Quantitative analysis of nuclear pore complex organization in <i>Schizosaccharomyces pombe</i>

Varberg, Joseph M.; Unruh, Jay R.; Bestul, Andrew J.; Khan, Azqa; Jaspersen, Sue L.

doi:10.1101/2021.08.11.455278

Cited by 7 publications

(7 citation statements)

References 128 publications

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“…To maintain homeostatic NPC densities, the insertion of newly synthesized complexes is tightly regulated. Due to the small size of the yeast nucleus, this could only recently be quantified using 3D SIM (Varberg et al, 2022). We asked whether we could obtain matching results using U-ExM and conventional microscopy.…”

Section: Resultsmentioning

confidence: 99%

Ultrastructure Expansion Microscopy reveals the nanoscale cellular architecture of budding and fission yeast

Hinterndorfer

Laporte

Mikus

et al. 2022

Preprint

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The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have served as invaluable model organisms to study various fundamental and highly conserved cellular processes. While super-resolution (SR) microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeast models has remained limited due to the specific know-how and specialized instrumentation required, contrasted with the relative ease of endogenous tagging and live cell fluorescence microscopy in these systems. To facilitate SR microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling 4-fold isotropic expansion in both systems. We demonstrate here that U-ExM allows the nanoscale imaging of the microtubule cytoskeleton and its associated spindle pole body (SPB), notably unveiling a conserved Sfi1p/Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by quantifying the homeostatic regulation of nuclear pore complex (NPC) number through the cell cycle. Combined with pan-labelling (NHS ester), which provides a global cellular context, U-ExM unveils the subcellular organization of the eukaryote yeast models S. cerevisiae and S. pombe. This easy-to-implement imaging with conventional microscopes provides nanoscale resolution and adds a powerful new method to the already extensive yeast toolbox.

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Section: Resultsmentioning

confidence: 99%

Ultrastructure Expansion Microscopy reveals the nanoscale cellular architecture of budding and fission yeast

Hinterndorfer

Laporte

Mikus

et al. 2022

Preprint

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“…However, it is difficult to estimate nuclear volume using fluorescence microscopy of DAPI signals because the nuclear boundary is ambiguous. A better strategy is to image a strain that expresses a fluorescent nuclear-envelope protein, measure the nucleus diameter, and then calculate the nuclear volume (Heun et al, 2001;Neumann and Nurse, 2007;Varberg et al, 2022). We therefore created an S. pombe strain in the wild-type background in which the nuclearpore protein Nup97 is tagged with eGFP at its endogenous locus (Figure S10).…”

Section: G0 Nucleus Shrinkage Is Inhibited By Tsa Treatmentmentioning

confidence: 99%

Macromolecular and cytological changes in fission yeast G0 nuclei

Cai

Nie

et al. 2020

Preprint

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Cells change their cytology in response to environmental cues and stress. Notably, large changes in nuclear architecture are accompanied by transcriptional reprogramming. When starved of nitrogen, Schizosaccharomyces pombe cells become rounder and they enter a quiescent "G0" state. These cells have smaller nuclei and undergo near-global transcriptional repression. Here we use electron cryotomography (cryo-ET) and cell-biology approaches to investigate the structural and biochemical changes of G0 S. pombe nuclei. We find that G0 cells have a denser nucleoplasm and fewer chromatin-associated multi-megadalton globular complexes (megacomplexes) than proliferating cells. These structural changes are correlated with mild histone deacetylation. Induced histone hyperacetylation in G0 results in cells that have larger nuclei and less condensed chromatin. However, these histone-hyperacetylated G0 cells still have repressed transcription, few megacomplexes, and a dense nucleoplasm. Like in budding yeast, S. pombe G0 nuclear phenotypes are controlled by multiple biochemical factors.

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“…This agrees with the observation of Wecke and Giesbrecht [cited in reference ( 45 )] that the nuclear pores of Prorocentrum are arranged in closely packed hexagonal groups. We found the nuclear envelope to contain 475 NPCs, which greatly exceeds that of unicellular fungal microorganisms: Saccharomyces cerevisiae (budding yeast) was found to have 65–182 NPCs per cell ( 46 ) and Schizosaccharomyces pombe (fission yeast) 100–150 NPCs depending on the phase in the cell cycle ( 47 ). P. cordatum has a greater complexity in metabolism and cell structure compared to yeast, which apparently requires more nuclear pores to afford efficient nuclear import and export.…”

Section: Resultsmentioning

confidence: 97%

The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum

Kalvelage,

Wöhlbrand,

Schoon

et al. 2023

mSphere

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The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum ) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum . High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4–6.7 µm 3 ), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

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Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe

Cited by 7 publications

References 128 publications

Ultrastructure Expansion Microscopy reveals the nanoscale cellular architecture of budding and fission yeast

Ultrastructure Expansion Microscopy reveals the nanoscale cellular architecture of budding and fission yeast

Macromolecular and cytological changes in fission yeast G0 nuclei

The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum

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