Images above are illuminated with laser only (488 nm; left panel), white light (right panel) or both (center panel). commensal fungus of humans. It is isolated frequently from your oral mucosa, gastrointestinal and genital tracts of normally healthy individuals. However, can overgrow its niche and cause disease when the normal microbiota is usually altered, or in cases of immune compromise. The close association between and its host is usually mediated in part by fungal factors, some of which are encoded COH29 by gene families (Jones et al., 2004). One example is the Als (agglutinin-like sequence) family of large cell-surface glycoproteins (examined by Hoyer et al., 2008). In cell wall. Als protein function is discussed most commonly in the context of adhesion to host and abiotic surfaces (examined in Hoyer et al., 2008). The presence of the ALS gene family raised many possibilities regarding the functional relationship between the Als proteins. For example, it was unknown whether one or more Als proteins are found at the same time on the surface of a cell. It was also unknown whether each Als protein is found in a similar relative large quantity or if some are more plentiful than others. Finally, it was also unclear whether the Als proteins are represented evenly over the cell surface, or have a specialized localization. The answers to these questions provide clues into the individual and collective function of the Als COH29 proteins. Study of ALS gene expression patterns provided some initial insight into these associations. Analysis of cells from disease models and human clinical specimens showed that transcription of all ALS genes could be detected, but that genes differed with respect to maximal expression levels (examined in Hoyer et al., 2008). Some genes could reach high transcriptional levels while others were usually relatively silent. In cultured cells, transcription of some ALS genes was affected by stage of culture growth and/or cellular morphology. Rabbit Polyclonal to 14-3-3 zeta (phospho-Ser58) Detecting simultaneous transcription of many ALS genes argued against the idea that a single Als protein is found on the surface at a time. To further explore these suggestions, as well as to determine the arrangement of Als proteins on individual cells in a populace, efforts shifted toward raising a monoclonal antibody (mAb) specific for each Als protein. Consistent with the strong transcriptional activity of in cultures of germ tubes and hyphae (Hoyer et al., 1998b; Argimon et al., 2007), anti-Als3 immunolabeling showed an intense covering of protein around the cell surface (Beucher et al., 2009; Coleman et al., 2009). This covering COH29 was also found on hyphae isolated from a murine model of disseminated candidiasis (Coleman et al., 2009). Anti-Als3 immunolabeling on yeast cells was not detectable, consistent with the low transcriptional activity in COH29 this morphological form. In contrast, anti-Als1 immunolabeled both yeast and germ tubes/hyphae (Coleman et al., 2010). transcription rises sharply as yeast cells from a saturated culture are placed into fresh medium, whether the cells are destined to grow as yeast or germ tubes. For yeast cultures, the inoculum cells become coated with Als1, except in the bud scar. The Als1 transmission weakens on the surface of cells from subsequent generations until the protein becomes undetectable by immunolabeling. Because Als1 is usually stable around the yeast cell surface, the culture populace is quite heterogeneous with respect to Als1 presence. On germ tubes in culture, Als1 is usually localized proximal to the mother yeast and persists as long hyphae form. In cells recovered from a mouse model of disseminated candidiasis, Als1 is found over a much more extensive area of the hypha surface compared to cultured cells (Coleman et al., 2010). In order to further these studies of Als protein localization, we developed a mAb specific for Als4. Here, we describe the mAb and use it to characterize the proteins localization on cultured cells of various morphologies and on fungal cells recovered from animal models of candidiasis. Materials and methods Production of mAbs The method for generating Als-specific mAbs was explained by Coleman et al. (2009, 2010) and.
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