Clockwise or anticlockwise? Turning the centriole triplets in the right direction!

Centrosomes are small cytoplasmic macromolecular assemblies composed from two major components, centrioles and pericentriolar material, each with its own complex architecture. This organelle is of interest because it plays a role in a number of fundamental cellular processes and defects in these processes have recently been correlated with variety of human disease. Increasingly, what is known about the structure of this organelle has been overshadowed by the increasing wealth of information on its biochemistry. In this short review, we highlight some of the common centriole structural errors found in the literature and define a set of rules that define centriole structure.     

The cell cycle and development: redundant roles of cell cycle regulators

The existence of families of cell cycle regulators reflects the need by a developing organism to precisely control proliferation of its cells and also suggests that family members may play redundant roles. Recent advances have shown redundancy to be a theme in development.     

Association of centrioles with clusters of apical vesicles in mitotic thyroid epithelial cells. Are centrioles involved in directing secretion?

Summary The ultrastructure of thyroid epithelial cells in mitosis has been investigated. A spatial association is described between clusters of apical vesicles (believed to contain thyroglobulin destined for secretion into the follicular lumen) and centrioles, in late prophase and late telophase cells. Quantitative techniques demonstrate the statistical significance of this association and suggest that it is not related to proximity of the Golgi apparatus or to the location of the centriole in the cell, which changes considerably during these phases of mitosis. The physical basis for this association remains uncertain, but microtubules emanating from the pericentriolar area may be involved.In interphase cells, centrioles are located very close to the follicular lumen, where the majority of apical vesicles are also found. The association of centrioles with clusters of apical vesicles also in mitotic cells suggests that in interphase cells the apically located centrioles may serve as a focus for apical vesicles, helping to direct these secretory vesicles toward the follicular lumen and to maintain cellular polarization. Previous studies demonstrating that centrioles can act as microtubule organizing centers in interphase cells and studies linking microtubules and secretion also tend to support this hypothesis.The author is grateful to Drs. Jan Wolff, Lars E. Ericson, and Seymour H. Wollman for useful discussions and to Mr. Franklin E. Reed for expert technical assistance.     

Microtubule organizing centers in plant cells: localization of a 49 kDa protein that is immunologically cross-reactive to a 51 kDa protein from sea urchin centrosomes in synchronized tobacco BY-2 cells

Summary A 49 kDa protein in tobacco BY-2 cells has been found to be cross-reactive with antibodies raised against a 51 kDa protein that was isolated from sea urchin centrosomes and identified as a microtubule-organizing center (MTOC) in animal cells. Tracing the fate of the 49 kDa protein during progression of the cell cycle in highly synchronized tobacco BY-2 cells revealed that this protein was colocalized with plant microtubules (MTs): the location of the 49 kDa protein coincided with preprophase bands (PPBs), mitotic spindles and phragmoplasts. Furthermore, between the M and G₁ phases, the 49 kDa protein was observed in the perinuclear regions, in which the initials of MTs are organizing to form cortical MTs. At the G₁ phase the location of the 49 kDa protein in the cell cortex coincided with that of the cortical MTs. It appeared that the 49 kDa protein in the cell cortex was transported as granules from the perinuclear regions. Thus, it is highly probable that the 49 kDa protein, which reacts with antibodies against the 51 kDa protein in sea urchin centrosomes, plays the role of an MTOC in plant cells. Thus, the mechanisms for organizing MTs in higher organisms appear to share a common protein, even though the organization of MTs is superficially very different in plant and animal cells.Abbreviations DAPI 4,6-diamidino-2-phenyl indole - MT microtubule - MTOC microtubule-organizing center - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PPB preprophase band - SDS sodium dodecylsulfate     

Biogenesis of the centrosome during mammalian gametogenesis and fertilization

Summary Mammalian gametogenesis results in the production of highly specialized cells, sperm and oocytes, that are complementary in their arsenal of organelles and molecules necessary for normal embryonic development. Consequently, some of the zygotic structures, as illustrated in this review on the centrosome, are a combination of complementary paternal and maternal contributions. Mammalian oocytes are deprived of their centrioles during oogenesis, yet at the same time they generate a huge cytoplasmic reserve of centrosomal proteins. The active centrosome of spermatogenic stem cells is reduced to a single centriole that does not possess microtubule-nucle-ating activity. This centrosomal activity is restored at fertilization, when the sperm centriole is released into the oocyte cytoplasm, from which it attracts the oocyte-derived proteins of pericentriolar material and ultimately converts itself into an active zygotic centrosome. Subsequently, the microtubules around the zygotic centrosome are organized into a radial array called the sperm aster, that guides the apposition of male and female pronuclei, and the union of paternal and maternal genomes in the cytoplasm of a fertilized oocyte. The original sperm centriole duplicates and gives rise to the first mitotic spindle. This biparental mode of centrosome inheritance is seen in most mammals, except for rodents, where both centrioles are degraded during spermiogenesis and the zygotic centrosome is organized without any paternal contributions. The studies of centrosomal inheritance at fertilization provide the platform for designing new safe methods of assisted-reproduction and infertility treatments in humans.     

