Nike Bharucha

Publications (2)10.59 Total impact

• Source

  Available from: PubMed Central

  Article: A large-scale complex haploinsufficiency-based genetic interaction screen in Candida albicans: analysis of the RAM network during morphogenesis.

  Nike Bharucha, Yeissa Chabrier-Rosello, Tao Xu, Cole Johnson, Sarah Sobczynski, Qingxuan Song, Craig J Dobry, Matthew J Eckwahl, Christopher P Anderson, Andrew J Benjamin, Anju Kumar, Damian J Krysan
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  ABSTRACT: The morphogenetic transition between yeast and filamentous forms of the human fungal pathogen Candida albicans is regulated by a variety of signaling pathways. How these pathways interact to orchestrate morphogenesis, however, has not been as well characterized. To address this question and to identify genes that interact with the Regulation of Ace2 and Morphogenesis (RAM) pathway during filamentation, we report the first large-scale genetic interaction screen in C. albicans.Our strategy for this screen was based on the concept of complex haploinsufficiency (CHI). A heterozygous mutant of CBK1(cbk1Δ/CBK1), a key RAM pathway protein kinase, was subjected to transposon-mediated, insertional mutagenesis. The resulting double heterozygous mutants (6,528 independent strains) were screened for decreased filamentation on SpiderMedium (SM). From the 441 mutants showing altered filamentation, 139 transposon insertion sites were sequenced,yielding 41 unique CBK1-interacting genes. This gene set was enriched in transcriptional targets of Ace2 and, strikingly, the cAMP-dependent protein kinase A (PKA) pathway, suggesting an interaction between these two pathways. Further analysis indicates that the RAM and PKA pathways co-regulate a common set of genes during morphogenesis and that hyperactivation of the PKA pathway may compensate for loss of RAM pathway function. Our data also indicate that the PKA–regulated transcription factor Efg1 primarily localizes to yeast phase cells while the RAM–pathway regulated transcription factor Ace2 localizes to daughter nuclei of filamentous cells, suggesting that Efg1 and Ace2 regulate a common set of genes at separate stages of morphogenesis. Taken together, our observations indicate that CHI–based screening is a useful approach to genetic interaction analysis in C. albicans and support a model in which these two pathways regulate a common set of genes at different stages of filamentation.
  PLoS Genetics 04/2011; 7(4):e1002058. · 8.69 Impact Factor
• Article: A small molecule-directed approach to control protein localization and function.

  Prasanthi Geda, Srikanth Patury, Jun Ma, Nike Bharucha, Craig J Dobry, Sarah K Lawson, Jason E Gestwicki, Anuj Kumar
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  ABSTRACT: Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus. The kinetics of this system are rapid: rapamycin-directed nucleo-cytoplasmic transport is evident 10-12 min post-treatment and the process is reversible upon removal of rapamycin. Accordingly, we envision this platform as a promising approach for the systematic construction of conditional loss-of-function mutants. As proof of principle, we used this system to direct nuclear export of the essential heat shock transcription factor Hsf1p, thereby mimicking the cell-cycle arrest phenotype of an hsf1 temperature-sensitive mutant. Our drug-induced localization platform also provides a method by which protein localization can be uncoupled from endogenous cell signalling events, addressing the necessity or sufficiency of a given localization shift for a particular cell process. To illustrate, we directed the nuclear import of the calcineurin-dependent transcription factor Crz1p in the absence of native stimuli; this analysis directly substantiates that nuclear translocation of this protein is insufficient for its transcriptional activity. In total, this technology represents a powerful method for the generation of conditional alleles and directed mislocalization studies in yeast, with potential applicability on a genome-wide scale.
  Yeast 09/2008; 25(8):577-94. · 1.89 Impact Factor