Kif15 Cooperates with Eg5 to Promote Bipolar Spindle Assembly

SummaryBackgroundThe formation of a bipolar spindle is an essential step during cell division. Bipolar spindle assembly is driven by the highly conserved microtubule motor Eg5 (kinesin-5), which can slide antiparallel microtubules apart to drive centrosome separation. However, it is currently unclear whether and how additional motors can contribute to centrosome separation and bipolar spindle formation.ResultsWe have developed a novel assay to identify motors involved in spindle bipolarity; via this assay, we identify Kif15/Hklp2 (kinesin-12, hereafter referred to as Kif15). Kif15 is not required for spindle bipolarity in cells with full Eg5 activity but becomes essential when Eg5 is partially inhibited. We show that the primary function of Kif15 is to promote spindle elongation and to ensure maintenance of spindle bipolarity. Nonetheless, ectopic expression of Kif15 can fully reconstitute bipolar spindle assembly in the absence of Eg5 activity, demonstrating that Kif15 can replace all essential functions of Eg5 in bipolar spindle assembly. Importantly, this activity of Kif15 depends on its interaction with the microtubule-associated protein TPX2, indicating that a Kif15-TPX2 complex promotes centrosome separation.ConclusionsThese findings show that, similar to Eg5, Kif15 can drive centrosome separation during bipolar spindle assembly. For this activity, Kif15 requires both its motor domain and its interaction with TPX2. Based on these data, we propose that a complex of Kif15 and TPX2 can crosslink and slide two antiparallel microtubules apart, thereby driving centrosome separation

Elevated serum levels of soluble CD14 in HBeAg-positive chronic HBV patients upon Peginterferon treatment are associated with treatment response

Pegylated IFNα (PEG-IFN) is one of the treatment options for chronic HBV (CHB) patients. However, the high patient treatment burden and limited response rate together clearly ask for biomarkers to predict PEG-IFN response. Soluble CD14 (sCD14) is considered a marker for immune activation and has been shown to predict clinical outcome of HIV infection. However, studies on sCD14 in CHB infection are inconclusive, and its relationship with clinical outcome is largely unknown. Here, we measured sCD14 levels in CHB patients and investigated whether changes in sCD14 level related to PEG-IFN response. Serum sCD14 levels were determined in 15 healthy controls, 15 acute self-limited HBV, 60 CHB patients in different disease phases and 94 HBeAg+ CHB patients at week 0 and week 12 of a 52-week PEG-IFN treatment. Response to PEG-IFN treatment was defined as HBeAg seroconversion or HBeAg loss at 26 weeks post-treatment. The mean sCD14 level in acute HBV patients (3.0 µg/mL) was significantly higher than in CHB patients (2.4 µg/mL) and healthy controls (2.4 µg/mL). In CHB patients receiving PEG-IFN, a significant increase in sCD14 was found after 12-week treatment (median week 0:2.1 µg/mL; week 12:3.7 µg/mL). After 12-week treatment, the fold change (FC = w12/w0) in sCD14 was significantly higher in responders compared to nonresponders (HBeAg seroconversion: median FCresponder = 2.1 vs FCnonresponder = 1.6; HBeAg loss: median FCresponder = 2.2 vs FCnonresponder = 1.5). Receiver operating characteristic curves demonstrated that FC-sCD14wk12/wk0 levels can be of significant value as a stopping rule to select patients at week 12 who are not likely to benefit from further PEG-IFN treatment

Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803

BACKGROUND: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product. RESULTS: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level. CONCLUSION: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription

Heterochromatin: Guardian of the Genome.

Constitutive heterochromatin is a major component of the eukaryotic nucleus and is essential for the maintenance of genome stability. Highly concentrated at pericentromeric and telomeric domains, heterochromatin is riddled with repetitive sequences and has evolved specific ways to compartmentalize, silence, and repair repeats. The delicate balance between heterochromatin epigenetic maintenance and cellular processes such as mitosis and DNA repair and replication reveals a highly dynamic and plastic chromatin domain that can be perturbed by multiple mechanisms, with far-reaching consequences for genome integrity. Indeed, heterochromatin dysfunction provokes genetic turmoil by inducing aberrant repeat repair, chromosome segregation errors, transposon activation, and replication stress and is strongly implicated in aging and tumorigenesis. Here, we summarize the general principles of heterochromatin structure and function, discuss the importance of its maintenance for genome integrity, and propose that more comprehensive analyses of heterochromatin roles in tumorigenesis will be integral to future innovations in cancer treatment

Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A.

Repair of DNA double-strand breaks (DSBs) must be orchestrated properly within diverse chromatin domains in order to maintain genetic stability. Euchromatin and heterochromatin domains display major differences in histone modifications, biophysical properties, and spatiotemporal dynamics of DSB repair. However, it is unclear whether differential histone-modifying activities are required for DSB repair in these distinct domains. We showed previously that the Drosophila melanogaster KDM4A (dKDM4A) histone demethylase is required for heterochromatic DSB mobility. Here we used locus-specific DSB induction in Drosophila animal tissues and cultured cells to more deeply interrogate the impact of dKDM4A on chromatin changes, temporal progression, and pathway utilization during DSB repair. We found that dKDM4A promotes the demethylation of heterochromatin-associated histone marks at DSBs in heterochromatin but not euchromatin. Most importantly, we demonstrate that dKDM4A is required to complete DSB repair in a timely manner and regulate the relative utilization of homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways but exclusively for heterochromatic DSBs. We conclude that the temporal kinetics and pathway utilization during heterochromatic DSB repair depend on dKDM4A-dependent demethylation of heterochromatic histone marks. Thus, distinct pre-existing chromatin states require specialized epigenetic alterations to ensure proper DSB repair

Intravital FRET Imaging of Tumor Cell Viability and Mitosis during Chemotherapy

<div><p>Taxanes, such as docetaxel, are microtubule-targeting chemotherapeutics that have been successfully used in the treatment of cancer. Based on data obtained from cell cultures, it is believed that taxanes induce tumor cell death by specifically perturbing mitotic progression. Here, we report on data that suggest that this generally accepted view may be too simplified. We describe a high-resolution intravital imaging method to simultaneously visualize mitotic progression and the onset of apoptosis. To directly compare <i>in vitro</i> and <i>in vivo</i> data, we have visualized the effect of docetaxel on mitotic progression in mouse and human colorectal tumor cell lines both <i>in vitro</i> and in isogenic tumors in mice. We show that docetaxel-induced apoptosis <i>in vitro</i> occurs via mitotic cell death, whereas the vast majority of tumor cells in their natural environment die independent of mitotic defects. This demonstrates that docetaxel exerts its anti-tumor effects <i>in vivo</i> through means other than mitotic perturbation. The differences between <i>in vitro</i> and <i>in vivo</i> mechanisms of action of chemotherapeutics may explain the limited response to many of the anti-mitotic agents that are currently validated in clinical trials. Our data illustrate the requirement and power of our intravital imaging technique to study and validate the mode of action of chemotherapeutic agents <i>in vivo</i>, which will be essential to understand and improve their clinical efficacy.</p></div