Mitotic Centromere–associated Kinesin Is Important for Anaphase Chromosome Segregation

Mitotic centromere–associated kinesin (MCAK) is recruited to the centromere at prophase and remains centromere associated until after telophase. MCAK is a homodimer that is encoded by a single gene and has no associated subunits. A motorless version of MCAK that binds centromeres but not microtubules disrupts chromosome segregation during anaphase. Antisense-induced depletion of MCAK results in the same defect. MCAK overexpression induces centromere-independent bundling and eventual loss of spindle microtubule polymer suggesting that centromere-associated bundling and/or depolymerization activity is required for anaphase. Live cell imaging indicates that MCAK may be required to coordinate the onset of sister centromere separation.     

Neuroendocrine correlates of behavioral polymorphism in white-throated sparrows

Interspecific differences in the neuropeptide systems of the lateral septum (LS) often parallel differences in social behavior. In rodents, some closely related species that differ in aggressive behavior also differ according to the level of vasopressin (VP) innervation of the LS. In songbirds, the neuropeptides vasotocin (VT) and vasoactive intestinal peptide (VIP) affect aggression when administered directly to the LS. Here, we tested whether the density of VT or VIP innervation of the LS reflects patterns of intraspecific behavioral polymorphism in male and female white-throated sparrows (Zonotrichia albicollis), in which the "white-stripe" (WS) morph behaves more aggressively than the "tan-stripe" (TS) morph. We found that the WS birds had more VT-immunoreactivity (IR) than the TS birds in the ventrolateral subdivision of the caudal LS (LSc.vl) and in the medial portion of the bed nucleus of the stria terminalis (BSTm). In addition, the TS birds had more densely stained VIP-IR in the LSc.vl than the WS birds. Males had more VT-IR than females in the LSc.vl and BSTm, and more VIP-IR in the LSc.vl. We also report sex and morph differences in VIP-IR in the basal hypothalamus, where VIP is synthesized and released into the portal vasculature. Males had nearly twice as many VIP-immunoreactive (ir) neurons in the infundibular nucleus than did females, and birds of the WS morph had more densely stained VIP-IR in the median eminence than TS birds. Our results support the hypothesis that differences in these neuropeptide systems underlie inter- and intraspecific differences in social behavior across vertebrates.     

Molecular dissection of the microtubule depolymerizing activity of mitotic centromere-associated kinesin

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only 30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest functional MCAK deletion constructs are monomers. These results define a highly conserved domain within MCAK and related (KIN I) kinesins that is critical for depolymerization activity and show that this depolymerization is not dependent on MCAK dimerization.     

Reactive oxygen species delay control of lymphocytic choriomeningitis virus

Cluster of differentiation (CD)8⁺ T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8⁺ T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1^−/−) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage.     

Evaluation of reference genes for quantitative real-time PCR in the brain,pituitary, and gonads of songbirds

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.     

Rapid effects of hearing song on catecholaminergic activity in the songbird auditory pathway

Catecholaminergic (CA) neurons innervate sensory areas and affect the processing of sensory signals. For example, in birds, CA fibers innervate the auditory pathway at each level, including the midbrain, thalamus, and forebrain. We have shown previously that in female European starlings, CA activity in the auditory forebrain can be enhanced by exposure to attractive male song for one week. It is not known, however, whether hearing song can initiate that activity more rapidly. Here, we exposed estrogen-primed, female white-throated sparrows to conspecific male song and looked for evidence of rapid synthesis of catecholamines in auditory areas. In one hemisphere of the brain, we used immunohistochemistry to detect the phosphorylation of tyrosine hydroxylase (TH), a rate-limiting enzyme in the CA synthetic pathway. We found that immunoreactivity for TH phosphorylated at serine 40 increased dramatically in the auditory forebrain, but not the auditory thalamus and midbrain, after 15 min of song exposure. In the other hemisphere, we used high pressure liquid chromatography to measure catecholamines and their metabolites. We found that two dopamine metabolites, dihydroxyphenylacetic acid and homovanillic acid, increased in the auditory forebrain but not the auditory midbrain after 30 min of exposure to conspecific song. Our results are consistent with the hypothesis that exposure to a behaviorally relevant auditory stimulus rapidly induces CA activity, which may play a role in auditory responses.