Aurora B SUMOylation Is Restricted to Centromeres in Early Mitosis and Requires RANBP2.

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.

Importin-ß and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function.

Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2-SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-ß and CRM1 play essential roles in localising the RANBP2-SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO-conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2.

CEC enantioseparations of carboxylic acids on silica-based monoliths modified with ergot alkaloid derivative.

The enantioseparation of carboxylic acids, including amino acid dansyl-derivatives, 2-arylpropionic acids and 2-aryloxypropionic acids, was tested by CEC on a porous silica sol-gel monolithic column that was prepared by polycondensation of tetramethoxysilane in acidic conditions and post-gelation heat treatment (120 degrees C for 3 h) in the presence of urea, and successively, by anchoring to the silica (+)-1-allyl-(5R,8S,10R)-terguride as the chiral selector. The bimodal structure of the sorbent showed through pores with a median value of 1.39 mum and a mesopore size distribution ranging between 6 and 12 nm (average pore size of 9.9 nm). To attain optimum separation conditions, the influence of the pH and the concentration of the buffer solution in the mobile phase on resolution were investigated. The monolithic column showed: (i) for the compounds studied resolution values two or three times higher in comparison with previously developed separation systems where the same chiral selector was used. For example, on the monolithic column Dns-serine enantiomers were much better separated (8 min with a selectivity factor of 1.34) than by HPLC (20 min, alpha=1.17); (ii) high chemical and mechanical stability as demonstrated by the use of such column for hundreds of analysis along about 1 year without significant variations of the resolution and the retention parameters.