Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.     

The tubulin-depolymerising agent combretastatin-4 induces ectopic aster assembly and mitotic catastrophe in lung cancer cells H460

The relationship between microtubular dynamics, dismantling of pericentriolar components and induction of apoptosis was analysed after exposure of H460 non-small lung cancer cells to anti-mitotic drugs. The microtubule destabilising agent, combretastatin-A4 (CA-4) led to microtubular array disorganization, arrest in mitosis and abnormal metaphases, accompanied by the presence of numerous centrosome-independent “star-like” structures containing tubulin and aggregates of pericentrosomal matrix components like γ-tubulin, pericentrin and ninein, whereas the structural integrity of centrioles was not affected by treatment. On the contrary, in condition of prolonged exposure or high concentrations of CA-4 such aggregates never formed. Treatment with 7.5 nM CA-4, which produced a high frequency “star-like” aggregates, was accompanied by mitotic catastrophe commitment characterized by translocation of the proapoptotic Bim protein to mitochondria activation of caspases-3/9 and DNA fragmentation as a result of either prolonged metaphase arrest or attempt of cells to divide. Drug concentrations which fail to block cells at mitosis were also unable to activate apotosis. A detailed time-course analysis of cell cycle arrest and apoptosis indicated that after CA-4 washout the number of metaphases with “star-like” structures decreased as a function of time and arrested cells proceeded in anaphase. After 4 h, the multiple α- and γ-tubulin aggregates coalesced into two well-defined spindles in a bipolar mitotic spindle organization. Overall, our findings suggest that the maintenance of microtubular integrity plays a relevant role in stabilising the pericentriolar matrix, whose dismantling can be associated with apoptosis after exposure to microtubule depolymerising agents.     

Identification of quantitative trait loci and candidate genes for an anxiolytic‐like response to ethanol in BXD recombinant inbred strains

Genetic differences in acute behavioral responses to ethanol contribute to the susceptibility to alcohol use disorder and the reduction of anxiety is a commonly reported motive underlying ethanol consumption among alcoholics. Therefore, we studied the genetic variance in anxiolytic‐like responses to ethanol across the BXD recombinant inbred (RI) mouse panel using the light–dark transition model of anxiety. Strain‐mean genetic mapping and a mixed‐model quantitative trait loci (QTL) analysis replicated several previously published QTL for locomotor activity and identified several novel anxiety‐related loci. Significant loci included a chromosome 11 saline anxiety‐like QTL (Salanq1) and a chromosome 12 locus (Etanq1) influencing the anxiolytic‐like response to ethanol. Etanq1 was successfully validated by studies with BXD advanced intercross strains and fine‐mapped to a region comprising less than 3.5 Mb. Through integration of genome‐wide mRNA expression profiles of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens and ventral midbrain) across the BXD RI panel, we identified high priority candidate genes within Etanq1, the strongest of which was Ninein (Nin), a Gsk3β‐interacting protein that is highly expressed in the brain.