FAJARDO, BATERÍAS Y CUARTEL DEFENSIVO DE

This strategy would allow us to find out which proteins were abnormally distributed in cells overex- pressing cfh3+ _{and, therefore, which alterations were}

associated with the multi-septation phenotype ob- served in the WT strain overexpressing this gene. In this way, physical interactions and functional relation- ship between the Cfh3p and other proteins involved in cell division could be envisaged. To undertake this approach, strains carrying GFP-tagged proteins (except for actin, see below) involved in different stages cytokinesis and overexpressing cfh3+ _{were ob-}

served under the fluorescence microscope. We ana- lyzed the distribution of CAR components (actin, the myosin light chains Cdc4p and Rlc1p, and Cdc15p), a protein that links the CAR to the plasma membrane (Chs2p), and proteins involved in cell separation (the septin Spn3p and the glucanases Agn1p and Eng1p). The results are detailed below.

3.1.1. Localization of actin in cells overexpressing

cfh3+

Actin takes part in the formation of the CAR and consequently in cytokinesis. Actin is necessary not only for the assembly of the ring, but also for the cor- rect positioning of many components of the CAR. Ad- ditionally, actin can be observed at the cell poles. In order to see the localization of actin in cells overex- pressing cfh3+_{, actin was stained with Rhodamine-}

Phalloidin and the cell wall was stained with Cal- cofluor. As shown in the figure 14, in the WT strain actin localized at the CAR and in the form of patches at the cell poles and equator. In the cells overexpress- ing cfh3+_{, actin patches accumulated all around the}

cell, and formed multiple rings in about 10% of the cells undergoing cytokinesis. This result shows that

cfh3+ _{overexpression produces alterations in the distri-}

bution of actin.

3.1.2. Localization of myosin components in cells overexpressing cfh3+

In S. pombe, type-II myosins are in a complex

of three proteins including a heavy chain (Myo2p or Myo3p), an essential light chain (Cdc4p), and a regu- latory chain (Rlc1p). These proteins are required for CAR assembly and maturation. We analyzed the local- ization of myosin components in cells overexpressing

cfh3+_{. Cdc4p, the essential light chain of the myosin,}

is associated to the heavy chains Myo2/Myo3 and lo- calizes early at the CAR. In the WT strain, and in agreement with previous results (McCollum et al., 1995), Cdc4p was only observed at the equator of the cells forming a ring that contracted until it formed a dot and disassembled after the septum was synthesized (figure 15). When cells overexpressed cfh3+_{, 60% of}

the cells (n=500) showed aberrant Cdc4p localizations,

Figure 14. cfh3+ _{overexpression disturbs actin localization.}

Calcofluor (left panels) and rhodamine-phalloidin (right panels) staining of wild-type cells or cells overexpressing cfh3+_.

which included protein that remained at the midzone after the septum had been synthesized (20% of the cells; see asterisk in figure 15), multiple rings (70% of the cells; dot in figure 15), and protein localized at the cell cortex (10% of the cases; arrows in figure 15). Similar results were obtained for Rlc1p (results not shown). In conclusion, cfh3+ _{overexpression produces}

alterations in the distribution of the type-II myosins at the CAR.

3.1.3. Localization of the Cdc15p in cells overex- pressing cfh3+

Cdc15p is one of the components of the CAR, and it is involved in the processes of polymerization and assembly of actin in this ring. Cdc15p can also be observed forming patches near the cellular poles dur- ing interphase, although it has been described that Cdc15p does not co-localize totally with the actin patches (Fankhauser et al., 1995). In the cells that are undergoing division, Cdc15p disappears from the poles and localizes at the medial region of the cell, forming a ring in the zone where the nucleus is placed. The ring, then, contracts towards the cellular interior and, finally, disassembles. After the disassembling of

the Cdc15p ring, the protein is observed in the form of patches in the zone of the old pole of the cells (Carnahan & Gould, 2003). When cfh3+ _{was overex-}

pressed, an aberrant distribution of this protein was observed in 80% of the cells (n= 500), including accu- mulation of the protein at the cell cortex as dots or thread-like structures (50% of the cells; see arrows in figure 16), multiple rings (20% of the cells, see the dot in figure 16) or asymmetric rings (30% of the cells; see the asterisk in figure 16). The results showed that

cfh3+ _{overexpression produces alterations in the distri-}

bution of Cdc15p.

3.1.4. Localization of the Chs2p in cell overexpress- ing cfh3+

Chs2p is a transmembrane protein that associ- ates with the CAR (Matsuo et al., 2004, Martin-Garcia & Valdivieso, 2006). Chs2p localizes at the CAR and is required for the stability of the ring. However, when cells overexpressed cfh3+ _{it was possible to observe}

septating cells with no Chs2p signal (20% of the cells, n=500; see arrows in figure 17) or cells in which the signal at the ring was asymmetric (35% of the cells, n= 500; asterisk in figure 17). Therefore, cfh3+ _overex-

pression produces alterations in the distribution of Chs2p.

