YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

<p>Abstract</p> <p>Background</p> <p>YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.</p> <p>Results</p> <p>We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly <it>in vitro</it>. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.</p> <p>Conclusion</p> <p>These results suggest that YB-1 may regulate microtubule assembly <it>in vivo </it>and that its interaction with tubulin may contribute to the control of mRNA translation.</p

Polyamine Sharing between Tubulin Dimers Favours Microtubule Nucleation and Elongation via Facilitated Diffusion

We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends). This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine) are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics

Etude fonctionnelle d'une structure conservée de la sous-unité d'enveloppe gp120 d'un isolat primaire du VIH-1

Le virus de l'immunodéficience humaine VIH-1 est responsable, chez l'homme, de la perte des lymphocytes T CD4+ et du syndrome de l'immunodéficience acquise (SIDA). L'entrée du virus dans les cellules cibles est effectuée par la glycoprotéine d'enveloppe composée de la sous-unité de surface gp120 et de la sous-unité transmembranaire gp41. La gp120 qui est la sous-unité la plus exposée permet la liaison du virus aux récepteurs cellulaires. Le récepteur CD4 initie l'interaction avec la gp120 et cette interaction provoque des changements conformationnels qui permettent l'interaction avec un co-récepteur appartenant à la famille des récepteurs aux chimiokines comme les récepteurs CXCR4 (virus lymphotropes) et CCR5 (virus monocytotropes). Dans le but d'étudier la gp120 de l'isolat primaire macrophage-tropique YU2, des mutations ont été effectuées dans une région très conservée de la gp120 chez les rétrovirus. Les résidus concernés appartiennent aux brins ?16 et ?17 qui composent une épingle à cheveux qui module la liaison aux récepteurs CD4 et CCR5. Ainsi, il semblerait que cet élément de structure de la gp120 joue un rôle charnière entre le site d'interaction au récepteur CD4 et le site supposé d'interaction avec le co-récepteur. La structure de la gp120 ainsi que sa liaison au récepteur CD4 ne sont pas affectées lorsque la substitution E381A est réalisée. Cependant, E381 joue un rôle critique dans la liaison au co-récepteur CCR5 par le biais d'un pont salin avec K207. Il a également été montré que d'autres résidus de l'épingle à cheveux ?16/?17 jouent un rôle important lors des interactions, comme les phénylalanines F376 et F383. Ainsi, les résultats obtenus suggèrent que l'épingle à cheveux ?16/?17 gouverne les interactions entre la gp120 et le co-récepteur CCR5. Les mutations effectuées ont un impact direct sur la surface du pont interdomaine et sur le site d'interaction avec le récepteur CD4 ainsi que sur le site supposé d'interaction avec le co-récepteur CCR5. Ces résultats éclaircissent le mécanisme de liaison au co-récepteur CCR5 et contribuent à la compréhension de l'entrée du VIH-1, ils pourraient être pris en compte pour la conception de molécules inhibitrices de l'entrée virale.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Atomic force microscopy reveals binding of mRNA to microtubules mediated by two major mRNP proteins YB-1 and PABP

International audienceA significant fraction of mRNAs is known to be asso-ciated in the form of mRNPs with microtubules for active trans-port. However, little is known about the interaction betweenmRNPs and microtubules and most of previous works were fo-cused on molecular motor:microtubule interactions. Here, wehave identified, via high resolution atomic force microscopyimaging, a significant binding of mRNA to microtubules medi-ated by two major mRNP proteins, YB-1 and PABP. This inter-action with microtubules could be of critical importance foractive mRNP traffic and for mRNP granule formation. A similarrole may be fulfilled by other cationic mRNA partners

Atomic Force Microscopy Imaging of DNA under Macromolecular Crowding Conditions

International audienceStudying the influence of macromolecular crowding at high ionic strengths on assemblies of biomolecules is of particular interest because these are standard intracellular conditions. However, up to now, no techniques offer the possibility of studying the effect of molecular crowding at the single molecule scale and at high resolution. We present a method to observe double-strand DNA under macromolecular crowding conditions on a flat mica surface by atomic force microscope. By using high concentrations of monovalent salt ([NaCl] > 100 mM), we promote DNA adsorption onto NiCl2 pretreated muscovite mica. It therefore allows analysis of DNA conformational changes and DNA compaction induced by polyethylene glycol (PEG), a neutral crowding agent, at physiological concentrations of monovalent salt

Mica Surface Promotes the Assembly of Cytoskeletal Proteins

International audienceWe report the surface-mediated polymerization of FtsZ protein, the prokaryote homologue of tubulin, by AFM.FtsZ protein can form filaments on mica whereas the bulk FtsZ concentration is orders of magnitude lower than thecritical concentration. Surface polymerization is favored by a local increase in protein concentration and requires ahigh mobility of proteins on the surface. To generalize to other cytoskeleton protein, we also show that mica can initiatethe formation of tubulin protofilaments. This study is of particular interest for studying cytoskeletal protein dynamicsby AFM but also for the surface autoassembly of nanostructures

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation

International audienc

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation

International audienceDevelopment of SpiCee, a genetically encoded calcium scavenger d SpiCee inhibits calcium-dependent downstream pathways d SpiCee enables cell-specific manipulation of calcium-dependent processes in vivo d Subcellular targeting confers cell compartment specificity to SpiCee In Brief Ros et al. develop SpiCee, a genetically encoded calcium chelator that enables the manipulation of this second messenger in single cells with subcellular specificity. SpiCee alters the migration of developing cortical neurons in vivo. Targeting of lipid rafts prevents axonal repulsion, whereas exclusion from this subcellular compartment does not

SponGee: A Genetic Tool for Subcellular and Cell-Specific cGMP Manipulation

International audiencecGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation