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Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles

Nature Cell Biology volume 16, pages 322–334 (2014)Cite this article

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Abstract

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

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Figure 1: Defects in actomyosin assembly kinetics during cellularization of septin mutants.
Figure 2: Defects in actin and Myo-II organization during cellularization of septin mutants.
Figure 3: Actin organization in DRhoGEF2 mutants.
Figure 4: Electron microscopy of FCs in wild-type and septin mutants.
Figure 5: F-actin molecular orientational order at the FC is decreased in septin mutants.
Figure 6: In vitro reconstituted actin filaments are bundled and curved in the presence of septins.
Figure 7: Negative-stain electron microscopy of in vitro reconstituted actin filaments in the presence of septins.
Figure 8: F-actin bundle formation in the presence of septin hexamers or preformed septin bundles.

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Acknowledgements

We thank the Lecuit and Lenne groups for discussions and for providing a fruitful environment during the course of this work. We thank J-P. Chauvin, A. Aouane and F. Richard at the IBDM electron microscopy facility for help with embryo processing and image acquisition, G. Pehau-Arnaudet at Imagopole (Institut Pasteur) and the electron microscopy facility at Imagif (Gif sur Yvette) for technical help with imaging of in vitro actin–septin assemblies, P. Ferrand, X. Wang, J. Duboisset and H. Rigneault (Institut Fresnel) for their contribution in the implementation of polarization-resolved fluorescence microscopy, C. Romier and C. Fernández-Tornero for discussions on septin purification, F. Maina for key advice on phosphoprotein westerns, M. Kuit-Vinkenoog for G-actin purification, and A. Kamor and C. Chandre for Matlab and POV-ray code for drawing FC tori. M.M., Y.A-G. and F.I. were supported by the ANR Blanc ARCHIPLAST (T.L.), the Fondation pour la Recherche Medicale (équipe labelisée, T.L.), the Association pour la Recherche contre la Cancer (Programme ARC, SL220120605305) and the CNRS. M.M., A.B., F-C.T., J.A. and G.H.K. were supported by two PHC Van Gogh grants (no. 25005UA and no. 28879SJ, ministères des Affaires étrangères et de l’Enseignement supérieur et de la Recherche), and G.H.K., F-C.T. and J.A. were supported by a VIDI grant from NWO and by the Foundation for Fundamental Research on Matter (FOM). A.K. and S.B. were supported by the CNRS, the ANR BLANC grants 150902 (ReceptORIENT) and 18818 (RADORDER) and the region Provence Alpes Côte d’Azur. This work was supported by the national infrastructure France Bio-Imaging and the Nikon Application Center Marseille.

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  1. Feng-Ching Tsai, José Alvarado and Aurélie Bertin: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut de Biologie du Développement de Marseille, CNRS UMR 7288, Aix-Marseille Université, 13288 Marseille, France

    Manos Mavrakis, Yannick Azou-Gros, Francois Iv & Thomas Lecuit

  2. FOM Institute AMOLF, 1098 XG Amsterdam, The Netherlands

    Feng-Ching Tsai, José Alvarado & Gijsje H. Koenderink

  3. Institut Curie, CNRS UMR 168, 75231 Paris, France

    Aurélie Bertin

  4. Institut Fresnel, CNRS UMR 7249, Aix-Marseille Université, Ecole Centrale Marseille, 13397 Marseille, France

    Alla Kress & Sophie Brasselet

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  1. Manos Mavrakis
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Contributions

Experiments were conceived and planned by M.M., G.H.K. and T.L. Experiments were performed by M.M., Y.A-G., F-C.T., J.A., A.B., F.I. and A.K. Data analysis was performed by M.M., Y.A-G., A.B., A.K. and S.B. M.M. wrote the first version of the manuscript, and T.L. and G.H.K. contributed to the writing of the final version. F-C.T., J.A., A.B. and S.B. contributed to the writing of the methods. All authors participated in the discussion of the data and in producing the final version of the manuscript.

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Correspondence to Manos Mavrakis or Thomas Lecuit.

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Integrated supplementary information

Supplementary Figure 1 FC membrane polarity is not affected during cellularization of septin mutants.

a, b, Confocal images of DSep1 (a, left) and DSep2 (b, left) in a top view of the FC in a wild-type embryo. Side views of the FC in wild-type and septin mutant depicting the localization of the lateral membrane marker, Scb, and DSep1 (a, right) and Scb and DSep2 (b, right). (c) Confocal images of the lateral membrane marker, Dlg, and the FC membrane markers, DPatj, Slam and DRhoGEF2, in side views of the FC during the fast phase of wild-type and septin mutant embryos. Notice the flat morphology of FC membranes in mutants (arrowheads). d, Confocal images of DPatj and Dlg in a top view of the FC during the fast phase of wild-type and septin mutant embryos. A higher magnification of a FC in the mutant (dashed box) shows the segregation of Dlg and DPatj. e, Localization of myosin heavy chain (MHC), Pnut/hSep7 and DSep1 at the FC of septin mutant embryos. A higher magnification of the FC region is depicted on the right. f, Two-photon images of UtrCHD::GFP during cellularization of wild-type (top panel) and septin mutant (bottom panel). Scale bars, 5 μm.

