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Structural insights into yeast septin organization from polarized fluorescence microscopy

Abstract

Septins are polymerizing GTPases1 that function in cortical organization and cell division2,3,4. In Saccharomyces cerevisiae they localize at the isthmus between the mother and the daughter cells, where they undergo a transition from a non-dynamic hourglass-shaped assembly5 to two separate rings, at the onset of cytokinesis6,7. Septins form filaments as pure protein8 and in vivo9, but the filament organization within the hourglass and ring structures is controversial9,10. Here, we use polarized fluorescence microscopy11 of orientationally constrained green fluorescent protein to determine septin filament organization and dynamics in living yeast. We found that the hourglass is made of filaments aligned along the yeast bud neck. During the transition from hourglass to rings the filaments rotate through 90° in the membrane plane and become circumferential. These data resolve a long-standing controversy in the field and provide strong evidence that septins have a mechanical function in cell division.

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Figure 1: The experimental system.
Figure 2: The average dipole direction.
Figure 3: The average filament direction.

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Acknowledgements

We thank I. Vrabioiu and colleagues at INCDMF-CEFIN, Romania, for designing and fabricating the rotating stage used for our experiments, and M. Volles for comments. This work was supported by a National Institutes of Health grant to T.J.M.

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Correspondence to Alina M. Vrabioiu.

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Supplementary information

Supplementary Notes

This file contains Supplementary Methods, Supplementary Discussion, Supplementary Notes, Supplementary Tables, Supplementary Figures and Supplementary Movie Legend. (DOC 1163 kb)

Supplementary Figure 1

Characterization of the Cdc12 strain. (JPG 24 kb)

Supplementary Figure 2

Hourglass area C quantitations. (JPG 17 kb)

Supplementary Figure 3

Cdc3 strain rearrangement time lapse. (JPG 22 kb)

Supplementary Movie

This movie indicates that the septin filaments change orientation during the hourglass to rings transition. (MOV 4949 kb)

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Vrabioiu, A., Mitchison, T. Structural insights into yeast septin organization from polarized fluorescence microscopy. Nature 443, 466–469 (2006). https://doi.org/10.1038/nature05109

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  • DOI: https://doi.org/10.1038/nature05109

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