RESUMO
Using correlative light and electron microscopy (CLEM), we studied the intracellular organization by of glucose-starved fission yeast cells (Schizosaccharomyces pombe) with regards to the localization of septin proteins throughout the cytoplasm. Thereby, we found that for cells carrying a deletion of the gene encoding septin-2 (spn2Δ), starvation causes a GFP-tagged version of septin-3 (spn3-GFP) and family members, to assemble into a single, prominent filamentous structure. It was previously shown that during exponential growth, spn2Δ cells form septin-3 polymers. However, the polymers we observed during exponential growth are different from the spn3p-GFP structure we observed in starved cells. Using CLEM, in combination with anti-GFP immunolabeling on plastic-sections, we could assign spn3p-GFP to the filaments we have found in EM pictures. Besides septin-3, these filamentous assemblies most likely also contain septin-1 as an RFP-tagged version of this protein forms a very similar structure in starved spn2Δ cells. Our data correlate phase-contrast and fluorescence microscopy with electron micrographs of plastic-embedded cells, and further on with detailed views of tomographic 3D reconstructions. Cryo-electron microscopy of spn2Δ cells in vitrified sections revealed a very distinct overall morphology of the spn3p-GFP assembly. The fine-structured, regular density pattern suggests the presence of assembled septin-3 filaments that are clearly different from F-actin bundles. Furthermore, we found that starvation causes substantial mitochondria fission, together with massive decoration of their outer membrane by ribosomes.
