Biology Reference
In-Depth Information
of membrane curvature changes (
Zimmerberg & Kozlov, 2006
), membrane scission
(
Klein, Lee, Frank, Marks, & Lemmon, 1998; Ross et al., 2011
), and assembly and
egress of viral proteins from the host cell (
Adu-Gyamfi, Digman, Gratton, &
Stahelin, 2012; Hoenen, Jung, Herwig, Groseth, & Becker, 2010; Murray et al.,
2005
). Because high-affinity interactions between these peripheral proteins regulate
lipid signaling and trafficking events from cellular membranes, accurately character-
izing lipid specificity and membrane affinity can establish how the membrane-
interface signals throughout the cell. Technologies aimed at measuring the assembly
and stoichiometry of lipid-protein and protein-protein interactions on or near the
membrane interface are important for revealing molecular details of the underlying
cellular architecture.
The biophysical approaches discussed earlier have been applied to investigate the
oligomerization state of the fission protein dynamin (
Ross et al., 2011
) as well as
oligomerization of the lipid-binding ENTH (
Yoon et al., 2010
) and BAR (
Yoon,
Zhang, & Cho, 2012
) domains involved in membrane curvature generation and sens-
ing. Additionally, they have been useful in yielding mechanistic information of as-
sembly and oligomerization of Annexin A4 (
Crosby et al., 2013
), oligomerization of
the
voltage-dependent anion channel in response to phosphatidylglycerol binding
(
Betaneli, Petrov, & Schwille, 2012
) as well as oligomerization, assembly, and
egress of the Ebola virus matrix protein (
Adu-Gyamfi et al., 2012, 2013; Soni,
Adu-Gyamfi, Yong, Jee, & Stahelin, 2013
). In this chapter, I will describe how
RICS and N&B can be used to study assembly of peripheral proteins on biological
membranes.
19.1
MATERIALS AND METHODS
19.1.1
Cell maintenance, transfection, and observation
Human embryonic kidney (HEK293) and Chinese hamster ovary-K1 (CHO-K1) cells
were used to study the assembly and oligomerization of the Ebola virus matrix protein
VP40 (
Adu-Gyamfi et al., 2012, 2013
). HEK293 cells were cultured and maintained
at 37
C in a 5% CO
2
humidified incubator supplemented with Dulbecco's Modified
Eagle Medium (DMEM) (low glucose) containing 10% FBS and 1% Pen/Strep. After
trypsinization, cells were transferred from a T-25 tissue culture flask to an 8-well plate
used for imaging. Cells were then grown to 50-80% confluency and transfected with
1
g DNA/dish using Lipofectamine 2000 according to the manufacturer's protocol.
Cells were imaged between 12 and 20 h posttransfection.
CHO-K1 cells were cultured and maintained at 37
C in a 5% CO
2
humidified
incubator supplemented with DMEM/F12 (low glucose) containing 10% FBS and
1% Pen/Strep. After trypsinization, cells were transferred from a T-25 tissue culture
flask to an 8-well plate used for imaging. Cells were then grown to 50-80% con-
fluency and transfected with 1
m
g DNA/dish using Lipofectamine LTX according
to the manufacturer's protocol. Both HEK293 and CHOK-1 cells were imaged using
a Zeiss LSM 710 confocal microscope using a Plan-Apochromat 63x 1.4 NA oil
m
