Anastasia Eskova

Publications

• Cell arrays and high-content screening.

  Holger Erfle, Anastasia Eskova, Jürgen Reymann, Vytaute Starkuviene

  Methods in molecular biology (Clifton, N.J.). 01/2011; 785:277-87.

  Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse -material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocyt... [more] Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse -material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocytosis pathways are described by now; however, their specificity for different cellular cargoes is poorly resolved. Only few of endocytosis regulators are well-characterised and even less are attributed to the specific cargo. That is very true for the integrin endocytosis pathway, which is a key process in cell migration, adhesion, and signalling. The recent advent of quantitative fluorescent microscopy and cell arrays opened an exciting possibility to systematically characterise molecules playing a role in this crucially important process. Here, we describe a fluorescent screening microscopy-based assay to identify regulators of integrin α2 internalisation. The experimental procedure is the best suited for a highly parallel screening format, such as cell arrays, albeit can be used in single experiments. We provide protocols for sample preparation, fabrication of cell arrays and quantification of integrin α2 internalisation. The approach can be modified to quantify endocytosis of other cargo, and can be used under the conditions of knock-down and knock-in as well as for chemical screening.
• 5.36

  Impact points

  Hap2 regulates the pheromone response transcription factor prf1 in Ustilago maydis.

  Artemio Mendoza-Mendoza, Anastasia Eskova, Carolin Weise, Robert Czajkowski, Regine Kahmann

  Molecular microbiology. 04/2009;

  In Ustilago maydis the pheromone signal is transmitted via a mitogen-activated protein kinase (MAP kinase) module to the transcription factor Prf1. Prf1 activates transcription of a and b mating type genes by binding to pheromone response elements (PREs) located in regulatory regions of these genes.... [more] In Ustilago maydis the pheromone signal is transmitted via a mitogen-activated protein kinase (MAP kinase) module to the transcription factor Prf1. Prf1 activates transcription of a and b mating type genes by binding to pheromone response elements (PREs) located in regulatory regions of these genes. Here we show that the CCAAT-box binding protein Hap2 from U. maydis regulates prf1 expression. Hap2 was initially identified as a potential interaction partner of the MAP kinase Kpp6 in yeast two-hybrid screens and was subsequently also shown to interact with the MAPK Kpp2. Deletion of hap2 in haploid cells abolished mating, resulting from a defect in pheromone-induced gene expression. Crosses of haploid hap2 deletion strains were completely impaired in pathogenicity. Constitutive expression of prf1 complemented the pheromone response defect in Deltahap2 strains. Chromatin immunoprecipitation assays indicated that Hap2 binds directly to CCAAT motifs in the prf1 promoter. Point mutations in two putative MAPK phosphorylation sites in Hap2 attenuated the pheromone response. In a solopathogenic strain hap2 deletion affected filamentation and the mutants showed reduced pathogenicity symptoms. These data suggest that Hap2 is a novel regulator of prf1 with additional functions after cell fusion.