As reported previously (Okuzaki et al., 2003) and shown in Fig. 3a, in the WT strain Meu14p was observed as four rings of different diameters that were situated in the vicinity of the nuclei, but apart from them. In the exo70Δ asci, the four Meu14p rings seemed to be attached to the nuclei
(Fig. 3b), suggesting that the LEP complex could not develop properly. 3MA It has been described that in meu14Δ mutants, in which the FSM do not develop properly, the SPBs seem to be fragmented (Okuzaki et al., 2003). We wished to know whether the same phenomenon was observed in the absence of Exo70p. To do so, asci carrying a Sad1-GFP protein were analyzed under the microscope. We observed that in the mutant strain, 34% of the asci exhibited multiple Sad1-GFP fluorescent dots (Fig. 3c), while this value was 11% for the WT strain. This result suggested that the SPBs are unstable in the exo70Δ mutant. Finally, we analyzed the distribution of the α-glucan synthase homologues Mok12p and Mok13p, which are required for the synthesis of the spore cell wall. Mok13p is expressed earlier than Mok12p (Garcia et al., 2006). As reported previously, in the WT strain, Mok13p localized to the FSM, forming cup-shaped structures and sacs around the nuclei (Garcia et al., 2006). The same result was obtained for the sec8-1 mutant (not shown). In the Proteases inhibitor exo70Δ mutant,
Mok13p formed amorphous structures or small sacs, like those formed by Psy1p, which did not surround the nuclei (not shown). This result was in agreement with an inability of the exo70Δ mutant to develop the FSM properly. The α-glucan synthase Mok12p localizes at the surface of the developing spores Resminostat (Garcia et al., 2006). Because the spore cell wall is not permeable to Hoechst, we analyzed the localization of the Mok12-GFP protein with respect to the spore surface photographed under a phase-contrast microscope. In the control strain, Mok12p was observed at the spore periphery (Fig. 4; WT). In the sec8-1 mutant, the distribution of this protein was heterogeneous; in those asci that had refringent spores, Mok12p localized at the spore surface (Fig. 4; sec8-1), while in those asci that exhibited immature spores, Mok12p
could not be observed. In the exo70Δ mutant, the signal corresponding to Mok12p was hardly observed in the asci interior (Fig. 4; exo70Δ). These results suggest that both exocyst subunits participate in the maturation of the spore cell wall. All the results described above confirmed that the exocyst was required for mating in S. pombe and that different steps of this process are differentially regulated by these exocyst subunits. In order to know whether the different requirements of Sec8p and Exo70p for agglutination and sporulation were a consequence of a different distribution of these proteins, cells carrying a GFP-tagged Sec8p and an RFP-tagged Exo70p were induced to mate in liquid medium and were observed under the microscope. As shown in Fig.