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Cited by 106Fungal and yeast genetics researchMicrotubule and mitosis dynamicsCellular transport and secretion

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Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae

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Bertin, Aurélie, McMurray, Michael A., Pierson, Jason, Thai, Luong, McDonald, Kent L., Zehr, Elena A., García, Galo, Peters, Peter, Thorner, Jeremy, Nogales, Eva·2012·Molecular Biology of the Cell
DOI 10.1091/mbc.e11-10-0850PMC PMC3268722
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Abstract

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

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Figures

FIGURE 1: Resin-embedded, 50-nm sections of wild-type S. cerevisiae bud necks. Left, sagittal (medial) section of a mother cell and its bud in schematic view (top) and an actual image (A) with the region (square) showing apparent cross sections of filament-like structures (arrowheads) closely apposed to the plasma membrane and interconnected by a continuous fibril. Scale bar, 0.2 μm. Right, grazing section of a mother cell and its bud in schematic view (top) and an actual image (B) with region (square) showing a banding pattern of apparent filament-like structures perpendicular to the mother–bud axis. Scale bar, 0.5 μm.
FIGURE 1: Resin-embedded, 50-nm sections of wild-type S. cerevisiae bud necks. Left, sagittal (medial) section of a mother cell and its bud in schematic view (top) and an actual image (A) with the region (square) showing apparent cross sections of filament-like structures (arrowheads) closely apposed to the plasma membrane and interconnected by a continuous fibril. Scale bar, 0.2 μm. Right, grazing section of a mother cell and its bud in schematic view (top) and an actual image (B) with region (square) showing a banding pattern of apparent filament-like structures perpendicular to the mother–bud axis. Scale bar, 0.5 μm.CC BY-NC-SA · PubMed Central
FIGURE 2: Tomographic visualization of resin-embedded thick sections of wild-type S. cerevisiae bud necks. (A) Grazing section. Top, slice from a tomogram showing circumferential filaments (green arrows) perpendicular to the mother–bud axis. Middle, magnified view of an area (square) from the top that shows both the circumferential filament-like densities (green arrows) and the orthogonal filament-like densities parallel to the mother–bud axis (red arrows). Bottom, segmentation model of complete tomogram with cell membrane (yellow), circumferential filaments (green), axial filaments (red), and densities connecting the circumferential filaments to the membrane (blue). (B) Medial section. Top, slice from a tomogram showing one side of the bud neck containing an axial filament (red arrow) parallel to the membrane running continuously above and periodically connected to (blue arrows) dot-like densities corresponding to cross sections through the circumferential filaments. Middle, section from the same tomogram, perpendicular to that in the top (with the common axis shown vertically), showing the double-fiber character of the circumferential filaments (green arrows). Bottom, a glancing (left) and edge-on (right) view of the cell membrane (yellow) in segmentation model of the tomogram with the circumferential filaments (green), axial filaments (red), and connecting densities from the filaments to the membrane (blue). Scale bars, 100 nm.
FIGURE 2: Tomographic visualization of resin-embedded thick sections of wild-type S. cerevisiae bud necks. (A) Grazing section. Top, slice from a tomogram showing circumferential filaments (green arrows) perpendicular to the mother–bud axis. Middle, magnified view of an area (square) from the top that shows both the circumferential filament-like densities (green arrows) and the orthogonal filament-like densities parallel to the mother–bud axis (red arrows). Bottom, segmentation model of complete tomogram with cell membrane (yellow), circumferential filaments (green), axial filaments (red), and densities connecting the circumferential filaments to the membrane (blue). (B) Medial section. Top, slice from a tomogram showing one side of the bud neck containing an axial filament (red arrow) parallel to the membrane running continuously above and periodically connected to (blue arrows) dot-like densities corresponding to cross sections through the circumferential filaments. Middle, section from the same tomogram, perpendicular to that in the top (with the common axis shown vertically), showing the double-fiber character of the circumferential filaments (green arrows). Bottom, a glancing (left) and edge-on (right) view of the cell membrane (yellow) in segmentation model of the tomogram with the circumferential filaments (green), axial filaments (red), and connecting densities from the filaments to the membrane (blue). Scale bars, 100 nm.CC BY-NC-SA · PubMed Central
FIGURE 3: Tomographic visualization of resin-embedded thick sections of the bud neck of wild-type S. cerevisiae undergoing mitosis. (A) Tomographic slice near the plasma membrane at the most constricted point of the bud neck, showing clear circumferential filaments (green arrows). (B) Deeper slice, clearly showing axial filaments (red lines follow their contour, at a slightly farther distance from the membrane), as well as circumferential filaments, which are seen in cross section (green arrows). The nuclear envelope is also indicated (NE). Scale bar, 100 nm. (See also corresponding Supplemental Movie.)
FIGURE 3: Tomographic visualization of resin-embedded thick sections of the bud neck of wild-type S. cerevisiae undergoing mitosis. (A) Tomographic slice near the plasma membrane at the most constricted point of the bud neck, showing clear circumferential filaments (green arrows). (B) Deeper slice, clearly showing axial filaments (red lines follow their contour, at a slightly farther distance from the membrane), as well as circumferential filaments, which are seen in cross section (green arrows). The nuclear envelope is also indicated (NE). Scale bar, 100 nm. (See also corresponding Supplemental Movie.)CC BY-NC-SA · PubMed Central
FIGURE 4: Tomography of cryo-sections of wild-type S. cerevisiae bud necks. (A) Slice from a tomogram of a 50-nm cryo-section of the bud neck (inset, diffraction pattern confirming vitrification of the sample). (B, C) Magnified views of the left (B) and right (C) sides of the image shown in A, with axial filaments parallel to the membrane (red arrows) and connectors from the axial filaments to dot-like densities corresponding to cross sections through the circumferential filaments and to the membrane (blue arrows). (D, E) Slices perpendicular to those shown in B and C, respectively (the common axis is vertical, as indicated) showing the double-fiber nature of each circumferential filament (green arrows).
FIGURE 4: Tomography of cryo-sections of wild-type S. cerevisiae bud necks. (A) Slice from a tomogram of a 50-nm cryo-section of the bud neck (inset, diffraction pattern confirming vitrification of the sample). (B, C) Magnified views of the left (B) and right (C) sides of the image shown in A, with axial filaments parallel to the membrane (red arrows) and connectors from the axial filaments to dot-like densities corresponding to cross sections through the circumferential filaments and to the membrane (blue arrows). (D, E) Slices perpendicular to those shown in B and C, respectively (the common axis is vertical, as indicated) showing the double-fiber nature of each circumferential filament (green arrows).CC BY-NC-SA · PubMed Central
FIGURE 5: Tomographic visualization of resin-embedded thick sections of Cdc10-less mutant cells. (A) Low-magnification view of a section from a bloc of the haploid cdc10Δ strain, showing diagnostic abnormal budding patterning (black arrows) and the region from which the tomograms were collected (white arrow). (B, C) Slices through tomograms of two different grazing sections, showing circumferential filaments perpendicular to the mother–bud axis (green arrows) and axial filaments parallel to the mother–bud axis (red arrows). (D) Segmentation model from the tomogram shown in C, with circumferential filaments (green) and axial filaments (red).
FIGURE 5: Tomographic visualization of resin-embedded thick sections of Cdc10-less mutant cells. (A) Low-magnification view of a section from a bloc of the haploid cdc10Δ strain, showing diagnostic abnormal budding patterning (black arrows) and the region from which the tomograms were collected (white arrow). (B, C) Slices through tomograms of two different grazing sections, showing circumferential filaments perpendicular to the mother–bud axis (green arrows) and axial filaments parallel to the mother–bud axis (red arrows). (D) Segmentation model from the tomogram shown in C, with circumferential filaments (green) and axial filaments (red).CC BY-NC-SA · PubMed Central
FIGURE 6: Schematic representation of the septin filament array at the yeast bud neck. Top, orientation of the lattice-like septin collar relative to the mother–bud axis (black arrow). Bottom, enlarged view depicting the 10-nm circumferential filaments composed of a pair of tightly associated septin filaments (green) with short linkers (blue) connecting them to the inner leaflet of the PM and the axial filaments (red) composed of a single septin filament that intersect the circumferential filaments with a 30-nm spacing that corresponds to the 30-nm length of each octameric repeat in the axial filaments.
FIGURE 6: Schematic representation of the septin filament array at the yeast bud neck. Top, orientation of the lattice-like septin collar relative to the mother–bud axis (black arrow). Bottom, enlarged view depicting the 10-nm circumferential filaments composed of a pair of tightly associated septin filaments (green) with short linkers (blue) connecting them to the inner leaflet of the PM and the axial filaments (red) composed of a single septin filament that intersect the circumferential filaments with a 30-nm spacing that corresponds to the 30-nm length of each octameric repeat in the axial filaments.CC BY-NC-SA · PubMed Central

