A mitotic lamin B matrix induced by RanGTP required for spindle assembly

Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.     

A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint

The spindle assembly checkpoint guards the fidelity of chromosome segregation. It requires the close cooperation of cell cycle regulatory proteins and cytoskeletal elements to sense spindle integrity. The role of the centrosome, the organizing center of the microtubule cytoskeleton, in the spindle checkpoint is unclear. We found that the molecular requirements for a functional spindle checkpoint included components of the large gamma-tubulin ring complex (gamma-TuRC). However, their localization at the centrosome and centrosome integrity were not essential for this function. Thus, the spindle checkpoint can be activated at the level of microtubule nucleation.     

A mitotic septin scaffold required for Mammalian chromosome congression and segregation

Coordination of cytokinesis with chromosome congression and segregation is critical for proper cell division, but the mechanism is unknown. Here, septins, a conserved family of polymerizing guanosine triphosphate-binding proteins, localized to the metaphase plate during mitosis. Septin depletion resulted in chromosome loss from the metaphase plate, lack of chromosome segregation and spindle elongation, and incomplete cytokinesis upon delayed mitotic exit. These defects correlated with loss of the mitotic motor and the checkpoint regulator centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes. Mammalian septins may thus form a mitotic scaffold for CENP-E and other effectors to coordinate cytokinesis with chromosome congression and segregation.     

A functional dosage compensation complex required for male killing in Drosophila

Bacteria that selectively kill males ("male-killers") were first characterized more than 50 years ago in Drosophila and have proved to be common in insects. However, the mechanism by which sex specificity of virulence is achieved has remained unknown. We tested the ability of Spiroplasma poulsonii to kill Drosophila melanogaster males carrying mutations in genes that encode the dosage compensation complex. The bacterium failed to kill males lacking any of the five protein components of the complex.     

Comment on "A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint"

Müller et al. (Reports, 27 October 2006, p. 654) proposed a role for microtubule nucleation in mitotic checkpoint signaling. However, their observations of spindle defects and mitotic delay after depletion of gamma-tubulin ring complex (gamma-TuRC) components are fully consistent with activation of the established pathway of checkpoint signaling in response to incomplete or unstable interactions between kinetochores of mitotic chromosomes and spindle microtubules.     

A centrosomal localization signal in cyclin E required for Cdk2-independent S phase entry

Excess cyclin E-Cdk2 accelerates entry into S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. We identified 20 amino acids in cyclin E as a centrosomal localization signal (CLS) essential for both centrosomal targeting and promoting DNA synthesis. Expressed wild-type, but not mutant, CLS peptides localized on the centrosome, prevented endogenous cyclin E and cyclin A from localizing to the centrosome, and inhibited DNA synthesis. Ectopic cyclin E localized to the centrosome and accelerated S phase entry even with mutations that abolish Cdk2 binding, but not with a mutation in the CLS. These results suggest that cyclin E has a modular centrosomal-targeting domain essential for promoting S phase entry in a Cdk2-independent manner.     

gridlock, an HLH gene required for assembly of the aorta in zebrafish

The first artery and vein of the vertebrate embryo assemble in the trunk by migration and coalescence of angioblasts to form endothelial tubes. The gridlock (grl) mutation in zebrafish selectively perturbs assembly of the artery (the aorta). Here it is shown that grl encodes a basic helix-loop-helix (bHLH) protein belonging to the Hairy/Enhancer of the split family of bHLH proteins. The grl gene is expressed in lateral plate mesoderm before vessel formation, and thereafter in the aorta and not in the vein. These results suggest that the arterial endothelial identity is established even before the onset of blood flow and implicate the grl gene in assignment of vessel-specific cell fate.     

Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection

Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.     

Stathmin-tubulin interaction gradients in motile and mitotic cells

The spatial organization of the microtubule cytoskeleton is thought to be directed by steady-state activity gradients of diffusible regulatory molecules. We visualized such intracellular gradients by monitoring the interaction between tubulin and a regulator of microtubule dynamics, stathmin, using a fluorescence resonance energy transfer (FRET) biosensor. These gradients were observed both during interphase in motile membrane protrusions and during mitosis around chromosomes, which suggests that a similar mechanism may contribute to the creation of polarized microtubule structures. These interaction patterns are likely to reflect phosphorylation of stathmin in these areas.     

A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.     

运用MTT比色法检测印楝素对果蝇S2细胞的毒力作用

采用MTT(四甲基偶氮唑蓝,methylthiazolyl-tetrazolium)法测定不同质量浓度印楝素(0.01,0.1,0.5,1.0,2.0mg·L-)作用不同时间(24,48,72,96h)对果蝇S2细胞的抑制率.结果表明,印楝素低质量浓度处理时,对果蝇S2细胞有促进生长的作用,表现为低质量浓度促进效应;在质量浓度为0.1～2mg·L-1处理剂量范围内,表现为明显的时间依赖性,随时间延长,抑制率增加.0.5mg·L-1处理48h抑制率达54％,为最佳作用条件.     

