Microtubule nucleation properties of single human γTuRCs explained by their Cryo-EM structure

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001163 and FC0010065), the UK Medical Research Council (FC001163 and FC0010065), and the Wellcome Trust (FC001163 and FC0010065) to T.S. and A.C. The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149). J. Rappsilber is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2008 – 390540038 – UniSysCat and 329673113. J. Roostalu. was supported by a Sir Henry Wellcome Postdoctoral Fellowship (100145/Z/12/Z) and M.A.C. is supported by a Marie Sk1odowska-Curie Postdoctoral Fellowship (agreement no. 845939). T.S. acknowledges support from the European Research Council (Advanced Grant, project 323042). A.C. receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 820102). T.C., J.A., J.W.M., and T.S. acknowledge also the support of the Spanish Ministry of Economy, Industry and Competitiveness to the CRG-EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme of the Generalitat de Cataluny

Biobased, Internally pH-Sensitive Materials: Immobilized Yellow Fluorescent Protein as an Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Matrices

The pH is fundamental to biological function and its measurement therefore crucial across all biosciences. Unlike homogenous bulk solution, solids often feature internal pH gradients due to partition effects and confined biochemical reactions. Thus, a full spatiotemporal mapping for pH characterization in solid materials with biological systems embedded in them is essential. In here, therefore, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is presented. A genetically encoded ratiometric pH sensor, the enhanced superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of different materials, including natural and synthetic organic polymers as well as silica frameworks. By using controlled, tailor-made immobilization, sYFP is homogenously distributed within these materials and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. Evolution of internal pH is monitored in consequence of a proton-releasing enzymatic reaction, the hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix. Unlike optochemical pH sensors, which often interfere with biological function, labeling with sYFP enables pH sensing without altering the immobilized enzyme’s properties in any of the materials used. Fast response of sYFP to pH change permits evaluation of biochemical kinetics within the solid materials. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of biology confined to the internally heterogeneous environment of solid matrices.Spanish Ministry of Economy and CompetitivenessRamón Areces FoundationDepto. de Ingeniería Química y de MaterialesFac. de Ciencias QuímicasTRUEpu

Selection and Characterization of Artificial Proteins Targeting the Tubulin α Subunit

International audienceMicrotubules are cytoskeletal filaments of eukaryotic cells made of αβ-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an αRep library that are specific to α-tubulin. Turbidity experiments indicate that these αReps impede microtubule assembly in a dose-dependent manner and total internal reflection fluorescence microscopy further shows that they specifically block growth at the microtubule (-) end. Structural data indicate that they do so by targeting the α-tubulin longitudinal surface. Interestingly, in one of the complexes studied, the α subunit is in a conformation that is intermediate between the ones most commonly observed in X-ray structures of tubulin and those seen in the microtubule, emphasizing the plasticity of tubulin. These α-tubulin-specific αReps broaden the range of tools available for the mechanistic study of microtubule dynamics and its regulation

Biobased, Internally pH-Sensitive Materials: Immobilized Yellow Fluorescent Protein as an Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Matrices

The pH is fundamental to biological function and its measurement therefore crucial across all biosciences. Unlike homogenous bulk solution, solids often feature internal pH gradients due to partition effects and confined biochemical reactions. Thus, a full spatiotemporal mapping for pH characterization in solid materials with biological systems embedded in them is essential. In here, therefore, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is presented. A genetically encoded ratiometric pH sensor, the enhanced superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of different materials, including natural and synthetic organic polymers as well as silica frameworks. By using controlled, tailor-made immobilization, sYFP is homogenously distributed within these materials and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. Evolution of internal pH is monitored in consequence of a proton-releasing enzymatic reaction, the hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix. Unlike optochemical pH sensors, which often interfere with biological function, labeling with sYFP enables pH sensing without altering the immobilized enzyme’s properties in any of the materials used. Fast response of sYFP to pH change permits evaluation of biochemical kinetics within the solid materials. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of biology confined to the internally heterogeneous environment of solid matrices

Biobased, Internally pH-Sensitive Materials: Immobilized Yellow Fluorescent Protein as an Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Matrices

The pH is fundamental to biological function and its measurement therefore crucial across all biosciences. Unlike homogenous bulk solution, solids often feature internal pH gradients due to partition effects and confined biochemical reactions. Thus, a full spatiotemporal mapping for pH characterization in solid materials with biological systems embedded in them is essential. In here, therefore, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is presented. A genetically encoded ratiometric pH sensor, the enhanced superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of different materials, including natural and synthetic organic polymers as well as silica frameworks. By using controlled, tailor-made immobilization, sYFP is homogenously distributed within these materials and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. Evolution of internal pH is monitored in consequence of a proton-releasing enzymatic reaction, the hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix. Unlike optochemical pH sensors, which often interfere with biological function, labeling with sYFP enables pH sensing without altering the immobilized enzyme’s properties in any of the materials used. Fast response of sYFP to pH change permits evaluation of biochemical kinetics within the solid materials. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of biology confined to the internally heterogeneous environment of solid matrices

Biobased, Internally pH-Sensitive Materials: Immobilized Yellow Fluorescent Protein as an Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Matrices

The pH is fundamental to biological function and its measurement therefore crucial across all biosciences. Unlike homogenous bulk solution, solids often feature internal pH gradients due to partition effects and confined biochemical reactions. Thus, a full spatiotemporal mapping for pH characterization in solid materials with biological systems embedded in them is essential. In here, therefore, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is presented. A genetically encoded ratiometric pH sensor, the enhanced superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of different materials, including natural and synthetic organic polymers as well as silica frameworks. By using controlled, tailor-made immobilization, sYFP is homogenously distributed within these materials and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. Evolution of internal pH is monitored in consequence of a proton-releasing enzymatic reaction, the hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix. Unlike optochemical pH sensors, which often interfere with biological function, labeling with sYFP enables pH sensing without altering the immobilized enzyme’s properties in any of the materials used. Fast response of sYFP to pH change permits evaluation of biochemical kinetics within the solid materials. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of biology confined to the internally heterogeneous environment of solid matrices