Rifka VLijm: Resolving a complicated web of filaments: STED nanoscopy unexpectedly reveals a highly periodic ring-anchored protein network at centrioles

The centrosome linker joints both centrosomes of a cell into one microtubule-organizing unit. Therefore, it plays important roles in cell organization, chromosome segregation, and cancer development. The proteins C-Nap1, rootletin, and CEP68 have been identified as linker proteins. However, little is known about the structure of the linker, despite earlier attempts using varying methods including Electron Microscopy. Here, we show by STimulated Emission Depletion Microscopy (STED) that the centrosome linker consists of a vast network of repeating rootletin units with a C-Nap1 ring at centrioles as organizer and CEP68 as filament modulator. Imaging the rootletin N and C terminus leads to an estimated minimal rootletin length of ∼110 nm. Rootletin filaments originating from the two centrosomes form a web-like, interdigitating filamentous network, explaining the flexible nature of the centrosome linker and the ability of the kinesin motor Eg5 to disrupt the linker function by force. I also describe how the STED concept in combination with newly developed protocols will allow imaging in living cells and Drosophila embryos at a resolution of 30 nm, to study both the centriolar cell division machinery as well as chromatin organization, a long-term interest of mine, in live settings.