Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

Background

Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.

Methodology/Principal Findings

We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.

Conclusions/Significance

These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.     

Synthesis of some epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives and evaluation of their biological activity

Steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives have been prepared using 3beta,17beta-dihydroxy-17alpha-picolyl-androst-5-ene (1), 3beta-acetoxy-17-picolinylidene-androst-5-ene (2), and 3beta-hydroxy-17-picolinylidene-androst-5-ene (3) as synthetic precursors. The compounds 2 and/or 3 were reacted with m-chloroperoxybenzoic acid (MCPBA). The compounds synthesized from 2 were 17-picolinylidene-N-oxide 4, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 5 and 6, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 7 and 8. Starting from compound 3, a mixture of 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene 9 and 10, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 11 and 12, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 13 and 14 were obtained. From compounds 15 and 18, obtained from 1 and 3 by the Oppenauer oxidation, the 4alpha,5alpha-epoxy and 4beta,5beta-epoxy derivatives 16, 17 and 20, 21 were prepared by oxidation with 30% H(2)O(2). Oxidation of 18 with MCPBA yielded only the N-oxide 19. The structures of compounds 15 and 18 were proved by the X-ray analysis. Compounds 1-6, 9, 15, 17, 18, and 21 were tested on activity against the enzyme aromatase. Antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Three tested compounds (1, 4, and 19) showed strong activity against PC3, the IC(50) values being in the range 0.55-10microM, whereas compound 17 showed strong activity against MDA-MB-231 (IC(50) 10.4microM).     

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in <Emphasis Type="Italic">Xenopus</Emphasis>

Background  

Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes.     

Perineurial Barrier Glia Physically Respond to Alcohol in an Akap200-Dependent Manner to Promote Tolerance

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Climate change,biological invasions,and the shifting distribution of Mediterranean fishes: A large‐scale survey based on local ecological knowledge

Climate change and biological invasions are rapidly reshuffling species distribution, restructuring the biological communities of many ecosystems worldwide. Tracking these transformations in the marine environment is crucial, but our understanding of climate change effects and invasive species dynamics is often hampered by the practical challenge of surveying large geographical areas. Here, we focus on the Mediterranean Sea, a hot spot for climate change and biological invasions to investigate recent spatiotemporal changes in fish abundances and distribution. To this end, we accessed the local ecological knowledge (LEK) of small‐scale and recreational fishers, reconstructing the dynamics of fish perceived as “new” or increasing in different fishing areas. Over 500 fishers across 95 locations and nine different countries were interviewed, and semiquantitative information on yearly changes in species abundance was collected. Overall, 75 species were mentioned by the respondents, mostly warm‐adapted species of both native and exotic origin. Respondents belonging to the same biogeographic sectors described coherent spatial and temporal patterns, and gradients along latitudinal and longitudinal axes were revealed. This information provides a more complete understanding of the shifting distribution of Mediterranean fishes and it also demonstrates that adequately structured LEK methodology might be applied successfully beyond the local scale, across national borders and jurisdictions. Acknowledging this potential through macroregional coordination could pave the way for future large‐scale aggregations of individual observations, increasing our potential for integrated monitoring and conservation planning at the regional or even global level. This might help local communities to better understand, manage, and adapt to the ongoing biotic transformations driven by climate change and biological invaders.