Molecular and functional characterization of a Rho GDP dissociation inhibitor in the filamentous fungus Tuber borchii.

BACKGROUND: Small GTPases of the Rho family function as tightly regulated molecular switches that govern important cellular functions in eukaryotes. Several families of regulatory proteins control their activation cycle and subcellular localization. Members of the guanine nucleotide dissociation inhibitor (GDI) family sequester Rho GTPases from the plasma membrane and keep them in an inactive form. RESULTS: We report on the characterization the RhoGDI homolog of Tuber borchii Vittad., an ascomycetous ectomycorrhizal fungus. The Tbgdi gene is present in two copies in the T. borchii genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tbgdi during the phase preparative to the symbiosis instauration, in particular after stimulation with root exudates extracts, that correlates with expression of Tbcdc42. In a translocation assay TbRhoGDI was able to solubilize TbCdc42 from membranes. Surprisingly, TbRhoGDI appeared not to interact with S. cerevisiae Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of Dictyostelium discoideum, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the D. discoideum RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response. CONCLUSION: T. borchii expresses a functional RhoGDI homolog that appears as an important modulator of cytoskeleton reorganization during polarized apical growth that antecedes symbiosis instauration. The specificity of TbRhoGDI actions was underscored by its inability to elicit a growth defect in S. cerevisiae or to compensate the loss of a D. discoideum RhoGDI. Knowledge of the cell signaling at the basis of cytoskeleton reorganization of ectomycorrhizal fungi is essential for improvements in the production of mycorrhized plant seedlings used in timberland extension programs and fruit body production.

Effects of formalin, methacarn, and fineFIX fixatives on RNA preservation.

Formalin-fixed tissues represent the most abundant clinical material for retrospective studies. However, formalin highly affects macromolecules, impairing their extraction and analysis. In this study, the suitability of some potential substitutes of formalin for RNA-based applications has been considered. Conventional formalin was compared with methacarn and the commercial FineFIX. Their impact on overall RNA preservation was investigated in a cell line-based model fixed during a time course treatment and in a series of fixed human tissues. RNA yield was detected by Nanodrop; ribosomal RNA (rRNA) integrity by electrophoresis and the Agilent Bioanalyzer; messenger RNA (mRNA) integrity by Northern blot and endpoint reverse transcription-polymerase chain reaction; and mRNA amount by real-time polymerase chain reaction. In the cell line model, formalin fixation showed time-dependent detrimental effects on overall RNA preservation. Methacarn and FineFIX were more conservative on both rRNA and mRNA preservation and their impact was time-independent. In tissues, high rRNA degradation levels were found in all fixed specimens, contrasting with the results found in the cells. Conversely, the effects of the fixatives on mRNA integrity reflected the observations shown in the cell line model. In methacarn-fixed samples mRNA amount was also preserved, whereas in formalin and FineFIX-fixed samples it was notably lower when compared with the fresh frozen control. Alcohol-based fixatives are a good solution for long-term fixation of both cytologic and tissue samples by virtue of their time-independent effects on mRNA preservation. In fixed tissue samples, however, the potential effects of preanalytical tissue-related factors should be considered when performing mRNA quantitative analysis.