Structural model for differential cap maturation at growing microtubule ends.

Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release.

Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland.

Staphylococcus aureus is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. S. aureus naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. Whether EVs play a role in a mastitis context is still unknown. In this work, we showed that S. aureus Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs in vitro with a designated protein content. Purified S. aureus N305-secreted EVs were not cytotoxic when tested in vitro on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similar to those induced by live S. aureus N305. The in vivo immune response to purified S. aureus N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live S. aureus N305, heat-killed S. aureus N305 and to S. aureus lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 h post-inoculation. Live S. aureus induced a significantly stronger inflammatory response than that of any other condition tested. Nevertheless, S. aureus N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary S. aureus N305-secreted EVs was comparable to that of heat-killed S. aureus N305 and, partly, by LTA. These results demonstrated that S. aureus N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen after intramammary injection. Overall, our combined in vitro and in vivo data suggest that EVs are worth to be investigated to better understand the S. aureus pathogenesis and are relevant tools to develop strategies against bovine S. aureus mastitis.

The 90-kDa heat shock protein Hsp90 protects tubulin against thermal denaturation.

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues.

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.