Figure 6.
ZBP1 Nuclear Transport and Accumulation at ß-actin Transcription
Sites
(A) Accumulation of endogenous ZBP1 at ß-actin transcription
sites in CEF nuclei. Serum was applied to chick embryo fibroblasts
for 5 min after synchronization by serum starvation. The cells were
immediately fixed and stained for ß-actin mRNA (red) by in situ
hybridizaton and ZBP1 (green) by immunofluorescence. ZBP1 and ß-actin
mRNA colocalize (yellow) at the transcription sites.
(B) Accumulation of ZBP1-GFP fusion at ß-actin transcription
sites. CEFs were transfected with ZBP1-GFP fusion DNA and were imaged
live after treatment as in (A). After identification and imaging of
transcription sites in live cells (see [E]), they were fixed and stained
by in situ hybridization for the ß-actin mRNA (red). The image
was subjected to deconvolution to yield the optical section shown.
(C) ZBP1-GFP fusion proteins move into the nucleus and bind to nascent
chains of ß-actin at the transcription site shortly after serum
induction. The transcription site in a ZBP1-GFP-transfected CEF was
photobleached, and fluorescence recovery was monitored by confocal
microscopy. Half-recovery occurred within 90 s. The panels shown are
at 30-s intervals. “Before,” before photobleaching; “0,”
after photobleaching for 20 s. See Movie 13
in the Supplementary Material.
(D) Time plot of ZBP1-GFP fluorescence recovery at a transcription
site. Relative intensity was calculated from raw imaging data and
was plotted as a fraction of intensity prior to bleaching. The time
in seconds after bleaching was complete is indicated on the horizontal
axis.
(E) The same cell as in (B) before fixation and in situ hybridization.
ZBP1 particle movement between the nuclear envelope and ß-actin
transcription site can be seen in live cells. The time between the
frames shown is 800 ms. See Movie 14 in the
Supplementary Material.
(F) Electron microscopy of ZBP1 at the nuclear pore (arrow). Sections
of cells treated in (A) were stained with anti-ZBP1 antibody and secondary
gold-conjugated antibody. N, nucleus; C, cytoplasm.
The scale bars in (A)–(C) represent 5 µm, the scale bar
in (E) represents 10 µm, and the scale bar in (F) represents
100 nm.
