Mica surface promotes the assembly of cytoskeletal proteins.

We 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 the critical concentration. Surface polymerization is favored by a local increase in protein concentration and requires a high mobility of proteins on the surface. To generalize to other cytoskeleton protein, we also show that mica can initiate the formation of tubulin protofilaments. This study is of particular interest for studying cytoskeletal protein dynamics by AFM but also for the surface autoassembly of nanostructures.

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.

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

BACKGROUND: 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. RESULTS: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. 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. CONCLUSION: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.

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

A significant fraction of mRNAs is known to be associated in the form of mRNPs with microtubules for active transport. However, little is known about the interaction between mRNPs and microtubules and most of previous works were focused on molecular motor:microtubule interactions. Here, we have identified, via high resolution atomic force microscopy imaging, a significant binding of mRNA to microtubules mediated by two major mRNP proteins, YB-1 and PABP. This interaction with microtubules could be of critical importance for active mRNP traffic and for mRNP granule formation. A similar role may be fulfilled by other cationic mRNA partners.

Atomic force microscopy imaging of DNA under macromolecular crowding conditions.

Studying 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 NiCl 2 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.

Highly conserved beta16/beta17 beta-hairpin structure in human immunodeficiency virus type 1 YU2 gp120 is critical for CCR5 binding.

Whereas gp120 CD4-induced structures have been largely documented and at least in part elucidated by crystallization, information about gp120 coreceptor-induced structures remains incomplete despite numerous studies. In this work, mutations were carried out in a selected internal region of HIV-1/YU2 gp120, proximal to the CD4-binding site, because of its highly conserved nature among retroviruses and its high structural stability. The targeted residues, belonging to the beta16/beta17 beta-hairpin, modulate gp120 binding to CD4 and gp120-CD4 complex binding to CCR5. Thus, it appears that this gp120 structure acts as a hinge between the CD4-binding site and the putative coreceptor binding structure. Substitution of amino acid residues like E381A did not affect gp120 binding to CD4 and did not induce significant structural changes in gp120, as demonstrated by epitope analysis, BIACORE analysis, and circular dichroism. Nevertheless, E381 has a critical influence on the maintenance of CCR5 coreceptor binding by forming a salt bridge with K207. Another important element of the beta-hairpin in this interaction is the probable hydrophobic link between F383 and I420. Altogether, these results suggest that the beta-hairpin structure likely governs interactions between the surface of gp120 with native CCR5 or the CCR5 amino-terminal domain (CCR5-Nt). The mutations within the beta-hairpin had a direct effect on the proximal surface of the bridging sheet, the putative CCR5 surface, and the gp120 YU2 HIV-1-CD4 binding site. These results on the gp120-CCR5-Nt binding mechanism contribute to our understanding of CCR5 and HIV-1 gp120 association and HIV-1 entry; they may also contribute to designing novel inhibitors.

A high-throughput fluorescence polarization assay specific to the CD4 binding site of HIV-1 glycoproteins based on a fluorescein-labelled CD4 mimic.

The three-dimensional structure of CD4M33, a mimic of the host-cell receptor-antigen CD4 and a powerful inhibitor of CD4-gp120 (viral envelope glycoprotein 120) interaction and HIV-1 entry into cells [Martin, Stricher, Misse, Sironi, Pugniere, Barthe, Prado-Gotor, Freulon, Magne, Roumestand et al. (2003) Nat. Biotechnol. 21, 71-76], was solved by 1H-NMR and its structure was modelled in its complex with gp120. In this complex, CD4M33 binds in a CD4-like mode and inserts its unnatural and prominent Bip23 (biphenylalanine-23) side-chain into the gp120 interior 'Phe43 cavity', thus filling its volume. CD4M33 was specifically labelled with fluorescein and shown by fluorescence anisotropy to bind to different gp120 glycoproteins with dissociation constants in the nanomolar range. Fluorescent CD4M33 was also used in a miniaturized 384-well-plate assay to study direct binding to a large panel of gp120 glycoproteins and in a competition assay to study binding of CD4 or other ligands targeting the CD4 binding site of gp120. Furthermore, by using the fluorescently labelled CD4M33 and the [Phe23]M33 mutant, which possesses a natural Phe23 residue and thus cannot penetrate the gp120 Phe43 cavity, we show that a recently discovered small-molecule-entry inhibitor, BMS-378806, does not target the CD4 binding site nor the Phe43 cavity of gp120. The fluorescently labelled CD4M33 mimic, its mutants and their derivatives represent useful tools with which to discover new molecules which target the CD4 binding site and/or the Phe43 cavity of gp120 glycoproteins in a high-throughput fluorescence-polarization assay and to characterize their mechanism of action.