Supplementary Figure 2 Defects in F-actin organization during cellularization of septin mutants.

a, c, d, Confocal images of Actin (phalloidin-stained) during the early slow phase (A, 1-3 depict three representative examples), the early fast phase (C, 1-2 depict two representative examples) and the late fast phase (D, right) of septin mutants. Actin in wild type embryos of the respective stages are shown in c and d, left for comparison. (b) Side view of FCs in a septin mutant stained for Actin, MHC and Pnut/hSep7. e, Quantification of FC perimeters from segmentation analysis of wild-type (N=32–144 FCs/timepoint/embryo) embryos. All the N numbers of FCs per timepoint per embryo are provided in Supplementary Table 1. Constriction rates are calculated from linear regression analysis in the regions depicted by the two black line segments. Error bars are mean ± s.d. f, Definition of circularity. Scale bars, 5 μm.

Supplementary Figure 3 Electron microscopy of FCs in wild-type and septin mutants.

(a) Electron micrograph of a FC section depicting how measurements of FC coat densities (quantified in Fig. 4g) were performed. I, Mean pixel intensity. (b) Examples of FC sections in wild-type and septin mutants at low magnification. (c) Electron micrograph of grazing sections through the side of a FC in a wild-type (left) and a septin mutant (right). The schematic depicts the grazing sections with respect to the FC geometry. Dashed black lines follow the FC membrane contour. White guidelines in wild-type are oriented parallel to the observed arrays of filaments. d, Electron micrograph of a top view of a wild-type FC depicting the curved electron-dense coat (white brackets) at the membrane e, Electron micrograph of a grazing section through the base of a wild-type FC. White guidelines are oriented parallel to the observed arrays of filaments.

Supplementary Figure 4 In vitro reconstituted actin filaments are bundled in the presence of septins.

a, b SDS-PAGE analysis (Coomassie blue stained) of fractions from low speed co-sedimentation of 1 μM F-actin co-polymerized with the indicated concentrations of Fascin (a) or Drosophila septins (b). T, total. S, supernatant. P, pellet.

Supplementary Figure 5 Characterization of DSep1 and DSep2 antibodies generated in this study.

a, b, Homology models of DSep1 (a) and DSep2 (b) structures depicting in red the amino acid (aa) sequence and respective regions in the structure that were used as epitopes for antibody production. c, d, Western blot of purified recombinant septin complexes (c) and wild-type embryo lysates (d) using the DSep1 and DSep2 antibodies generated in this study. e, SDS-PAGE analysis of recombinant septin complexes of His6-hSep2, hSep6 and hSep7-Strep.

Supplementary Figure 6 Representative entire images of immunoblotting and SDS-PAGE.

Boxed areas were cropped for designated figures. Dotted lines denote separations of membranes for dual labeling.

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Supplementary Information (PDF 1032 kb)

Supplementary Table 1

Supplementary Information (XLSX 40 kb)

Side view of membrane invagination during cellularization in a MRLC::GFP expressing wild-type and septin mutant embryo, imaged with two-photon microscopy.

Time interval between acquired frames was 2 min and the acquisition length was 90 min (wild-type) and 98 min (mutant). Video is 706 × faster than real-time. (MOV 825 kb)

Top view of a MRLC::GFP expressing wild-type and septin mutant embryo during the slow phase of cellularization.

Images were collected at the FC with spinning disk confocal microscopy. Time interval between acquired frames was 10 s and the acquisition length was 44 min. Video is 149 × faster than real-time. (MOV 11781 kb)

Time-lapse imaging of actin bundle formation in the presence of 0.1 μM fly septins.

Time interval between acquired frames was 0.5 s and the acquisition length was 5 min. Video is 7.5 × faster than real-time. (MOV 8893 kb)

Time-lapse imaging of thermally undulating single actin filaments.

Time interval between acquired frames was 0.5 s and the acquisition length was 1 min. Video is 7.5 × faster than real-time. (MOV 889 kb)

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Mavrakis, M., Azou-Gros, Y., Tsai, FC. et al. Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles. Nat Cell Biol 16, 322–334 (2014). https://doi.org/10.1038/ncb2921

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