Biophysical Studies

 

Back to Main Research page

 

 

Sarah Veatch

Probing membrane heterogeneity in cell membranes
 

I am interested in understanding how the physical chemistry of lipids and lipid mixtures influences biological functions at the plasma membrane. I am currently investigating this topic using complimentary approaches: through simulations, and experiments in model membranes, plasma membrane vesicles, and intact cells.

Model membranes: My PhD and early postdoctoral work was aimed at understanding the thermodynamics of the miscibility transition in ‘simple’ model membranes. We recently characterized critical fluctuations in model membranes (1).
Giant plasma membrane vesicles (GPMVs): GPMVs are shed from the plasma membranes of living RBL cells after a mild chemical treatment. These vesicles phase separate at low temperature, and exhibit critical fluctuations near room temperature (~22°C; Figure A). Based on these experiments, we predict <50nm sized fluctuations in cell membranes at physiological temperatures (37°C) (2).
Simulations: Since cell plasma membranes appear to have critical compositions, it may be possible to capture important aspects of cell plasma membrane organization through fairly simple 2D Ising model Monte Carlo Simulations (Figure B). I am working with members of the Sethna group in Cornell’s physics department to investigate the impact of receptor clustering and the actin cytoskeleton on membrane heterogeneity.
Intact cells: We are testing the hypothesis generated from our plasma membrane vesicle experiments and Monte Carlo simulations by imaging cell surface protein distributions using experimental techniques sensitive to nanometer-scale ordering. I am currently working with Ethan Chiang to understand results of scanning electron microscopy experiments (Figure C), and with Ethan Chiang and Amit Singhai on implementing a sub-optical resolution fluorescence imaging in the Baird/Holowka Laboratory.

(above) (A). Micron-sized and dynamic critical fluctuations are found at the surface of a giant plasma membrane vesicle (GPMV) at multiple temperatures near the critical temperature. Figure reproduced from (2). Vesicles are roughly 10 micrometers in diameter. (B) Monte Carlo simulation of blue ‘lipids’ clustering around fixed white ‘proteins,’ mimicking what may occur after cross-linking of FceRI with multivalent antigen. (C) Reconstructed SEM micrograph demonstrating that two different proteins co-cluster on the surface of stimulated RBL mast cells (image courtesy of Ethan Chiang). Protein clusters are roughly 200nm.
1. A. R. Honerkamp-Smith, P. Cicuta, M. Collins, S. L. Veatch, M. Schick, M. den Nijs, and S. L. Keller, Line tensions, correlation lengths, and critical exponents in lipid membranes near critical points. Biophys. J. 95(1) 236-46 (2008).
2. S. L. Veatch, P. Sengupta, A. Honerkamp-Smith, D. Holowka, B. Baird. Critical Fluctuations in Plasma Membrane Vesicles. ACS Chem. Bio. 3(5):287-93 (2008).
 

 

 

Sarah Veatch and Amit Singhai

Super-resolution fluorescent imaging of the cell surface
 

Several groups have recently developed and characterized a novel microscopy technique capable of obtaining electron-microscopy resolution images using fluorescently labeled samples (1-3). This technique, called STORM (1) or PALM (2,3), utilizes photo-activated fluorescent probes and single molecule measurements to break the optical resolution limit. We are currently implementing a PALM/STORM microscope in the Baird/Holowka research group, and will use it to visualize IgE-FceRI and its signaling partners at the plasma membrane with nanometer-scale lateral resolution. We also hope to use this technique to characterize <50nm-size heterogeneities in resting cell membranes (often referred to as ‘lipid rafts.’)

(above) Unstimulated RBL cell surface as imaged using total internal reflection fluorescence (TIRF) microscopy (Figure A) or STORM (Figure B). IgE-FceRI complexes are secondary antibody labeled with a Cy3-Cy5 switch and are imaged using TIRF microscopy. The STORM image is reconstructed from 1000 individual images using automatic image processing. In both images, puncta are present due to insufficient labeling of the cell surface, and not due to nano-scale structure. Nonetheless, this preliminary example demonstrates the power of the PALM/STORM technique. Neighboring fluorophores that cannot be distinguished in using standard epi-fluorescence microscopy (inset; Figure A) are clearly resolved in the reconstructed STORM image (inset; Figure A). The inset a magnified reproduction of the white square and is roughly 1mm by 1mm.
1. Rust, M. J., M. Bates, et al. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10): 793-5.
2. Betzig, E., G. H. Patterson, et al. (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793): 1642-5.
3. Hess, S. T., T. P. Girirajan, et al. (2006). Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 91(11): 4258-72.
Home · Research Interests · People

Publications · Links