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Show all 64 references

Research works printed but not linked: 3

  1. Garcia G, 2011, J Cell Biol
  2. McMurray MA, 2008, The Septins, 49
  3. Pringle JR, 2008, The Septins, 7

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Metadata

Authors
Bertin, A., McMurray, M. A., Pierson, J., Thai, L., McDonald, K. L., Zehr, E. A., García, G., Peters, P., Thorner, J., & Nogales, E.
Affiliations
  • University of California, Berkeley (US)
  • The Netherlands Cancer Institute (NL)
  • Howard Hughes Medical Institute (US)
Corresponding author
Nogales, Eva
ORCID
Venue
Molecular Biology of the Cell
Publisher
American Society for Cell Biology (ASCB)
Published
2012-02
Volume
23
Issue
3
Pages
423-432
ISSN
  • 1059-1524 (ISSN-L)
  • 1939-4586
DOI
10.1091/mbc.e11-10-0850
PMC
PMC3268722
OpenAlex
W2013196716
Cited by
106
References
64
Citations by year
2026: 5 · 2025: 5 · 2024: 5 · 2023: 5 · 2022: 5 · 2021: 10 · 2020: 8 · 2019: 3 · 2018: 8 · 2017: 4 · 2016: 14 · 2015: 12 · 2014: 9 · 2013: 7 · 2012: 6
FWCI
2.85
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • Molecular Biology of the Cell (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
  • Howard Hughes Medical Institute
  • National Institute of General Medical Sciences (R00GM086603, K99 GM86603, R01GM21841, R01GM101314, K99GM086603, R01GM021841)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
septins, Saccharomyces cerevisiae, bud neck, membrane compartmentalization, electron tomography, cryo-electron tomography, thin section electron microscopy, filament polymerization
MeSH terms
Cytoskeleton, Mutation, Saccharomyces cerevisiae, Imaging, Three-Dimensional, Saccharomyces cerevisiae Proteins, Septins

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