Improved technique for electron microscope visualization of yeast membranestructure

Yeast cells represent a powerful model system in cell biology mainly due to their amenability to genetic manipulations. Increasingly, studies focus on mutant genes resulting in alterations of cellular structures and organelle s, To ascertain the phenotypic changes involved, it is often desirable to u se the resolving power of electron microscopy. In contrast to higher eukary otic cells, yeast cells are particularly difficult to preserve mainly due t o the presence of a thick cell wall that acts as a barrier against diffusio n of fixatives. Although several procedures are targeted to overcome these difficulties, none of them have become established as a standard procedure. As a consequence, electron microscopy is still not used routinely as a too l in yeast cell biology. This prompted us to develop an easy-to-follow prot ocol for yeast transmission electron microscopy that should be useful in al l cases where membrane integrity and organelle morphology is emphasized. On e means of making the yeast cytoplasm more attainable to fixation and stain ing solutions is by enzymatic digestion of the cell wall. Following this ap proach, we were able to reliably preserve yeast cells and their cellular or ganelles. Enzymatic treatment with zymolyase 20T to partially remove the ye ast cell wall allowed the fixation, preservation, and visualization of the yeast cytoplasm revealing detailed ultrastructure. The advancement of this technique is demonstrated with mitochondria as a model organelle. Our studi es on various yeast mutants clearly show the power of the enzymatic digesti on technique in visualizing subtle changes of membrane structure and organe lle morphology.