Biomedical Engineering Reference
In-Depth Information
Fig. 3
Colocalization of proteins on dynamically moving endosomes. (
a
) Dual-color acquisition with msALEX of
hypha expressing RrmC and Pab1G. Kymographs were generated from a movie that was acquired with a 63×
objective and 80 ms exposure time per fl uorophore. (
b
) Dual-color acquisition with local photobleaching fol-
lowed by msALEX of hypha expressing Rrm4G and Rab5aC. Kymographs were generated from a movie that
was acquired with a 100× objective, 80 ms exposure time per fl uorophore, and camera chip binning 2; prior
to acquisition, the indicated area was photobleached
5. Adjust laser intensity (
see
Note 15
).
6. Convert resulting movies into kymographs (Fig.
3
;
see
Note 8
).
7. Analyze kymographs for protein colocalization (
see
Note 16)
.
3.4 RNA Live
Imaging
1. Generate the
N
*
-Gfp
2
fusion construct (Fig.
4a, b
;
see
Note 17
).
2. Insert
boxB
hairpins into the 3
ʻ
′
UTR of your gene of interest
(Fig.
4a, b
;
see
Note 18
).
3. Transform both constructs successively into
U. maydis
. Verify
correct strain generation by Southern blot analysis.
4. Inoculate 3 ml of CM-glc (1 % f.c.) in a glass reaction tube for
20-24 h at 28 °C on an incubation wheel.
5. Dilute cell culture 1:2,500 in 30 ml of CM-ara (1 % f.c.;
see
Notes 3
and
19
), and regrow in a baffl ed fl ask for 18 h at
200 rpm and 28 °C.
