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This Week's Colloquium: Don Lamb, Mar. 8, 2012

Don Lamb
Ludwig-Maximilians-Universität München

Thursday, Mar. 8, 2012
102 JFB

Refreshments: 3:30 pm in 219 JFB
Lecture 4:00pm (102 JFB)

Title: Physics in Biology: Advanced Fluorescence Methodologies for Investigating Viruses & Single Proteins

Abstract:

Developments in fluorescence techniques have lead beyond the mere detection of single particles and molecules to studying their individual behavior. In my talk, I will describe various single molecule methods we have developed and how we apply them to investigate the function of viruses and proteins.

In the first example, single particle tracking was used to investigate the assembly and release of HIV particles [1]. The main structural protein of HIV (GAG) was fluorescently labeled with the green fluorescent protein (GFP). The kinetics show three phases. The first phase, due to recruitment of Gag to the assembly site, is surprisingly fast, on the order of 200 s. After a second phase with roughly constant fluorescence intensity, a decrease in fluorescence intensity marks phase III. Occasionally, the start of phase III coincides with change in mobility of the budding sites or disappearance of the virus, signifying virus release and occurs on the timescale of ~1500 +/- 700 s.

Upon characterizing the assembly process of HIV, we have analyzed the interaction of cellular proteins with HIV [2]. For virus release, the virus hijacks the ESCRT machinery that is used by cells for vesicle formation. For these studies, the ESCRT protein, VPS4, was labeled with GFP. We observed short fluorescence bursts from GFP-labeled VPS4 molecules that assembled into complexes at HIV assembly sites. The bursts where observed before viral release and occurred mainly during phase II. Using image correlation spectroscopy, we could show that multiple dodecamers are present during bursts at the individual viral budding sites.

To investigate the fusion process necessary for virus entry, we developed a new method that combines single particle TRacking with Image Correlation spectroscopy (TRIC). Using TRIC, we discovered an intermediate stage during the fusion process that has never been observed before where the virus envelope and capsid are separated but still coupled together.

In the last example, I will switch to methods that have been developed to improve the accuracy of Förster Resonance Energy Transfer (FRET) experiments on single molecules. We have combined pulsed interleaved excitation (PIE) [3], a method developed in my laboratory, with multiparameter fluorescence detection (MFD) [4] to allow accurate single pair FRET experiments. The advantage of MFD is that it utilizes all available information from the fluorescence such as fluorescence intensity, wavelength, lifetime and polarization to distinguish multiple subspecies in a burst analysis experiment. By combining MFD with PIE, we can also gather stoichiometry information on the labeling of the sample. With the information available in the MFD-PIE experiment, all calibration factors and data necessary for performing an accurate spFRET experiment can be collected in a single measurement. In addition, the information available in a PIE-MFD measurement can also be used to perform additional checks to ensure good alignment of the detection volumes, remove bursts where the acceptor has photobleached and distinguish multiple fluorescence states of the acceptor.

[1] Ivanchenko, S., Godinez, W.J., Lampe, M., Kräusslich, H.-G., Eils, R., Rohr, K., Bräuchle, C., Müller, B., and Lamb, D.C. (2009). PLoS Pathogens 5, e1000652.
[2] Baumgärtel, V., Ivanchenko, S., Dupont, A., Sergeev, M., Wiseman, P.W., Kräusslich, H.-G., Bräuchle, C., Müller, B., and Lamb, D.C. (2011). Nature Cell Biology 13, 469-474.
[3] Muller, B. K., Zaychikov, E., Brauchle, C., and Lamb, D. C. (2005) Biophys J 89, 3508-3522
[4] Widengren, J., Kudryavtsev, V., Antonik, M., Berger, S., Gerken, M., Seidel, C.A.M. (2006) Anal. Chem. 78, 2039-2050

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