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University of Minnesota Department of Chemistry and Biochemistry
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Friday, April 24, 2015; 3:00 p.m.; Chem 200 (Kobilka Lecture Hall)

 

Department of Chemistry & Biochemistry Welcomes:

Spencer Gardeen

Master Student, Department of Chemistry & Biochemistry, University of Minnesota Duluth,

Research Advisor – Dr. Joseph Johnson & Dr. Ahmed Heikal

Presents:

“Fluorescence Micro-spectroscopy Assessment of the in vitro Dimerization of BACE1-GFP Fusion Protein in Cultured Cells”

 

Alzheimer’s Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid (Aβ) plaques in the brain, which triggers known symptoms in AD patients such as memory loss. Aβ plaques are formed via the β-secretase pathway, in which amyloid precursor protein (APP) is cleaved by the proteases BACE1 and γ-secretase. This enzyme-facilitated cleavage leads to the production of Aβ fragments that are believed to form plaques, which cause neuronal cell death. Recent detergent protein extraction studies suggest that the native BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Currently, however, there are no studies that support the dimerization of BACE1 hypothesis in living cells. In this contribution, we describe our ongoing effort to examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells transfected with a BACE1-GFP fusion protein construct were first imaged using confocal and DIC microscopy for monitoring labeled BACE1 localization within the cell morphology. Subsequently, single-molecule fluctuation analysis allowed us to test the dimerization hypothesis of the labeled BACE1, using florescence fluctuation measurements of the diffusion coefficient (size-dependent observable) and the molecular brightness, as a function of BACE1 inhibitors and substrate binding. Complementary two-photon fluorescence lifetime and anisotropy imaging enabled us to probe any changes in BACE1 conformation and its local environment as a function of substrate binding. These studies will ultimately help elucidate BACE1 structure function relationships and the role of dimerization on its in vivo activity.

 

 

Friday, April 24, 2015; 3:00 p.m.; Chem 200 (Kobilka Lecture Hall)

 

Department of Chemistry & Biochemistry Welcomes:

Rochelle Warner

Master Student, Department of Chemistry & Biochemistry, University of Minnesota Duluth,

Research Advisor – Dr. Erin Sheets

Presents:

Molecular and Mechanical Manipulation of Membrane Domains in Planar Supported Bilayers”

 

Biomembranes are dynamic, two-dimensional fluids that actively participate in biological functions such as signaling, membrane trafficking, endocytosis and exocytosis. They are composed of thousands of lipid species and hundreds of proteins in cells, and the membrane itself is constantly remodeling. Nano-­-membrane domains are hypothesized to play an integral role in many cell-signaling pathways. Their transient nature and biocomplexity underlies a myriad of fundamental questions about lipid-­-lipid and lipid-­-protein interactions and their roles in cellular functions. As a result, there is a need for innovative approaches for understanding different biophysical aspects of membrane assemblies and their underlying, multiscale dynamics. 
 
Membranes in living cells are very complex and highly dynamic making it difficult to manipulate crosslinking at the molecular level. We propose to overcome this issue by using light (optical trapping) to crosslink proto-rafts in a well controlled, yet non-invasive manner and quantitative fluorescence microscopy to follow the subsequent dynamics. To simplify the investigations of specific molecular interactions and their dynamics, we use biomimetic, or model, membranes that are chemically well defined; that is, they are composed of only a few molecular species. The goal is to integrate dynamic holographic optical trapping (HOT) and fluorescence imaging with fluorescence correlation spectroscopy (FCS) to characterize membrane domain nucleation in biomimetic planar supported bilayers. Our hypothesis is that by trapping multiple microsphere-­-bound receptors, the associated heterogeneous lipid domains will nucleate a larger domain upon interaction in a manner that depends on the lipid type, cholesterol and protein content. To this end, I have pursued two parallel paths: preparing planar supported bilayers that contain the ganglioside GM1 and covalently conjugating microspheres with cholera toxin B (CTxB), which specifically binds to GM1. The CTB-microsphere acts as a “handle” for the optical trap and will allow proto-rafts to interact with one another. Fluorescence imaging is used to visualize membrane integrity, and subsequent lateral diffusion of lipid species is measured with FCS as a function of trap-­-induced confinement. These results will ultimately lead to new insights into domain formation in membranes.