SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae.

The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Only sla2 strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis of C. albicans infections.

Dynein-dependent nuclear dynamics affect morphogenesis in Candida albicans by means of the Bub2p spindle checkpoint.

Candida albicans, the most prevalent fungal pathogen of humans, grows with multiple morphologies. The dynamics of nuclear movement are similar in wild-type yeast and pseudohyphae: nuclei divide across the bud neck. By contrast, in hyphae, nuclei migrate 10-20 microm into the growing germ tube before dividing. We analyzed the role of the dynein-dynactin complex in hyphal and yeast cells using time-lapse fluorescence microscopy. Cells lacking the heavy chain of cytoplasmic dynein or the p150(Glued) subunit of dynactin were defective in the position and orientation of the spindle. Hyphal cells often failed to deliver a nucleus to the daughter cell, resulting in defects in morphogenesis. Under yeast growth conditions, cultures included a mixture of yeast and pseudohyphal-like cells that exhibited distinctive defects in nuclear dynamics: in yeast, nuclei divided within the mother cell, and the spindle position checkpoint protein Bub2p ensured the delivery of the daughter nucleus to the daughter cell before cytokinesis; in pseudohyphal-like cells, pre-mitotic nuclei migrated into the daughter and no checkpoint ensured return of a nucleus to the mother cell before cytokinesis. Analysis of double mutants indicated that Bub2p also mediated the pre-anaphase arrest and polarization of pseudohyphal-like cells. Thus, Bub2p has two distinct roles in C. albicans cells lacking dynein: it mediates pre-anaphase arrest and it coordinates spindle disassembly with mitotic exit.

Microtubules in Candida albicans hyphae drive nuclear dynamics and connect cell cycle progression to morphogenesis.

Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 microm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.

The mitotic cyclins Clb2p and Clb4p affect morphogenesis in Candida albicans.

The ability of Candida albicans to switch cellular morphologies is crucial for its ability to cause infection. Because the cell cycle machinery participates in Saccharomyces cerevisiae filamentous growth, we characterized in detail the two C. albicans B-type cyclins, CLB2 and CLB4, to better understand the molecular mechanisms that underlie the C. albicans morphogenic switch. Both Clb2p and Clb4p levels are cell cycle regulated, peaking at G2/M and declining before mitotic exit. On hyphal induction, the accumulation of the G1 cyclin Cln1p was prolonged, whereas the accumulation of both Clb proteins was delayed when compared with yeast form cells, indicating that CLB2 and CLB4 are differentially regulated in the two morphologies and that the dynamics of cyclin appearance differs between yeast and hyphal forms of growth. Clb2p-depleted cells were inviable and arrested with hyper-elongated projections containing two nuclei, suggesting that Clb2p is not required for entry into mitosis. Unlike Clb2p-depleted cells, Clb4p-depleted cells were viable and formed constitutive pseudohyphae. Clb proteins lacking destruction box domains blocked cell cycle progression resulting in the formation of long projections, indicating that both Clb2p and Clb4p must be degraded before mitotic exit. In addition, overexpression of either B-type cyclin reduced the extent of filamentous growth. Taken together, these data indicate that Clb2p and Clb4p regulate C. albicans morphogenesis by negatively regulating polarized growth.

The mouse secreted frizzled-related protein 5 gene is expressed in the anterior visceral endoderm and foregut endoderm during early post-implantation development.

The anterior visceral endoderm (AVE) plays an important role in anterior-posterior axis formation in the mouse. The AVE functions in part by expressing secreted factors that antagonize growth factor signaling in the proximal epiblast. Here we report that the Secreted frizzled-related protein 5 (Sfrp5) gene, which encodes a secreted factor that can antagonize Wnt signaling, is expressed in the AVE and foregut endoderm during early mouse development. At embryonic day (E) 5.5, Sfrp5 is expressed in the visceral endoderm at the distal tip region of the embryo and at E6.5 in the AVE opposite the primitive streak. In Lim1 embryos, which lack anterior neural tissue and sometimes form a secondary body axis, Sfrp5-expressing cells fail to move towards the anterior and remain at the distal tip of E6.5 embryos. When compared with Dkk1, which encodes another secreted Wnt antagonist molecule present in the visceral endoderm, Sfrp5 and Dkk1 expression overlap but Sfrp5 is expressed more broadly in the AVE. Between E7.5 and 8, Sfrp5 is expressed in the foregut endoderm underlying the cardiac mesoderm. At E8.5, Sfrp5 is expressed in the ventral foregut endoderm that gives rise to the liver. Additional domains of Sfrp5 expression occur in the dorsal neural tube and in the forebrain anterior to the optic placode. These findings identify a gene encoding a secreted Wnt antagonist that is expressed in the extraembryonic visceral endoderm and anterior definitive endoderm during axis formation and organogenesis in the mouse.