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downregulate the formation and release of new virus (
Hoenen et al., 2010
). N&B
analysis demonstrated that although oligomers of VP40 were detected on the plasma
membrane, the majority of plasma membrane associated VP40 was monomeric
(see
Figs. 19.1 and 19.2
)(
Adu-Gyamfi et al., 2012
). This was not particularly sur-
prising as the monomers may continuously be recruited from the cytosol to serve as
building blocks for self-multimerization into larger structures required for membrane
bending and plasma membrane egress. We also observed that oligomers (hexamers,
octamers, and larger oligomers) were found enriched at the tips of the cells in fila-
mentous protruding structures (see
Fig. 19.2
)(
Adu-Gyamfi et al., 2012, 2013
).
Filamentous protrusion sites are thought to be the sites of egress for Ebola and other
filamentous viruses such as Marburg. N&B analysis of an oligomerization-deficient
mutation supported this notion as when oligomerization was reduced, detectable
membrane protrusion sites were no longer observed (
Adu-Gyamfi et al., 2012, 2013
).
N&B analysis can also be applied to peripheral protein interactions
in vitro
.
Cho and colleagues have used giant unilamellar vesicles (GUVs) to investigate
the oligomerization of BAR and ENTH domains (
Yoon et al., 2010, 2012
) on the
surface of membranes. Here, they were able to observe large oligomeric complexes
that are required to induce membrane tubulation from GUVs (see
Fig. 19.3
).
Multiple fluorophores also can be used in live cells and provide a robust oppor-
tunity to dissect molecular complexes with the RICS and N&B methods (
Digman
et al., 2009, 2013
), which would be a boon to monitoring protein-protein interactions
or oligomerization induced by lipid membranes. This method has already been ap-
plied to resolve the interactions between endophilin and dynamin in live cells
(
Digman et al., 2013
).
In closing, raster scanning allows for stacks of images to be generated to study
assembly of fluorescently labeled proteins or observation and stoichiometry of
complexes to be determined when two or more fluorescently labeled molecules
are used. Because fluorescent fluctuation analysis is performed among pixels and
frames in a long scan, data on particle diffusion, number of fluorescent molecules
in a pixel, and their brightness can be determined. Perhaps most notably, these types
of data can be collected on commercial laser-scanningmicroscopes sans modification
of the hardware or software. The growing availability of these commercial
FIGURE 19.3—Cont'd (
20-mer) was superimposed onto the image of GUV.
(F) A representative image of POPC/POPE/POPS/PtdIns(4,5)P
2
/Rh-PE GUV after a 10-min
treatment with epsin 1 ENTH K23E/E42K. (G) Distribution of ENTH K23E/E42K (monomer
to 20-mer) was superimposed onto the image of GUV. (H) Distribution of ENTH K23E/
E42K aggregates (
>
20-mer) was superimposed onto the image of GUV. All measurements
were performed at 37
C using POPC/POPE/POPS/PtdIns(4,5)P
2
/Rh-PE
(46.5:30:20:3:0.5) GUV in 20 mm Tris-HCl buffer, pH 7.4, with 0.16 m KCl solution. Protein
concentration was 0.5
>
m.
This research was originally published in
Yoon et al. (2010)
.
#
2010 The American
Society for Biochemistry and Molecular Biology.
m
m. White bars, 10
m
