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doi: 10.1242/10.1242/dev.00303

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The Dictyostelium prestalk cell inducer DIF regulates nuclear accumulation of a STAT protein by controlling its rate of export from the nucleus

Masashi Fukuzawa^*, Tomoaki Abe^* and Jeffrey G. Williams

School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK

View larger version (19K): [in a new window]   Fig. 1. Photobleaching analysis of the efflux of GFP:STATc from the nucleus. (A) Principle of the photobleaching method. Isolated cells transformed with GFP:STATc, a blasticidin-based, `single copy' vector (Fukuzawa et al., 2001) in which there was a low to moderate fluorescence signal in the nucleus, were photobleached to reduce only the cytoplasmic fluorescence (the nucleus was masked) and then incubated in either the presence or the absence of 100 nM DIF for 5 minutes. Many of the cells in clumps, and a sub-set (approx. 10%) of the spatially separated cells, show a very high intrinsic level of Dd-STATc nuclear fluorescence. There is a DIF-independent, stress-induced mechanism that directs nuclear accumulation of Dd-STATc (T. Araki, M. Tsujioka, T. A. and J. G. W., unpublished data) and this may account for these `high background' cells. In such cells, nuclear fluorescence is retained irrespective of the presence or absence of DIF and they were not therefore included in the analysis. (B,C) Analysis of nuclear efflux rates of (B) GFP:STATc and (C) GFP:STATa in the presence and absence of DIF. Cells transformed with GFP:STATc or GFP:STATa were photobleached as described in A. The fluorescence signal from the cytoplasm and nucleus was monitored over 5 minutes. In order to maintain constant conditions, the `contrast-stretch' function of the microscope was turned off. In each experiment a total of at least 30 cells was analysed and the graph shows the ratio (nuclear signal — background)/(cytoplasmic signal — background) x 100 (%) ± s.d.

View larger version (28K): [in a new window]   Fig. 2. Identification of NESs within Dd-STATc. (A) Alignment of potential NES sequences. Dd-STATc (middle) contains, near its centre, a leucine/isoleucine rich region (red letters) (the EXP region). A part of Dd-STATa (top) that contains the 50 a.a. region homologous to EXP has been shown to function as an LMB-sensitive NES (see text) and is displayed here for comparison. Identities between Dd-STATa and Dd-STATc are shown in orange. The NES of rad24 is shown (bottom) for comparison, aligned below the leucine rich region near the C terminus of EXP (marked as B). Another candidate NES, i.e. a leucine-rich region, nearer the N terminus is also marked as A. (B) Nuclear accumulation of a Dd-STATc mutant protein lacking EXP. The GFP:STATc construct comprises the entire Dd-STATc protein, with GFP fused at its N terminus. It is a blasticidin-based, `single copy' vector (Fukuzawa et al., 2001). GFP:STATcEXP is an equivalent construct with an internal deletion (residues 505 to 554) that removes just the EXP region. Both these constructs were introduced into cells and stable transformant clones were selected. In order to rule out homologous gene conversion, a frequent event with Dd-STATc, the structure of the two GFP fusion proteins was checked by western blotting and were as expected. Cells transformed with these constructs were developed in shaken suspension for 4 hours and then analysed for nuclear GFP after a further 5 minutes of incubation in either the presence or absence of 100 nM DIF.

View larger version (15K): [in a new window]   Fig. 3. Analysis of nuclear efflux rates of GFP:STATcEXP in the presence and absence of DIF. Cells transformed with GFP:STATcEXP (Fig. 2B) were photobleached exactly as described in Fig. 1A,B.

View larger version (58K): [in a new window]   Fig. 4. Demonstration that EXP is an LMB-sensitive NES. The EXP region (residues 505-554) of Dd-STATc was inserted at the N terminus of GFP, to yield EXP:GFP, under transcriptional control of the Actin 15 promoter. This construct contains a G418 resistance cassette and was transformed into Dictyostelium. Clones of expressing cells containing multiple copies of EXP:GFP were selected using G418. GFP diffuses freely into the nucleus but in EXP:GFP-transformed cells, here shaken for 3 hours under starvation conditions, the fusion protein is selectively excluded from the nucleus (the positions of the nuclei are indicated by yellow arrows). When, however, cells are incubated for 90 minutes with LMB (20 nM) exclusion is reversed and there is a uniform GFP signal over the cell or, occasionally, even a slight nuclear enrichment [we have previously shown that there is sometimes a low level of nuclear enrichment of GFP itself when it is expressed in Dictyostelium (Ginger et al., 2000)].

View larger version (40K): [in a new window]   Fig. 5. Analysis of the N-terminal-proximal region of Dd-STATc. Identification of nuclear import signals in Dd-STATc. The approximate N-terminal half (residues 1-504) of Dd-STATc was fused upstream of GFP, to produce GFP:STATc1-504 and introduced into Dictyostelium cells. Stably expressing clones were selected and western transfer analysis confirmed that the fusion gene produced a fusion protein of the expected size. The cells were then analysed as in Fig. 4. (B) Analysis of the effect of the EXP region on the N-terminal-proximal import region. Construct GFP:STATc1-554 differs from construct GFP:STATc1-504 only in that it contains the EXP region. GFP:STATc1-554 was transformed into cells and they were analysed as described in Fig. 4. The panel at the right shows cells treated with LMB for 90 minutes, resulting in nuclear accumulation.

View larger version (28K): [in a new window]   Fig. 6. Mapping nuclear import signals in Dd-STATc. (A) Analysis of an N to C deletion series of Dd-STATc. A set of constructs, bearing the indicated N to C deletions, was constructed by PCR. The indicated regions were amplified and cloned immediately downstream of the Actin 15 promoter:GFP fusion present in GFP:STATc replacing the original Dd-STATc sequences (Fukuzawa et al., 2001). The constructs were transformed into cells and analysed for DIF inducibility as in Fig. 2B. (B) Identification of a nuclear import signal at the N terminus of Dd-STATc. Construct IMP:GFP contains the N-terminal-proximal 46 residues of Dd-STATc fused upstream of GFP. This construct contains a G418 resistance cassette and was transformed into Dictyostelium. Clones of expressing cells containing multiple copies of IMP:GFP were selected using G418. GFP accumulation in the nucleus was monitored in growing cells (left) and, in parallel, with an identical construct expressing GFP alone (right).

View larger version (13K): [in a new window]   Fig. 7. A model for DIF-induced nuclear accumulation of Dd-STATc. The Dd-STATc protein is proposed to be predominantly cytosolic in the absence of DIF, because the NESs in the EXP region predominate over the NLSs present within the IMP region (illustrated by the number of crosses). The model proposes a conformational change when Dd-STATc monomers dimerise, in reponse to DIF treatment, that masks the activity of EXP. The increased relative activity of the nuclear import signal(s) in the IMP regions then causes Dd-STATc to accumulate within the nucleus.