Partners in Crime:
Projects Current
Position
|
Andrew Lytle ’06 (Mo. Bio.) |
Lipid Rafts in GUVs WGM in liposomes |
M.D./Ph.D. student Jefferson Medical
College |
|
Izzy Smith ’05 (Physics) |
WGMs in dye-coated,
biotinylated microspheres |
Ph.D. student (Biophysics),
U. Wisconsin |
|
Robby Gerrity ’06
(Math. and Chem. Minor) |
WGMs in qdot-coated
microspheres and liposomes |
Law student, NYU |
|
Perry Schiro ’04
(Physics and Chem. Minor) |
WGM’s in dye
monolayer Capture Range of
laser tweezer |
Ph.D. student
(Chemistry), U. Washington |
|
Chris Dubois ’06
(Math) |
Capture range of
laser tweezer |
|
|
J. Steven Ross ’03
(Physics) |
Dual-tweezer setup |
Ph.D. student
(Applied Science), UC Davis |
|
Anders Knospe ’02
(Physics) |
Lasing-SRS in
microdroplets |
Ph.D. student
(Physics), Yale |
Listed
in reverse chronological order GUV
– Giant Unilamellar Vesicles
(in lab participation) WGM – Whispering GalleryMode SRS – Stimulated Raman Scattering
Ongoing Research (at
Pomona):
Characterization of Lipid
Domains in Model Membranes
The plasma
membrane of cells contains a variety of lipids and proteins. The 9-year old lipid raft model of cell
membranes hypothesis that rather than being a homogeneous mosaic of fluid,
cholesterol and sphingolipids assemble to form microdomain, or “rafts” in a
“liquid-ordered” lo phase that float within
the rest of the membrane that is in a “liquid-disordered” ld
phase. Certain membrane proteins
essential for signal transduction such as GPI-anchored proteins can be
sequestered in these rafts. Early experiments
supporting the existence for lipid rafts focused on using detergent
insolubility. In the past five years,
various groups have used fluorescent-conjugated lipid to directly visualize lo
domains in supported bilayers and giant unilamellar vesicles (GUV’s). 
After
months of following various publications with incomplete details, Andrew Lytle
(Molecular Biology ’06) was finally able to use fluorescence microscopy to
observe phase segregation of Oregon Green DHPE in GUVs that consist of DOPC,
brain sphinogomyelin and cholesterol.
The main concern with using a fluorescent probe is that the probe may be inducing phase segregation. We will therefore attempt to observe lipid phase segregation in vivo by using Raman imaging to obtain spatially-resolved Raman spectra of GUVs and supported bilayers consisting of raft-forming lipid mixtures. Equipment available for this research in my lab includes a Nikon TE-2000 inverted microscope, a Melles-Griot Argon-ion laser (488 nm), a Power Technology diode laser (785 nm), a 0.5-m Acton imaging spectrograph, a Princeton Instrument Spec-10 liquid-nitrogen-cooled CCD camera. We also have access to a Varian Eclipse fluorimeter, two Cary 300 spectrometers, a Nicolet FTIR spectrometer with a resolution of 0.1 cm-1in the Chemistry department. In addition, a postdoc. with NMR experience will also have access to the Chemistry department’s 400 MHz Bruker NMR spectrometer.
Sabbatical Research(at
UVA): Single Particle Tracking of t-SNARE’s
Neurotransmitters are usually stored in synaptic vesicles, which fuse with the membrane of a presynaptic neuron before the neurotransmitter is released. The well-supported SNARE hypothesis postulates that each type of transport vesicle has a distinct v-SNARE that pairs up with a unique t-SNARE at the appropriate target membrane. Although it has been established that Rabs mediate the early stage of vesicle targeting and tethering, we do not know what the SNARE components are doing in the time between firm docking and fusion pore formation.
The lateral distribution of t-SNAREs in membranes has been shown to affect vesicle docking. Syntaxin and SNAP-25 concentrate in 200 nm cholesterol-dependent clusters in planar membranes formed from ruptured PC12 cells where secretory vesicles preferentially dock and fuse. However, syntaxin does not co-patch with typical raft markers like GPI-linked proteins and thus seem to be distinct from cholesterol-dependent membrane rafts. On the other hand, Chamberlain et. al. have shown that syntaxin and SNAP-25 are highly enriched in lipid rafts in PC12 cells. However, Bacia et. al. have recently shown that SNAREs prefer the liquid-disordered phase over liquid-ordered raft domain when reconstituted into giant unilamellar vesicles. Single-particle tracking of t-SNAREs near the boundaries of lipid raft domains in supported bilayers will elucidate some of these inconsistencies.
Future Research:
Single Particle Tracking of
Gint3 (GDI-Gint3 Intreaction)
After spending the year at U VA studying presynaptic membrane fusion at U VA, I will step back in vesicle-fusion time and apply single particle tracking techniques to study Rab proteins and vesicle tethering and docking. Cris Cheney, a developmental biologist at Pomona, and her students have identified a 50kD protein, Gint3, that interacts with Rab GDP dissociation inhibitor (GDI) in a Matchmaker 2 (Clonetech) LexA-based yeast two-hybrid screen. Students in Cris’s lab will be studying the membrane-association of Gint3 with immunohistochemical localization to explore Gint3’s role in vesicle transport in the next two years. To complement these studies, we will use SPT to track the trajectory of a Rab molecule as it is released from a GDI-Rab complex upon GDI-Gint3 interaction and diffuses towards a bilayer.
Force Production in Dynein
The dynein molecular motor is a multisubunit ATPase that participates in several important microtubule[based motilities in eukaryotic cells. David Asai’s lab at neighboring Harvery Mudd college studies non-axonemal or “cytoplasmic” dynein of the ciliated protozoan, Tetrahymena thermophila. We will collaborate with Asai’s lab and use our laser tweezer to study the force generated by these non-axonemal dyneins under different loading condtions.
Past Research Corner: