Separating the effects of nucleotide and EB binding on microtubule structure

Zhang, Rui; LaFrance, Benjamin; Nogales, Eva

doi:10.1073/pnas.1802637115

Cited by 132 publications

(229 citation statements)

References 84 publications

(147 reference statements)

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“…5D, right), and (2) subsequent destruction of the stable 4-GTP-tubulin pocket binding site by hydrolysis of GTP-tubulin into GDP-P i (Fig. 5D, center), and, ultimately, GDP-tubulin, which may be accelerated by the binding of EB1 into its 4-tubulin pocket binding configuration (Maurer et al, 2014;Zhang et al, 2015;Zhang et al, 2018).…”

Section: Eb1 Tip Tracking By Microtubule Structure Recognitionmentioning

confidence: 99%

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

Reid

Coombes

Mukherjee

et al. 2019

Preprint

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The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends.Microtubule end targeting of EB1 has been attributed to high affinity binding of EB1 to GTPtubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, singlemolecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the conversion of growing microtubule ends from a tapered into a blunt configuration resulted in reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends. P a g e | 3

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Section: Eb1 Tip Tracking By Microtubule Structure Recognitionmentioning

confidence: 99%

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

Reid

Coombes

Mukherjee

et al. 2019

Preprint

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“…This value lies between those of the compact tubulin lattice of GDP-microtubules (with or without EB protein decoration) (81.7 -81.5 Å) 51 and the loose lattice of GTP analog GMPCPPmicrotubules (83.9 Å) 51 reconstituted in vitro. Interestingly, intermediate values between GDPand GTP-bound states can be achieved in vitro by using a combination of non-hydrolyzable GTP analogs and EB1 (81.9 Å tubulin dimer repeat) 51 . These numbers suggest that in primary cilia, EB1 microtubule decoration stabilizes a specific lattice configuration which is an intermediate between the GTP-and the GDP-bound state.…”

Section: Discussionmentioning

confidence: 87%

“…We measured that the tubulin dimer repeat of primary cilia microtubules is about 82.5 Å. This value lies between those of the compact tubulin lattice of GDP-microtubules (with or without EB protein decoration) (81.7 -81.5 Å) 51 and the loose lattice of GTP analog GMPCPPmicrotubules (83.9 Å) 51 reconstituted in vitro. Interestingly, intermediate values between GDPand GTP-bound states can be achieved in vitro by using a combination of non-hydrolyzable GTP analogs and EB1 (81.9 Å tubulin dimer repeat) 51 .…”

Section: Discussionmentioning

confidence: 88%

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The molecular structure of primary cilia revealed by cryo-electron tomography

Kiesel

Viar

Tsoy

et al. 2020

Preprint

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Primary cilia are microtubule-based organelles involved in key signaling and sensing processes in eukaryotic cells. Unlike motile cilia, which have been thoroughly studied, the structure and the composition of primary cilia remain largely unexplored despite their fundamental role in development and homeostasis. They have for long been falsely regarded as simplified versions of motile cilia because they lack distinctive elements such as dynein arms, radial spokes, and central pair complex. However, revealing the detailed molecular composition and 3D structure of primary cilia is necessary in order to understand the mechanisms that govern their functions. Such structural investigations are so far being precluded by the challenging preparation of primary cilia for cryo-electron microscopy. Here, we developed an enabling method for investigating the structure of primary cilia at molecular resolution by cryo-electron tomography. We show that the well-known "9+0" arrangement of microtubule doublets is present only at the base of the primary cilium. A few microns away from the base the ciliary architecture changes into an unstructured bundle of EB1-decorated microtubule singlets and some actin filaments. Our results suggest the existence of a previously unobserved crosstalk between actin filaments and microtubules in the primary cilium. Our work provides unprecedented insights into the molecular structure of primary cilia and a general framework for uncovering their molecular composition and function in health and disease. This opens up new possibilities to study aspects of this important organelle that have so far been out of reach.We would like to thank the Electron Microscopy Facility (in particular Tobias Fürstenhaupt, Weihua Leng, Michaela Wilsch-Bräuninger) and the Light Microscope Facility from the Services and Facilities of the MPI-CBG for their support. We are thankful to Helin Rägel and Cécilie Martin-Lemaitre for their tips on MDCKII cell culture, Noreen Walker for the Imaris tutorial, and Tim-Oliver Buchholz for denoising cryo-tomography data. We thank Pavel Tomancak, Florian Jug, Dennis Diener, and Jan Brugues for the fruitful discussions and suggestions to the manuscript. We thank Oscar Gonzales for IT support. . developed the cryo-peel-off method, prepared samples for FM and EM imaging, acquired and reconstructed room temperature and cryo-tomograms, contributed to FM data acquisition, analysed EM and FM data, prepared figures, interpreted results, and contributed to writing and revising the manuscript. G.A.V. prepared samples and contributed to data acquisition of the room temperature tomography, analyzed cryo-EM data with subtomogram averaging and tomogram segmentation, analysed EM and FM data, prepared figures, interpreted results, and contributed to writing and revising the manuscript. N.T. analyzed cryo-ET data to average the microtubule singlets, contributed to supplementary figure preparation, contributed to the interpretation of data, and contributed to writing and revising the manuscript. R.M. pre...

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“…The consistency between studies allays two concerns about the MD calculations. First, our simulation results used as a starting point a tubulin structure with kinesin bound, which may have some modest effects on the initial structure of tubulin (76). We note, however, that the equilibration MD runs reached steady-state in ~50 ns.…”

Section: Discussionmentioning

confidence: 99%

Multi-scale Computational Modeling of Tubulin-Tubulin Lateral Interaction

Hemmat

Castle

Sachs

et al. 2019

Preprint

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Microtubules are multi-stranded polymers in eukaryotic cells that support key cellular functions such as chromosome segregation, motor-based cargo transport, and maintenance of cell polarity. Microtubules self-assemble via "dynamic instability," where the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e. what is different between the GTP-and GDP-tubulin states that enables microtubule growth and shortening, respectively? More generally, a major challenge in biology is how to connect theoretical frameworks across length-time scales, from atoms to cellular behavior. In this study, we describe a multi-scale model by linking atomistic molecular dynamics (MD), molecular Brownian dynamics (BD), and cellular-level thermokinetic (TK) modeling of microtubules. Here we investigated the underlying interaction energy landscape when tubulin dimers associate laterally by performing all-atom molecular dynamics simulations. We found that the lateral free energy is not significantly different among three nucleotide states of tubulin, GTP, GDP, and GMPCPP, and is estimated to be ≅-11 kBT. Furthermore, using MD potential energy in our BD simulations of tubulin dimers in solution confirms that the lateral bond is weak on its own with a mean lifetime of ~0.1 μs, implying that the longitudinal bond is required for microtubule assembly. We conclude that nucleotide-dependent lateral bond strength is not the key mediator microtubule dynamic instability, implying that GTP acts elsewhere to exert its stabilizing influence on microtubule polymer. Furthermore the estimated bond strength is well-aligned with earlier estimates based on thermokinetic (TK) modeling and light microscopy measurements (VanBuren et al., PNAS, 2002). Thus, we have computationally connected atomistic level structural information, obtained by cryoelectron microscopy, to cellular scale microtubule assembly dynamics using a combination of MD, BD, and TK models to bridge from Ångstroms to micrometers and from femtoseconds to minutes.

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Separating the effects of nucleotide and EB binding on microtubule structure

Cited by 132 publications

References 84 publications

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

The molecular structure of primary cilia revealed by cryo-electron tomography

Multi-scale Computational Modeling of Tubulin-Tubulin Lateral Interaction

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