3.1.5. Localization of septins in cells overexpressing

cfh3+

Septins are a family of proteins that bind GTP and are conserved from yeasts to animal cells. They are involved in cellular separation, polarity, and secre- tion and membrane deposition. In S. pombe, septins

Figure 15. cfh3+ _{overexpression disturbs myosins at the}

CAR. Hoechst (left panels) and GFP (right panels) fluorescence of wild-type cells or cells overexpressing cfh3+_{. The asterisk}

marks a cell in which Cdc4p remains after the septum is com- plete, the dot marks a cell with two rings and the arrows mark cells in which Cdc4p accumulates at the cell cortex.

Figure 16. cfh3+ _{overexpression disturbs the distribution of}

Cdc15p. Hoechst (left panels) and GFP (right panels) fluores- cence of wild-type cells or cells overexpressing cfh3+_{. The ar-}

rows mark cells in which Cdc15p accumulates at the cell cortex, the dot marks a cell with two rings and the asterisk marks an asymmetric ring.

Figure 17. cfh3+ _{overexpression disturbs the distribution of}

Chs2p. Hoechst (left panels) and GFP (rigt panels) fluorescence of wild-type cells or cells overexpressing cfh3+_{. The arrows}

mark cells in which Chs2p is not observed in septating cells and the asterisk marks an asymmetric ring.

are involved in the localization of glucanases and therefore in cell separation. Septins localize as a ring that does not contract during cytokinesis at the mid- zone of WT cells (An et al., 2004). In cells overex- pressing cfh3+_{, 80% of the cells (n=500) carrying a}

GFP-tagged Spn3p showed fluorescent patches at the cell periphery or in the cytoplasm (65% of the cells; arrows in figure 18) or a signal that was at the leading edge of the growing septum (15% of the cases; lower panels in figure 18). The results showed that cfh3+

overexpression produces alterations in the distribution of the Spn3p.

3.1.6. Localization of glucanases in cell overex- pressing cfh3+

Agn1p and Eng1p glucanases are enzymes lo- cated in the zone of division and play a role in cell separation (Alonso-Nunez et al., 2005, Martin- Cuadrado et al., 2005). We wanted to know whether overexpression of cfh3+ _{had any effect in the localiza-}

tion of glucanases. To do this, we observed cells bear- ing GFP-fused Agn1p or Eng1p that overexpressed

cfh3+_{. We found that 70% of the cells (n=500) show-}

ing at least one septum did not exhibit the Agn1-GFP or the Eng1-GFP signal in some of them (figure 19 and results not shown). These results indicated that

cfh3+ _{overexpression produces alterations in the distri-}

bution of the glucanases.

3.1.7. Localization of Bgs1p in cell overexpressing

cfh3+

Bgs1p is a β-glucan synthase that is required for the synthesis of linear β-glucan. Bgs1p is a trans- membrane protein and in the WT strain localizes at the septum and the cell poles (Cortes, 2002). We wanted to know whether overexpression of cfh3+ _{had any ef-}

fect in the localization of the Bgs1p. Cells bearing a GFP-fused Bgs1 protein and overexpressing cfh3+

showed an aberrant localization of Bgs1p. In those cells showing a mild overexpression phenotype, Bgs1p localized in the poles but extended to the lateral mem- brane. In those cells showing a strong phenotype, Bgs1p localized at the membrane all around the cells (figure 20). In conclusion, cfh3+ _{overexpression pro-}

duces alterations in the distribution of the Bgs1p.

Figure 18. cfh3+ _{overexpression disturbs the distribution of}

septins. Hoechst (left panels) and GFP (right panels) fluores- cence of wild-type cells or cells overexpressing cfh3+_{. The ar-}

rows mark cells in which Spn3 accumulates at the cell cortex. The panels on the bottom are enlarged to allow a view of the septal area of a cell with multiple septa; Hoechst, GFP and the merge images are shown.

Figure 19. cfh3+ _{overexpression disturbs the distribution of}

glucanases. Hoechst (left panels) and GFP (right panels) fluo- rescence of wild-type cells or cells overexpressing cfh3+_{. The}

dots mark septa in which Agn1 cannot be observed or has a weak fluorescence.

Figure 20. cfh3+ _{overexpression disturbs the distribution of}

Bgs1p. Hoechst (left panels) and GFP-Bgs1 fluorescence (right panels) of wild-type cells or cells overexpressing cfh3+_.

Collectively, all the results described above show that a deregulated amount of Cfh3p interferes with different stages of cytokinesis and suggest that Cfh3p might be a component of a multiprotein com- plex.