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
