- Poly-D-lysine solution (0.1 mg/ml).
- Milli-Q water.
- Coverslip holder: serves as a chamber to hold cover slips for cell
imaging (for details,see[16]).
2.2 Fluorescence
Microscopy Imaging
and Analysis
- Physiological stimulant (such as 2-deoxyglucose (2-DG)):
1000 stock solution should be prepared according to manu-
facturer’s instructions (seeNote 3). - Imaging medium: phenol red-free DMEM with 25 mM
HEPES, pH 7.4 under 5–10% CO 2 environment (seeNotes
4–6). - Fluorescence microscopy: In our laboratory, live cell measure-
ments are performed using an epifluorescence microscope.
CFP and YFP excitation are carried out by an X-Cite Series
120Q mercury-vapor lamp and processed through appropriate
filter cubes. Images are taken using a 63objective (Plan-
Apochromat, NA¼1.4) mounted on an inverted Axiovert
135 TV microscope and are captured by a QIClick charge-
coupled device camera. The microscope is operated by the
MetaMorph software package. CFP and YFP channels are con-
figured to use the respective excitation and emission filter
(427/10 nm excitation and 472/30 nm emission for CFP,
504/12 nm excitation and 542/27 nm emission for YFP),
while the FRET channel is configured to use CFP as excitation
and YFP as emission. - Image analysis software (e.g., MetaMorph imaging software)
(seeNote 7).
3 Methods
3.1 osABKAR Design
and Preparation
- Selecting the appropriate OTS is critical for proper osABKAR
function. OTSs that were used in previously developed osAB-
KARs are summarized in Fig.2 (seeNote 8). For targeting to
relatively small subcellular compartments (e.g., centrosome,
basal body of primary cilia, etc.), it is essential to identify the
shortest sequence that effectively targets the sensor to the
appropriate compartment. Furthermore, the compartment-
specific targeting sequence needs to be as short as possible
without degrading the FRET efficiency of ABKAR and is
tagged to either the N-terminal or the C-terminal end of
ABKAR (seeNote 9). The information on protein localization
sequences can be easily obtained online as it is now universally
available (e.g., The Human Protein Atlas, http://www.
proteinatlas.org/). - Prepare the plasmids according to standard subcloning proto-
cols (seeNote 10). Validate all plasmids by sequencing.
262 Takafumi Miyamoto et al.