Articles by Joseph D. Unsay in JoVE
Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers Joseph D. Unsay1,2,3, Katia Cosentino1,2, Ana J. García-Sáez1,2 1Interfaculty Institute for Biochemistry, 2Max Planck Institute for Intelligent Systems, 3German Cancer Research Center We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.
Other articles by Joseph D. Unsay on PubMed
Cardiolipin Effects on Membrane Structure and Dynamics Langmuir : the ACS Journal of Surfaces and Colloids. Dec, 2013 | Pubmed ID: 23962277 Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.
Scanning Fluorescence Correlation Spectroscopy in Model Membrane Systems Methods in Molecular Biology (Clifton, N.J.). 2013 | Pubmed ID: 23996179 Fluorescence correlation spectroscopy (FCS) is an emerging technique employed in biophysical studies that exploits the temporal autocorrelation of fluorescence intensity fluctuations measured in a tiny volume (in the order of fL). The autocorrelation curve derived from the fluctuations can then be fitted with diffusion models to obtain parameters such as diffusion time and number of particles in the diffusion volume/area. Application of FCS to membranes allows studying membrane component dynamics, which includes mobility and interactions between the components. However, FCS encounters several difficulties like accurate positioning and stability of the setup when applied to membranes. Here, we describe the theoretical basis of point FCS as well as the scanning FCS (SFCS) approach, which is a practical way to address the challenges of FCS with membranes. We also list materials necessary for FCS experiments on two model membrane systems: (1) supported lipid bilayers and (2) giant unilamellar vesicles. Finally, we present simple protocols for the preparation of these model membrane systems, calibration of the microscope setup for FCS, and acquisition and analysis of point FCS and SFCS data so that diffusion coefficients and concentrations of fluorescent probes within lipid membranes can be calculated.
A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope The Journal of Biological Chemistry. Mar, 2015 | Pubmed ID: 25605719 Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission.