Figure 1.
EAST can associate with Polytene Chromosomes.
(A) Live intact salivary glands expressing EAST-GFP (green) were exposed to photobleaching. A rapid recovery (see Figure 3E) indicates a high mobility in the nucleoplasm. (B-D) Larval salivary glands expressing EAST-GFP (green) were fixed and counterstained with TOPRO3 (white) to visualize DNA. (B) In untreated cells, EAST-GFP is localized to extrachromosomal nuclear regions. (C) In larval salivary permeabilized with a saponin containing detergent buffer, EAST-GFP rapidly relocates to the polytene chromosomes. (D) In detergent treated and squashed preparations of polytene chromosomes, EAST-GFP shows a high degree of co-localization with DNA bands. The chromocenter is indicated (arrow head). EAST-GFP represents full length EAST with a C-terminal eGFP tag that was expressed by the GAL4 system using the smid-Gal4 driver. Bars correspond to 10 µm in all panels.
Figure 2.
EAST-GFP accumulates in chromosome regions of low transcriptional activity.
(A) EAST-GFP (green) is excluded from highly transcribed chromosome regions (arrow) in detergent treated larval salivary glands cells. Run-on transcription was visualized using BrUTP (red). TOPRO3 (white) was used to label DNA. (B) In easthop7 mutants, like in wildtype, regions of high BrUTP incorporation (arrow) are found in interbands. Note that the banding pattern shows less contrast compared to EAST-GFP overexpressing or wildtype (not shown) cells. Bars correspond to 10 µm in all panels.
Figure 3.
Characterization of EAST-GFP localization and mobility in larval salivary glands.
(A) Varying the salt concentration can modulate localization of EAST-GFP. At 100 mM, the distribution is mostly chromosomal, at 150 mM chromosomal and nucleoplasmic and at 200 mM no chromosomal-like pattern is detectable. (B-D) The mobility of EAST-GFP was assessed by FRAP. (B, C) Increasing the salt concentration lowers the affinity to chromatin. An increase in salt from 50 mM (B) to 100 mM (C) leads to a faster recovery of fluorescence after bleaching chromosome regions bound by EAST-GFP. (D) Removing the C-terminal residues 1535-2301 of EAST leads to an increase in mobility. At a salt concentration of 50 mM, the truncated version of EAST associates with a lower affinity than the full-length version. (E) The diagram shows the recovery in seconds after bleaching the indicated nuclear regions for 4 seconds at a laser intensity of 100%. The two different variants of EAST-GFP were expressed using the ftz-GAL4 driver. Cells were permeabilized in buffers containing 50 mM NaCl supplemented with varying amounts of KCl to reach the indicated salt concentrations. The recovery of the non detergent treated cell (non-perm) in Figure 1A is indicated for comparison. The Bar in A represents 10 µm and applies to all panels.
Figure 4.
east corresponds to suppressor of white-spotted.
All flies shown are homozygous for wsp1. (A) In male pharates dissected out of pupal cases, the mutations easthop7 and su(wsp)1 suppress the loss of eye pigmentation resulting from the white mutation wsp1. (B) The mutations easthop7 and su(wsp)1 show complementation. Like su(wsp)1/su(wsp)1, the combination easthop7/su(wsp)1 restores eye pigmentation to almost wildtype levels. Heterozygous flies of each mutation display intermediate eye pigmentation.
Figure 5.
Change of localization of EAST-GFP during apoptosis.
(A) In a 13 h APF old pupa, EAST-GFP (green) shows mainly extrachromosomal localization. The nuclear lamina (red) is still intact. DNA (blue) was labeled with TOPRO-3. (B) Co-localization of EAST-GFP with intact polytene chromosomes can be observed in a 19 hour APF old pupa. The destruction of the lamina indicates that part of the apoptotic program is executed. Note the barely detectable anti-lamin staining in salivary glands compared to neighboring diploid cells (arrow). The Bar in A represents 10 µm and also applies to B.
Figure 6.
EAST might act as a salt sensor that modulates gene expression in response to changing ion (Na+/K+) concentrations.
A change in EAST dosage may lead to the sensing of incorrect ion levels, which may result in inappropriate physiological responses.