Importance of the Fixative for Reliable Ultrastructural Preservation of Poikilohydric Plant Tissues. Observations on Dry, Partially, and Fully Hydrated Tissues ofSelaginella lepidophylla

Leaves of desiccated ‘resurrection plants’,Selaginellalepidophylla, were hydrated either through the roots of intactplants or as isolated organs. Air-dry tissue and samples at1, 4, 8 and 24 h (both detached and intact) of hydration wereprepared for electron microscopy using aldehyde fixatives ofdifferent osmotic strengths. Both dry and hydrated tissues werealso prepared using freeze substitution. Significant differencesin the ultrastructural preservation of these different sampleswere noted. There was a direct correlation between the osmolalityof both the fixative and the tissue with the quality of ultrastructuralpreservation. When the osmolality of the fixative was slightly(or even considerably) higher than that of the tissue, optimalpreservation was achieved. Freeze substitution, however, gavethe most faithful preservation of all subcellular compartments,despite the frequent presence of small ice crystals. Additionally,hydration of detached leaves for more than 4 h resulted in swellingdamage of the organelles and cytoplasm, regardless of the fixationprotocol. Broadly interpreted, the results of this study indicate thatan optimal preservation of plant cell and organelle ultrastructurecan be achieved by the use of high osmolality fixatives or,preferably, freeze substitution. These results are also importantin determining the method of hydration of poikilohydric samplesfor physiological studies and for interpretation of functionalchanges as related to the structural condition of the organelles.Copyright1997 Annals of Botany Company Selaginella; fixation; ultrastructure; dry; hydrated     

Dimerization of Corticotropin-Releasing Factor Receptor Type 1 Is Not Coupled to Ligand Binding

As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF₁R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF₁R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF₁R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.