Coincident Gene Conversion during Mitosis in Saccharomyces

During mitosis, gene conversion events at the TRP5 locus on chromosome VII are coupled with conversion events at LEU1 , a locus 18 cM away, 1200 times more frequently than would be expected for two independent acts of recombination. Such coincident conversion events that occur over relatively long distances could be due to several mechanisms. We discuss these possibilities and describe an experiment that indicates that a portion of coincident events is due to extensive heteroduplexes. The phenomenon of coincident gene conversion is discussed in relation to our earlier evidence that spontaneous recombination between homologues occurs prereplicationally in mitosis.     

Translocation of SenP5 from the Nucleoli to the Mitochondria Modulates DRP1-dependent Fission during Mitosis

The mechanisms that ensure an equal inheritance of cellular organelles during mitosis are an important area of study in cell biology. For the mitochondria fragment during mitosis, however, the cellular links that signal these changes are largely unknown. We recently identified a SUMO protease, SenP5, that deSUMOylates a number of mitochondrial targets, including the dynamin-related fission GTPase, DRP1. In interphase, SenP5 resides primarily within the nucleoli, in addition to a cytosolic pool. Here we report the relocalization of SenP5 from the nucleoli to the mitochondrial surface at G₂/M transition prior to nuclear envelope breakdown. The recruitment of SenP5 results in a significant loss in mitochondrial SUMOylation, and a concomitant increase in the labile pool of DRP1 that drives mitochondrial fragmentation. Importantly, silencing of SenP5 leads to an arrest in the cell cycle precisely at the time when the protease is translocated to the mitochondria. These data indicate that transition of SenP5 to the mitochondria plays an important role in mitochondrial fragmentation during mitosis. The altered intracellular localization of SenP5 represents the first example of the mitochondrial recruitment of a SUMO protease and provides new insights into the mechanisms of interorganellar communication during the cell cycle.     

Chromosome Dynamics during Mitosis

The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic machineries, cohesin and condensins, that ensure their faithful segregation during mitosis. Cohesin holds sister chromatids together immediately after their synthesis during S phase until the establishment of bipolar attachments to the mitotic spindle in metaphase. Condensins, on the other hand, attempt to “resolve” sister chromatids by counteracting cohesin. The products of the balancing acts of cohesin and condensins are metaphase chromosomes, in which two rod-shaped chromatids are connected primarily at the centromere. In anaphase, this connection is released by the action of separase that proteolytically cleaves the remaining population of cohesin. Recent studies uncover how this series of events might be mechanistically coupled with each other and intricately regulated by a number of regulatory factors.In eukaryotic cells, genomic DNA is packaged into chromatin and stored in the cell nucleus, in which essential chromosomal processes, including DNA replication and gene expression, take place (Fig. 1, interphase). At the onset of mitosis, the nuclear envelope breaks down and chromatin is progressively converted into a discrete set of rod-shaped structures known as metaphase chromosomes (Fig. 1, metaphase). In each chromosome, a pair of sister kinetochores assembles at its centromeric region, and their bioriented attachment to the mitotic spindle acts as a prerequisite for equal segregation of sister chromatids. The linkage between sister chromatids is dissolved at the onset of anaphase, allowing them to be pulled apart to opposite poles of the cell (Fig. 1, anaphase). At the end of mitosis, the nuclear envelope reassembles around two sets of segregated chromatids, leading to the production of genetically identical daughter cells (Fig. 1, telophase).Open in a separate windowFigure 1.Overview of chromosome dynamics during mitosis. In addition to the crucial role of kinetochore–spindle interactions, an intricate balance between cohesive and resolving forces acting on sister chromatid arms (top left, inset) underlies the process of chromosome segregation. See the text for major events in chromosome segregation.Although the centromere–kinetochore region plays a crucial role in the segregation process, sister chromatid arms also undergo dynamic structural changes to facilitate their own separation. Conceptually, such structural changes are an outcome of two balancing forces, namely, cohesive and resolving forces (Fig. 1, top left, inset). The cohesive force holds a pair of duplicated arms until proper timing of separation, otherwise daughter cells would receive too many or too few copies of chromosomes. The resolving force, on the other hand, counteracts the cohesive force, reorganizing each chromosome into a pair of rod-shaped chromatids. From this standpoint, the pathway of chromosome segregation is regarded as a dynamic process, in which the initially robust cohesive force is gradually weakened and eventually dominated by the resolving force. Almost two decades ago, genetic and biochemical studies for the behavior of mitotic chromosomes converged productively, culminating in the discovery of cohesin (Guacci et al. 1997; Michaelis et al. 1997; Losada et al. 1998) and condensin (Hirano et al. 1997; Sutani et al. 1999), which are responsible for the cohesive and resolving forces, respectively. The subsequent characterizations of these two protein complexes have not only transformed our molecular understanding of chromosome dynamics during mitosis and meiosis, but also provided far-reaching implications in genome stability, as well as unexpected links to human diseases. In this article, I summarize recent progress in our understanding of mitotic chromosome dynamics with a major focus on the regulatory networks surrounding cohesin and condensin. I also discuss emerging topics and attempt to clarify outstanding questions in the field.     

