Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid

Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2 alpha by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.This work was supported by Fundacao para a Ciencia e Tecnologia and COMPETE/QREN/EU (PTDC/BIA-MIC/114116/2009), and by the Canadian Institute for Health Research (MOP 89737 to MH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Novel 2.5D Culture Platform to Investigate the Role of Stiffness Gradients on Adhesion-Independent Cell Migration

Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration

Microarrays as research tools and diagnostic devices

Molecular diagnostics comprises a main analytical division in clinical laboratory diagnostics. The analysis of RNA or DNA helps to diagnose infectious diseases and identify genetic determined disorders or even cancer. Starting from mono-parametric tests within the last years, technologies have evolved that allow for the detection of many parameters in parallel, e.g., by using multiplex nucleic acid amplification techniques, microarrays, or next-generation sequencing technologies. The introduction of closed-tube systems as well as lab-on-a-chip devices further resulted in a higher automation degree with a reduced contamination risk. These applications complement or even stepwise replace classical methods in clinical microbiology like virus cultures, resistance determination, microscopic and metabolic analyses, as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel biomarkers. This article provides an overview of microarrays as diagnostics devices and research tools. Introduced in 1995 for transcription analysis, microarrays are used today to detect several different biomolecules like DNA, RNA, miRNA, and proteins among others. Mainly used in research, some microarrays also found their way to clinical dia- gnostics. Further, closed lab-on-a-chip devices that use DNA microarrays as detection tools are discussed, and additionally, an outlook toward applications of next-generation sequencing tools in diagnostics will be given