Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in α-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail. INTRODUCTIONThe mitotic spindle, a unique cellular apparatus assembled at the beginning of mitosis, plays a pivotal role in regulating mitosis and cytokinesis. Although tubulins are the most abundant protein in the mitotic spindle, many additional proteins are involved in regulating its formation and function. Most prominent among these are members of the kinesin and dynein families of MT-based motor proteins (Mandelkow and Mandelkow, 2002;Vale, 2003), which generate directional movement along microtubules. The kinesin proteins share a conserved, ϳ340 amino acid, motor domain that utilizes ATP to fuel their movement along MTs. Outside the motor domain, kinesins also contain different "stalk" and "tail" domains that mediate oligomerization, regulation of motor activity, and interactions with their specific cargos (Vale, 2003).The roles of kinesins in mitosis and cytokinesis have been extensively studied in Saccharomyces cerevisiae, in which there are six kinesin genes belonging to five subfamilies. Gene disruption experiments have demonstrated that five of these kinesins play essential roles in regulating the formation, orientation, and elongation of the mitotic spindle and the segregation of chromosomes in mitosis (Hildebrandt and Hoyt, 2000). In addition, one cytoplasmic dynein motorcontaining polypeptide, the dynein heavy chain (DHC), has also been implicated in mitotic spindle function (Hildebrandt and Hoyt, 2000). Using RNAi techniques, Goshima and Vale recently examined the mitotic functions of cytoplasmic dynein and all 25 fly kinesins in cultured Drosophila cells (Goshima and Vale, 2003). They found that four kinesins are needed for bipolar spindle assembly and four are crucial for metaphase chromosome alignment. Dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is required for cytokinesis.Th...

The spindle midzone, a conspicuous network of antiparallel interdigitating nonkinetochore microtubules between separating chromosomes, plays a crucial role in regulating the initiation and completion of cytokinesis. In this study, we report the use of time-lapse microscopy and a human kinesin endoribonucleases RNase III-prepared short interfering RNA (esiRNA) library to identify Kif4 as a motor protein that translocates PRC1, a spindle midzone-associated cyclin-dependent kinase substrate protein, to the plus ends of interdigitating spindle microtubules during the metaphase-to-anaphase transition. We show that Kif4 binds to PRC1 through its ''stalk plus tail'' domains and Kif4 and PRC1 colocalize on the spindle midzone͞midbody during anaphase and cytokinesis. Suppression of Kif4 expression by Kif4 esiRNA results in the inhibition of PRC1 translocation, a block of the midzone formation, and a failure of cytokinesis. PRC1 translocation and midzone formation can be restored, and the cytokinetic defects can be rescued in Kif4 esiRNA-treated cells by coexpression of Kif4 but not its motor dead mutant Kif4md. Furthermore, we show that cyclin-dependent kinase phosphorylation of PRC1 controls the timing of PRC1 translocation by Kif4. These results, in light of the crucial role of PRC1 in midzone formation, indicate that cell cycledependent translocation of PRC1 by Kif4 is essential for midzone formation and cytokinesis.kinesin ͉ cargo ͉ cyclin-dependent kinase ͉ phosphorylation ͉ mitosis T he spindle midzone, a conspicuous network of antiparallel interdigitating nonkinetochore microtubules (MTs) between separating chromosomes, has been shown to play an essential role in regulating the initiation and completion of cytokinesis in animal cells (1). Midzone assembly occurs during the metaphase-to-anaphase transition, at the time of cleavage furrow initiation. Recent studies from cultured mammalian cells, Caenorhabditis elegans and Drosophila, have begun to reveal factors that are involved in the midzone assembly process. These factors include the kinesin-like motors and the associated proteins, chromosomal passenger proteins, kinases, phosphatase, and the spindle midzone bundling protein PRC1 (2-7).The kinesins are a family of MT-based motor proteins that generate directional movement along MTs and are involved in many crucial cellular processes including cell division (8). Gene disruption experiments in Saccharomyces cerevisiae indicated that five of six kinesins play crucial roles in regulating formation of the spindle and segregation of chromosomes in mitosis (9). In higher eukaryotes, several studies have shown that a number of kinesins are crucial for spindle assembly and function, chromosome segregation, mitotic checkpoint control, and cytokinesis (10-12). For instance, the midzone-associated kinesin MKLP-1 was reported to play an essential role in regulating midzone͞ midbody formation and cytokinesis in various organisms (5, 7). Klp3A, a Drosophila homologue of human kinesin-4 member Kif4, was shown to be essential fo...