Synthesis of DNA during Mitosis

Growing roots of Zea mays in tritiated thymidine for brief periodsconfirms that G₁ may be eliminated from the mitotic cycle andDNA synthesis may be advanced into telophase but no further,in the fastest dividing cells of the cap initials, but not inthe stele.     

Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

Genetic Studies of Mei-1 Gene Activity during the Transition from Meiosis to Mitosis in Caenorhabditis Elegans

Genetic evidence suggests that the mei-1 locus of Caenorhabditis elegans encodes a maternal product required for female meiosis. However, a dominant gain-of-function allele, mei-1(ct46), can support normal meiosis but causes defects in subsequent mitotic spindles. Previously identified intragenic suppressors of ct46 lack functional mei-1 activity; null alleles suppress only in cis but other alleles arise frequently and suppress both in cis and in trans. Using a different screen for suppressors of the dominant ct46 defect, the present study describes another type of intragenic mutation that also arises at high frequency. These latter alleles appear to have reduced meiotic activity and retain a weakened dominant effect. Characterization of these alleles in trans-heterozygous combinations with previously identified mei-1 alleles has enabled us to define more clearly the role of the mei-1 gene product during normal embryogenesis. We propose that a certain level of mei-1 activity is required for meiosis but must be eliminated prior to mitosis. The dominant mutation causes mei-1 activity to function at mitosis; intragenic trans-suppressors act in an antimorphic manner to inactivate multimeric mei-1 complexes. We propose that inactivation of meiosis-specific functions may be an essential precondition of mitosis; failure to eliminate such functions may allow ectopic meiotic activity during mitosis and cause embryonic lethality.     

Cisternal Organization of the Endoplasmic Reticulum during Mitosis

The endoplasmic reticulum (ER) of animal cells is a single, dynamic, and continuous membrane network of interconnected cisternae and tubules spread out throughout the cytosol in direct contact with the nuclear envelope. During mitosis, the nuclear envelope undergoes a major rearrangement, as it rapidly partitions its membrane-bound contents into the ER. It is therefore of great interest to determine whether any major transformation in the architecture of the ER also occurs during cell division. We present structural evidence, from rapid, live-cell, three-dimensional imaging with confirmation from high-resolution electron microscopy tomography of samples preserved by high-pressure freezing and freeze substitution, unambiguously showing that from prometaphase to telophase of mammalian cells, most of the ER is organized as extended cisternae, with a very small fraction remaining organized as tubules. In contrast, during interphase, the ER displays the familiar reticular network of convolved cisternae linked to tubules.     

