The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p. INTRODUCTIONBud formation in the yeast Saccharomyces cerevisiae has served as an important model for studies of eukaryotic cell polarization (Hall, 1992;Nelson, 2003). In wild-type cells, nonrandom bud sites are selected by a system that involves cortical marker proteins and a signaling pathway based on the Ras-type GTPase Rsr1p Kang et al., 2004;Pruyne et al., 2004). GTP-bound Rsr1p then promotes the localization and/or localized activation of Cdc24p, the guanine-nucleotide-exchange factor (GEF) for the Rho-type GTPase Cdc42p, and of Cdc42p itself; the localized activation of Cdc42p then causes polarization of the cytoskeletal and secretory systems, which leads to the polarized growth of the bud Kozminski et al., 2003;Pruyne et al., 2004;Shimada et al., 2004).Among the proteins recruited early to the presumptive bud site are the septins. This widely conserved family of GTP-binding, filament-forming proteins functions in cytokinesis and other processes, many of which involve the organization of specialized regions of the cell cortex (Longtine et al., 1996;Gladfelter et al., 2001b;Longtine and Bi, 2003;Hall and Russell, 2004). S. cerevisiae has seven septins, five of which (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p/Sep7p) are expressed in vegetative cells, where they form heterooligomeric complexes and localize interdependently to the bud site (Kim et al., 1991;Longtine et al., 1996;Frazier et al., 1998;Mortensen et al., 2002;Vrabioiu et al., 2004). About 10 min before bud emergence, the septins form a ring at the cell cortex. The bud then emerges through this ring, which concurrently reorganizes into an hourglassshaped collar that spans the mother-bud neck. This reorganization coincides with a major decrease in the exchangeability of septin subunits, presumably reflecting the formation of more stable higher-order structures at this time (Caviston et al., 2003;Dobbelaere et al., 2003;. The septin collar remains at the neck until cytokinesis, when it splits into two rings as the actomyosin ring contracts an...

Background: The most diverse marine ecosystems, coral reefs, depend upon a functional symbiosis between cnidarian hosts and unicellular dinoflagellate algae. The molecular mechanisms underlying the establishment, maintenance, and breakdown of the symbiotic partnership are, however, not well understood. Efforts to dissect these questions have been slow, as corals are notoriously difficult to work with. In order to expedite this field of research, we generated and analyzed a collection of expressed sequence tags (ESTs) from the sea anemone Aiptasia pallida and its dinoflagellate symbiont (Symbiodinium sp.), a system that is gaining popularity as a model to study cellular, molecular, and genomic questions related to cnidarian-dinoflagellate symbioses.

In Saccharomyces cerevisiae, Bud8p and Bud9p are homologous plasma membrane glycoproteins that appear to mark the distal and proximal cell poles, respectively, as potential sites for budding in the bipolar pattern. Here we provide evidence that Bud8p is delivered to the presumptive bud site (and thence to the distal pole of the bud) just before bud emergence, and that Bud9p is delivered to the bud side of the mother-bud neck (and thence to the proximal pole of the daughter cell) after activation of the mitotic exit network, just before cytokinesis. Like the delivery of Bud8p, that of Bud9p is actin dependent; unlike the delivery of Bud8p, that of Bud9p is also septin dependent. Interestingly, although the transcription of BUD8 and BUD9 appears to be cell cycle regulated, the abundance of BUD8 mRNA peaks in G2/M and that of BUD9 mRNA peaks in late G1, suggesting that the translation and/or delivery to the cell surface of each protein is delayed and presumably also cell cycle regulated. The importance of time of transcription in localization is supported by promoter-swap experiments: expression of Bud8p from the BUD9 promoter leads to its localization predominantly to the sites typical for Bud9p, and vice versa. Moreover, expression of Bud8p from the BUD9 promoter fails to rescue the budding-pattern defect of a bud8 mutant but fully rescues that of a bud9 mutant. However, although expression of Bud9p from the BUD8 promoter fails to rescue a bud9 mutant, it also rescues only partially the budding-pattern defect of a bud8 mutant, suggesting that some feature(s) of the Bud8p protein is also important for Bud8p function. Experiments with chimeric proteins suggest that the critical element(s) is somewhere in the extracytoplasmic domain of Bud8p.

As sea surface temperatures increase worldwide due to climate change, coral bleaching events are becoming more frequent and severe, resulting in reef degradation. Leveraging the inherent ability of reef-building corals to acclimatize to thermal stress via pre-exposure to protective temperature treatments may become an important tool in improving the resilience of coral reefs to rapid environmental change. We investigated whether historical bleaching phenotype, coral host genotype, and exposure to protective temperature treatments would affect the response of the Hawaiian coral Montipora capitata to natural thermal stress. Fragments were collected from colonies that demonstrated different bleaching responses during the 2014-2015 event in Kāne'ohe Bay (O'ahu, Hawai'i) and exposed to four different artificial temperature pre-treatments (and a control at ambient temperature). After recovery, fragments experienced a natural thermal stress event either in laboratory conditions or their native reef environment. Response to thermal stress was quantified by measuring changes in the algal symbionts' photochemical efficiency, community composition, and relative density. Historical bleaching phenotype was reflected in stable differences in symbiont community composition, with historically bleached corals containing only Cladocopium symbionts and historically non-bleached corals having mixed symbiont communities dominated by Durusdinium. Mixed-community corals lost more Cladocopium than Cladocopium-only corals during the natural thermal stress event and preferentially recovered with Durusdinium. Laboratory pre-treatments exposed corals to more thermal stress than anticipated, causing photochemical damage that varied significantly by genotype. While none of the treatments had a protective effect, temperature variation during treatments had a significant detrimental effect on photochemical efficiency during the thermal stress event. These results show that acclimatization potential is affected by fine-scale differences in temperature regime, host genotype, and relatively stable differences in symbiont community composition that underpin historical bleaching phenotypes in M. capitata.

In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.