By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.

Candida albicans, the most prevalent fungal pathogen of humans, causes superficial mycoses, invasive mucosal infections, and disseminated systemic disease. Many studies have shown an intriguing association between C. albicans morphogenesis and the pathogenesis process. For example, hyphal cells have been observed to penetrate host epithelial cells at sites of wounds and between cell junctions. Ras-and Rho-type GTPases regulate many morphogenetic processes in eukaryotes, including polarity establishment, cell proliferation, and directed growth in response to extracellular stimuli. We found that the C. albicans Ras-like GTPase Rsr1p and its predicted GTPase-activating protein Bud2p localized to the cell cortex, at sites of incipient daughter cell growth, and provided landmarks for the positioning of daughter yeast cells and hyphal cell branches, similar to the paradigm in the model yeast Saccharomyces cerevisiae. However, in contrast to S. cerevisiae, CaRsr1p and CaBud2p were important for morphogenesis: C. albicans strains lacking Rsr1p or Bud2p had abnormal yeast and hyphal cell shapes and frequent bends and promiscuous branching along the hypha and were unable to invade agar. These defects were associated with abnormal actin patch polarization, unstable polarisome localization at hyphal tips, and mislocalized septin rings, consistent with the idea that GTP cycling of Rsr1p stabilizes the axis of polarity primarily to a single focus, thus ensuring normal cell shape and a focused direction of polarized growth. We conclude that the Rsr1p GTPase functions as a polarity landmark for hyphal guidance and may be an important mediator of extracellular signals during processes such as host invasion.Candida albicans, an important fungal pathogen, causes infections in both healthy and ill persons ranging from superficial mucosal diseases (thrush and vaginitis) to life-threatening disseminated disease involving the bloodstream (sepsis), central nervous system (meningitis), and vital organs (renal, hepatic, or brain abscess, for example). C. albicans exists in three major morphologies: ellipsoid yeast-form (YF) cells, pseudohyphal cells (chains of elongated yeast cells, separated by constrictions), and extremely elongated filamentous hyphal-form (HF) cells. YF cells are thought to be the predominant morphology found during colonization at epithelial surfaces, whereas HF cells have been associated with invasion into and through epithelial and endothelial cell layers (40). Indeed, C. albicans strains with defects in hyphal morphogenesis appear to be less virulent in animal and tissue-based models of candidiasis (9,36,45,48), leading to the idea that morphogenesis is required for virulence. On the one hand, this conclusion is complicated by the realization that many of these avirulent strains lack proteins (for example, transcription factors) that likely affect not only cell shape but also many other cellular processes. Thus, it remains unclear if cell shape per se is important for the pathogenesis process or if morphology is s...

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P MET 3 -GFP, P GAL1 -GFP, and P PCK 1 -GFP ), linked to a selectable nutritional marker gene (URA3 ). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5 -end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an aminoterminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.