E.coli的Prlc基因产物对OmpA前体蛋白翻译后转位的功能

使用OmpA为指示蛋白,我们证实了prlc基因产物主要定位在胞液中,在分泌蛋白的转位过程中,它不是一个必需因子,但它的确涉及OmpA前体的转位定域。使用印迹试验证明它可能是一个信号肽水解酶。     

sgf73基因敲除对粟酒裂殖酵母细胞有丝分裂的影响

sgf73基因编码的Sgf73蛋白对SAGA复合物的功能具有重要作用。为探究sgf73基因缺失后sgf73Δ菌株有丝分裂中纺锤体、染色体、肌动蛋白的动力学变化,以粟酒裂殖酵母（Schizosaccharomyces pombe）为材料,对sgf73Δ菌株进行生长曲线和减数分裂产孢实验;采用荧光蛋白标记和活细胞成像的方法,对sgf73Δ菌株有丝分裂进行观察。生长曲线结果分析发现,sgf73基因敲除极显著影响粟酒裂殖酵母的生长,并导致子囊孢子数目显著减少。活细胞成像结果分析发现,在有丝分裂间期,sgf73Δ菌株微管数量显著增加、微管长度趋于增长但无显著差异;sgf73Δ菌株在分裂期纺锤体出现组装缺陷,单极纺锤体极显著增加,前期和中期纺锤体伸长速率显著下降、后期纺锤体伸长速率极显著下降、分裂中期和后期时间分别显著和极显著延长,有丝分裂总时间极显著延长,细胞长度也增长。肌动蛋白分裂环在有丝分裂中维持及收缩时间出现极显著和显著延长,总速率显著降低。同时,染色体分离出现缺陷。以上结果表明,sgf73基因缺失会导致菌株微管、染色体、肌动蛋白缺陷。     

Membrane Expansion Increases Endocytosis Rate during Mitosis

Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis. We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels. Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added. Although the compounds could produce a variety of different effects, we have found a strong correlation with a physical alteration in the membrane tension as measured by the laser tweezers. Plasma membrane tethers formed by latex beads pull back on the beads with a force that was related to the in-plane bilayer tension and membrane– cytoskeletal adhesion. We found that as cells enter mitosis, the membrane tension rises as the endocytosis rate decreases; and as cells exited mitosis, the endocytosis rate increased as the membrane tension decreased. The addition of amphyphilic compounds decreased membrane tension and increased the endocytosis rate. With the detergent, deoxycholate, the endocytosis rate was restored to interphase levels when the membrane tension was restored to interphase levels. Although biochemical factors are clearly involved in the alterations in mitosis, we suggest that endocytosis is blocked primarily by the increase in apparent plasma membrane tension. Higher tensions inhibit both the binding of the endocytic complex to the membrane and mechanical deformation of the membrane during invagination. We suggest that membrane tension is an important regulator of the endocytosis rate and alteration of tension is sufficient to modify endocytosis rates during mitosis. Further, we postulate that the rise in membrane tension causes cell rounding and the inhibition of motility, characteristic of mitosis.     

P16^INK4、Rb基因表达在肺癌发生中协调性研究

采用mRNA原位双杂交和免疫组织化学方法对31例非小细胞肺癌组织进行P16^INK、Rb基因、Rb基因表达水平及其相关性的研究。结果显示,以Dig-碱性磷酸酶－NBT／BCIP系统检测P16^INK4基因转录,阳性结果呈蓝色,阴性杂交率为22．6％（7／31）;以Bio－辣根过氧化物酶－AEC系统检测Rb基因转录,阳性结果为红色,阴性率为16．1％（5／31）。免疫组织化学检测显示P16^INK4蛋白质阴性率为42％（13／31）;Rb蛋白表达阴性率为19．4％（6／31）。Rb、P16^INK4基因在非小细胞肺癌发生中起协同调控作用,以P16^INK4基因表达异常为主。     

Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH₂-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.     

Raptor is Phosphorylated by cdc2 during Mitosis

Background

The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.

Methodology/Principal Findings

We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.

Conclusions/Significance

This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis.