Glucose starvation triggers filamentous septin assemblies in an S. pombe septin-2 deletion mutant

Abstract

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.

Hibernating ribosomes tether to mitochondria as an adaptive response to cellular stress during glucose depletion

“…Besides, our cryo-ET data showed notable morphological rearrangements including increased mitochondrial fragmentation and their reshaping into spheres, confirming previous observations of similar growth conditions ( 13 , 18 ). The recruitment of cytosolic ribosomes to the OMM occurred 6-7 days after glucose depletion and may have been triggered by an underlying signaling mechanism elicited 48-72 hours after the shutdown of protein synthesis (fig.…”

“…It is possible that tethered ribosomes perform a moonlighting function promoting cell survival by protecting fragmented mitochondria from autophagy and preventing apoptosis by stabilizing the mitochondrial membrane potential, thus averting cell death. Finally, since the effects of glucose starvation are reversible ( 11 , 12 , 18 ), we speculate that upon the reinstatement of high-nutrient conditions and the resurgence of protein synthesis, OMM-bound ribosomes would be ideally placed to synthesize mitochondrial proteins ( 34 ) to jumpstart mitochondrial activity. Upon the completion of one cycle of mitochondrial protein synthesis, OMM-bound ribosomes may then be released from mitochondria to translate new mRNA molecules.…”

“…It enables various processes such as localized signaling, fatty acid oxidation for ATP production and mitophagy for the removal of damaged mitochondria ( 13 , 16 , 17 ). In yeast, prolonged glucose depletion induces mitochondrial fragmentation ( 13 , 18 ) and the sequestration of cytosolic ribosomes on mitochondria ( 18 ). The underlying molecular mechanisms of this adaptive response and how it could promote cell survival during stress are not yet known.…”

Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

“…The existence of naturally occurring bundled septin structures in cells suggest that cdc12-6 bundles are not necessarily only a gain-of-function mutation but that the C-terminus of Cdc12 could be relevant for bundling septins under certain contexts (DeMay et al, 2009;Liu et al, 2019). However, given their abundance in cdc12-6 mutants, the needles could act like a sponge, sequestering septin complexes from the cytoplasm into "function-less" structures.…”

Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

“…Importantly, certain septins have already been connected to autophagy in non-mammalian cells. Fungal septin-1 and 3 (spn1 and spn3, not orthologs of mammalian septins) form a filamentous bundle in response to glucose starvation, a well-known autophagy-inducing method [ 72 ]. An additional relevant work that probably strengthens our conclusions is the research of Barve and colleagues [ 73 , 74 ].…”