GRK2-dependent S1PR1 desensitization is required for lymphocytes to overcome their attraction to blood

Lymphocytes egress from lymphoid organs in response to sphingosine-1-phosphate (S1P); minutes later they migrate from blood into tissue against the S1P gradient. The mechanisms facilitating cell movement against the gradient have not been defined. Here, we show that heterotrimeric guanine nucleotide-binding protein-coupled receptor kinase-2 (GRK2) functions in down-regulation of S1P receptor-1 (S1PR1) on blood-exposed lymphocytes. T and B cell movement from blood into lymph nodes is reduced in the absence of GRK2 but is restored in S1P-deficient mice. In the spleen, B cell movement between the blood-rich marginal zone and follicles is disrupted by GRK2 deficiency and by mutation of an S1PR1 desensitization motif. Moreover, delivery of systemic antigen into follicles is impaired. Thus, GRK2-dependent S1PR1 desensitization allows lymphocytes to escape circulatory fluids and migrate into lymphoid tissues.     

Phosphatidylserine receptor is required for clearance of apoptotic cells

Cells undergoing apoptosis during development are removed by phagocytes, but the underlying mechanisms of this process are not fully understood. Phagocytes lacking the phosphatidylserine receptor (PSR) were defective in removing apoptotic cells. Consequently, in PSR-deficient mice, dead cells accumulated in the lung and brain, causing abnormal development and leading to neonatal lethality. A fraction of PSR knockout mice manifested a hyperplasic brain phenotype resembling that of mice deficient in the cell death-associated genes encoding Apaf-1, caspase-3, and caspase-9, which suggests that phagocytes may also be involved in promoting apoptosis. These data demonstrate a critical role for PSR in early stages of mammalian organogenesis and suggest that this receptor may be involved in respiratory distress syndromes and congenital brain malformations.     

Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit

Ribosomes are self-assembling macromolecular machines that translate DNA into proteins, and an understanding of ribosome biogenesis is central to cellular physiology. Previous studies on the Escherichia coli 30S subunit suggest that ribosome assembly occurs via multiple parallel pathways rather than through a single rate-limiting step, but little mechanistic information is known about this process. Discovery single-particle profiling (DSP), an application of time-resolved electron microscopy, was used to obtain more than 1 million snapshots of assembling 30S subunits, identify and visualize the structures of 14 assembly intermediates, and monitor the population flux of these intermediates over time. DSP results were integrated with mass spectrometry data to construct the first ribosome-assembly mechanism that incorporates binding dependencies, rate constants, and structural characterization of populated intermediates.     

Cohesin cleavage by separase required for anaphase and cytokinesis in human cells

Cell division depends on the separation of sister chromatids in anaphase. In yeast, sister separation is initiated by cleavage of cohesin by the protease separase. In vertebrates, most cohesin is removed from chromosome arms by a cleavage-independent mechanism. Only residual amounts of cohesin are cleaved at the onset of anaphase, coinciding with its disappearance from centromeres. We have identified two separase cleavage sites in the human cohesin subunit SCC1 and have conditionally expressed noncleavable SCC1 mutants in human cells. Our results indicate that cohesin cleavage by separase is essential for sister chromatid separation and for the completion of cytokinesis.     

RAD51C is required for Holliday junction processing in mammalian cells

During genetic recombination and the recombinational repair of chromosome breaks, DNA molecules become linked at points of strand exchange. Branch migration and resolution of these crossovers, or Holliday junctions (HJs), complete the recombination process. Here, we show that extracts from cells carrying mutations in the recombination/repair genes RAD51C or XRCC3 have reduced levels of HJ resolvase activity. Moreover, depletion of RAD51C from fractionated human extracts caused a loss of branch migration and resolution activity, but these functions were restored by complementation with a variety of RAD51 paralog complexes containing RAD51C. We conclude that the RAD51 paralogs are involved in HJ processing in human cells.     

Cloning of rat kangaroo (PTK2) cells following laser microirradiation of selected mitotic chromosomes

Mitotic chromosomes of rat kangaroo cells were irradiated with a green argon laser microbeam without prior dye sensitization. Deoxyribonucleic acid-negative chromosome paling was observed. The irradiated cells were isolated and cloned into viable populations.     

果蝇硫化氢合成相关基因在不同虫态的转录水平

构建了23个物种的甲硫氨酸腺苷转移酶、甘氨酸N甲基转移酶、胱硫醚β合成酶和胱硫醚γ裂解酶的氨基酸序列的系统进化树,进化分析表明这4类蛋白均具有基本一致的系统进化关系,并且均被归为昆虫和哺乳动物两大类群。通过实时荧光定量PCR方法分析了黑腹果蝇不同发育阶段编码甲硫氨酸腺苷转移酶、甘氨酸N甲基转移酶、胱硫醚β合成酶和胱硫醚γ裂解酶的M(2)21AB、CG6188、CG1753和Eip55E基因的mRNA水平。结果显示:M(2)21AB基因mRNA水平在各发育阶段间无显著差异;CG6188基因mRNA的表达量在卵期较低,到了幼虫期显著升高,尤其在2龄幼虫期达到最高,而在蛹期和成虫期明显下降;CG1753基因mRNA的表达量在幼虫期明显上升,至蛹期达到最高水平,而在成虫期又显著降低;Eip55E基因mRNA水平在幼虫期、蛹期及成虫期均比卵期显著提高。以上结果表明,内源性H2S在黑腹果蝇中具有重要的生理学功能。