3D-structured illumination microscopy provides novel insight into architecture of human centrosomes.
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TLDR
This study provides novel insights into the architecture of human centrosomes and illustrates the power of super-resolution microscopy in revealing the relative localization of centriole and PCM proteins in unprecedented detail.Abstract:
Centrioles are essential for the formation of cilia and flagella. They also form the core of the centrosome, which organizes microtubule arrays important for cell shape, polarity, motility and division. Here, we have used super-resolution 3D-structured illumination microscopy to analyse the spatial relationship of 18 centriole and pericentriolar matrix (PCM) components of human centrosomes at different cell cycle stages. During mitosis, PCM proteins formed extended networks with interspersed γ-Tubulin. During interphase, most proteins were arranged at specific distances from the walls of centrioles, resulting in ring staining, often with discernible density masses. Through use of site-specific antibodies, we found the C-terminus of Cep152 to be closer to centrioles than the N-terminus, illustrating the power of 3D-SIM to study protein disposition. Appendage proteins showed rings with multiple density masses, and the number of these masses was strongly reduced during mitosis. At the proximal end of centrioles, Sas-6 formed a dot at the site of daughter centriole assembly, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but Cep135 was associated mostly with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation. Our study provides novel insights into the architecture of human centrosomes and illustrates the power of super-resolution microscopy in revealing the relative localization of centriole and PCM proteins in unprecedented detail.read more
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Super-resolution microscopy demystified
Lothar Schermelleh,Alexia Ferrand,Thomas R Huser,Christian Eggeling,Markus Sauer,Oliver Biehlmaier,Drummen Gpc. +6 more
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The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin
Jeffrey B. Woodruff,Beatriz Ferreira Gomes,Per O. Widlund,Julia Mahamid,Alf Honigmann,Anthony A. Hyman +5 more
TL;DR: It is found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM.
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Centrosome function and assembly in animal cells
TL;DR: Advances should ultimately allow the in vitro reconstitution of functional centrosomes from their component proteins to unlock the secrets of these enigmatic organelles.
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Reversible centriole depletion with an inhibitor of Polo-like kinase 4
Yao Liang Wong,John V. Anzola,Robert L. Davis,Michelle Yoon,Amir Motamedi,Ashley V. Kroll,Chanmee P. Seo,Judy E. Hsia,Sun K. Kim,Jennifer W. Mitchell,Brian J. Mitchell,Arshad Desai,Timothy C. Gahman,Andrew K. Shiau,Karen Oegema +14 more
TL;DR: Centrinone, a reversible inhibitor of Polo-like kinase 4 (Plk4), a serine-threonine protein kinase that initiates centriole assembly is developed, which reveals that cancer cells but not normal cells can divide in the absence of centrosomes.
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Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material
Steffen Lawo,Steffen Lawo,Monica Hasegan,Gagan D. Gupta,Laurence Pelletier,Laurence Pelletier +5 more
TL;DR: It is found that PCM components occupy separable spatial domains within mitotic PCM that are maintained in the absence of microtubule nucleation complexes and further implicate PCNT and CDK5RAP2 in the organization and assembly of PCM.
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