Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.
How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne CH-1015, Switzerland.
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Guichard
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne CH-1015, Switzerland.
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V
Hamel
Department of Cell Biology, Sciences III, University of Geneva, Geneva CH-1211, Switzerland.
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M
Le Guennec
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne CH-1015, Switzerland.
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Banterle
Institute of Anatomy, University of Bern, Bern CH-3012, Switzerland.
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I
Iacovache
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne CH-1015, Switzerland.
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Nemčíková
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne CH-1015, Switzerland.
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I
Flückiger
Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel CH-4058, Switzerland.
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Goldie
Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel CH-4058, Switzerland.
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0000-0002-1185-4592
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Stahlberg
Institut Curie, PSL Research University, UMR 168, Centre de Recherche, 26 rue d'ULM, Paris 75231, France.
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Lévy
Institute of Anatomy, University of Bern, Bern CH-3012, Switzerland.
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0000-0001-7725-5579
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Zuber
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne CH-1015, Switzerland.
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Gönczy
D020418:Algal Proteins / Q000378:metabolism* / Q000648:ultrastructure; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000648:ultrastructure; D002502:Centrioles / Q000378:metabolism* / Q000648:ultrastructure; D016825:Chlamydomonas reinhardtii / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy / Q000379:methods; D055032:Electron Microscope Tomography / Q000379:methods; D008954:Models, Biological; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure
D020418:Algal Proteins; D018797:Cell Cycle Proteins