and beta-spectrin (Tse et al., 2001) in the nucleus indicates that spectrin is likely to be more versatile than previously assumed.

Researchers: Ruby MacDonald, Julie Ruffatti, Tina Byun

A regular progression of polymorphic phase behavior was observed for mixtures of the anionic phospholipid, cardiolipin, and the cationic phospholipid derivative, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine. As revealed by freeze-fracture electron microscopy and small angle X-ray diffraction, whereas the two lipids separately assume only lamellar phases, their mixtures exhibit a symmetrical (depending on charge ratio and not polarity) sequence of non-lamellar phases. The
inverted hexagonal phase, HII, formed from equimolar mixtures of the two lipids, i.e., at net charge neutrality (charge ratio = CR(+/ ) = 1:1). When one type of lipid was in significant excess
(CR(+/ )=2:1 or CR(+/ ) = 1:2), a bicontinuous cubic structure was observed. These cubic phases were very similar to those sometimes present in cellular organelles that contain cardiolipin.
Increasing the excess of cationic or anionic charge to CR(+/ ) = 4:1 or CR(+/ ) = 1:4, led to the appearance of membrane bilayers with numerous interlamellar contacts, i.e., sponge structures. It is evident that interactions between cationic and anionic moieties can influence the packing of polar heads and hence control polymorphic phase transitions. The facile isothermal, polymorphic interconversion of these lipids may have biological and technical implications for cellular function, gene therapy, and membrane protein crystallization.

Researchers: Yury Tarahovsky