Visualization of Chromatin in the Yeast Nucleus and Nucleolus Using Hyperosmotic Shock

Abstract

Unlike in most eukaryotic cells, the genetic information of budding yeast in the exponential growth phase is only present in the form of decondensed chromatin, a configuration that does not allow its visualization in cell nuclei conventionally prepared for transmission electron microscopy. In this work, we studied the distribution of chromatin and its relationships to the nucleolus using different cytochemical and immunocytological approaches applied to yeast cells subjected to hyperosmotic shock. Our results show that osmotic shock induces the formation of heterochromatin patches in the nucleoplasm and intranucleolar regions of the yeast nucleus. In the nucleolus, we further revealed the presence of osmotic shock-resistant DNA in the fibrillar cords which, in places, take on a pinnate appearance reminiscent of ribosomal genes in active transcription as observed after molecular spreading ("Christmas trees"). We also identified chromatin-associated granules whose size, composition and behaviour after osmotic shock are reminiscent of that of mammalian perichromatin granules. Altogether, these data reveal that it is possible to visualize heterochromatin in yeast and suggest that the yeast nucleus displays a less-effective compartmentalized organization than that of mammals.

Keywords: chromatin; electron cytochemistry; immunoelectron microscopy; nucleolus; nucleus; perichromatin granules; yeast.

Figure 1

Nuclei of exponentially growing yeast cells, as revealed after the application of the cytochemical method of acetylation (A,B) combined with the immunocytological technique of detecting the DNA by the transferase of terminal deoxyribonucleotides (C) or with the staining of the argyrophilic proteins of the nucleolar organizer (D). The nucleolar region is delimited by a dotted line. It contains fibrillar strands (F) surrounded by clusters of granules (G). In addition, heterogeneous areas (asterisks) can be observed within nucleoli. The nucleoplasmic region contains numerous small granules (arrowheads) and rare large granules (thick arrow). Bars = 0.5 µm (A,C) or 0.25 (B,D).

Figure 2

Nuclei of exponentially growing yeast cells exposed to hyperosmotic shock as revealed after the application of the cytochemical method of acetylation (A–E) combined with the immunological technique of DNA detection by the transferase of terminal deoxyribonucleotides (F). (A,B) Dense masses (Ch, chromatin) are mainly observed in the nucleoplasmic region. Heterogeneous areas (asterisks) in the nucleolar region also contain patches of condensed chromatin. Some dense masses conspicuously associated with the nuclear envelope (thick arrows). (C,D) Some portions of the fibrillar strands (F) of the nucleolus have a pennate appearance with remarkable alignment (thin arrows). (E) Cell in division showing the presence of dense masses of compacted chromatin in the two forming nuclei. (F) Gold particles labelling DNA are observed essentially on the nucleoplasmic and intranucleolar dense masses. F, fibrillar strands; G, granular zone. Bars = 0.5 (A,B,E) or 0.1 µm (C,D,F).

Figure 3

Nuclei of exponentially growing yeast cells subjected to hyperosmotic shock as revealed after the application of the cytochemical method of acetylation combined with the technique of regressive EDTA staining to visualize RNP-containing structures. Dense masses (Ch) present in nucleoplasmic region and in the heterogenous areas of the nucleolus (asterisks) are “bleached” indicating they consisted mostly of DNA. The fibrillar strands (F) and granular masses (G) of the nucleoli remain contrasted as well as the small (arrowheads) and large extranucleolar granules (thick arrows). The latter are always associated with dense masses. Bars = 0.5 (A) or 0.25 µm (B).