Biology subjects

Olieric, N.

Publications and source records attributed to Olieric, N..

5 recordsLinked to original sources

bioRxiv2021

Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

Paclitaxel (Taxol(R)) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drugs mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 [A] resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the {beta}M loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, binding of the taxane core to the taxane site displaces the S9-S10 loop of {beta}-tubulin resulting in microtubule expansion. Our results provide detailed new insights into the mechanism of microtubule-stabilization by taxanes.

biophysics↗

bioRxiv2020

The mechanism of selective kinesin inhibition by kinesin binding protein.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, crescent-shaped right-handed -solenoid that sequesters the tubulin-binding surface of the kinesin motor domain, structurally distorting the motor domain and sterically blocking MT attachment. KBP uses its -solenoid concave face and edge loops to bind the kinesin motor domain and selective mutation of this extended binding surface disrupts KBP inhibition of kinesin transport in cells. The KBP-interacting surface of the motor domain contains motifs exclusively conserved in KBP-interacting kinesins, providing a basis for kinesin selectivity.

biochemistry↗

bioRxiv2020

The inner scaffold protects from centriole fracture

Centrioles are characterized by a nine-fold arrangement of long-lived microtubule triplets that are held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy (U-ExM) for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the centriolar microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein with a predicted structurally homology with the ciliary inner junction protein FAP20. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in both human and Chlamydomonas cells. Altogether, this work highlights that POC16/WDR90 is a crucial evolutionary conserved molecular player participating in centriole architecture integrity.

cell biology↗

bioRxiv2019

Photoswitchable microtubule inhibitors enabling robust, GFP-orthogonal optical control over the tubulin cytoskeleton

Here we present GFP-orthogonal optically controlled reagents for reliable and repetitive in cellulo modulation of microtubule dynamics and its dependent processes. Optically controlled reagents (\"photopharmaceuticals\") have developed into powerful tools for high-spatiotemporal-precision control of endogenous biology, with numerous applications in neuroscience, embryology, and cytoskeleton research. However, the restricted chemical domain of photopharmaceutical scaffolds has constrained their properties and range of applications. Styrylbenzothiazoles are an as-yet unexplored scaffold for photopharmaceuticals, which we now rationally design to feature potent photocontrol, switching microtubule cytoskeleton function off and on according to illumination conditions. We show more broadly that this scaffold is exceptionally chemically and biochemically robust as well as substituent-tolerant, and offers particular advantages for intracellular biology through a range of desirable photopharmaceutical and drug-like properties not accessible to the current classes of photoswitches. We expect that these reagents will find powerful applications enabling robust, high precision, optically controlled cell biological experimentation in cytoskeleton research and beyond.

cell biology↗

bioRxiv2019

Mechanisms of motor-independent membrane remodeling driven by dynamic microtubules

Microtubule-dependent organization of membranous organelles, such as the endoplasmic reticulum, occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. How highly transient protein-protein interactions occurring at growing microtubule tips can induce load-bearing processive motion is currently unclear. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, End-Binding (EB) proteins and a membrane-targeted protein that interacts with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation by growing microtubule ends, motor-independent membrane sliding along microtubule shafts and pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Force spectroscopy revealed that attachments to growing and shrinking microtubule ends can sustain forces of [~]0.5 and [~]5 pN, respectively. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear sufficient load to induce membrane deformation and motility.

cell biology↗