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one induces cell cycle arrest and apoptosis in HeLa cells by preventing microtubule polymerization

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is known as a specific inhibitor of soluble guanylyl cyclase (sGC). Previously, however, ODQ was reported to induce cell death via sGC-dependent and sGC-independent means in a variety of cell types. The aim of this study was to investigate the mechanism by which ODQ induces cell death in HeLa cells.Treatment of HeLa cells with ODQ induced a concentration-dependent decrease in cell viability over the range from 10 to 100 μM. DNA fragmentation and fluorescence-activated cell sorting analysis using annexin V and propidium iodide staining revealed that ODQ triggered apoptosis at concentrations of 50 and 100 μM within 24 to 48 h. The addition of 8-Br-cGMP in the presence of ODQ failed to rescue HeLa cells from death, suggesting that the inhibition of sGC was not responsible for the pro-apoptotic action of ODQ. ODQ arrested the cell cycle at the G2/M phase and caused disassembly of the microtubule network. This process was reversed by dithiothreitol. In addition, ODQ was shown to inhibit the polymerization of purified tubulin, and this was also prevented by dithiothreitol. These results indicate that ODQ inhibits microtubule assembly by direct oxidation of tubulin, induces cell cycle arrest at the G2/M phase, and triggers apoptosis in HeLa cells.     

缺血缺氧对体外培养星形胶质细胞细胞活化和细胞周期的影响

目的观察缺血缺氧损伤对星形胶质细胞细胞活化和细胞周期的影响。方法用流式细胞仪及BrdU掺入法检测缺血缺氧后不同时间点星形胶质细胞细胞周期变化和细胞的增殖活力;用荧光免疫细胞化学技术测定胶质细胞纤维酸性蛋白(GFAP)及细胞周期蛋白cyclinD1的表达水平。结果体外缺血缺氧损伤后星形胶质细胞S期较正常组明显增高,6h达高峰,BrdU掺入法显示损伤后6h星形胶质细胞的增殖活力最高,而随后S期细胞数目及细胞增殖活力都呈下降趋势。在缺血缺氧早期,GFAP阳性染色增强,6h最高;缺血缺氧12h后GFAP阳性染色变弱,而cyclinD1的表达在损伤后逐渐增加,在24h时达高峰。结论缺血缺氧损伤激活星形胶质细胞,使其进入新的细胞周期,出现细胞的增殖反应;cyclinD1参与了损伤后星形胶质细胞的修复和增殖;细胞周期事件与星形胶质细胞的增殖活化密切相关。     

Functional characterization of the microtubule-binding and -destabilizing domains of CPAP and d-SAS-4

We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423–607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311–422). Site-specific mutagenesis revealed that the mutations that either disrupt the α-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved  20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4′s MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.     

Cell proliferation drives neural crest cell invasion of the intestine

A general mathematical model of cell invasion is developed and validated with an experimental system. The model incorporates two basic cell functions: non-directed (diffusive) motility and proliferation to a carrying capacity limit. The model is used here to investigate cell proliferation and motility differences along the axis of an invasion wave. Mathematical simulations yield surprising and counterintuitive predictions. In this general scenario, cells at the invasive front are proliferative and migrate into previously unoccupied tissues while those behind the front are essentially nonproliferative and do not directly migrate into unoccupied tissues. These differences are not innate to the cells, but are a function of proximity to uninvaded tissue. Therefore, proliferation at the invading front is the critical mechanism driving apparently directed invasion. An appropriate system to experimentally validate these predictions is the directional invasion and colonization of the gut by vagal neural crest cells that establish the enteric nervous system. An assay using gut organ culture with chick-quail grafting is used for this purpose. The experimental results are entirely concordant with the mathematical predictions. We conclude that proliferation at the wavefront is a key mechanism driving the invasive process. This has important implications not just for the neural crest, but for other invasion systems such as epidermal wound healing, carcinoma invasion and other developmental cell migrations.     

One-pot synthesis and biological evaluation of N-(aminosulfonyl)-4-podophyllotoxin carbamates as potential anticancer agents

A series of N-(aminosulfonyl)-4-podophyllotoxin carbamates were synthesized via the Burgess-type intermediate, and their antiproliferative activities were evaluated. Most of them possessed more potent cytotoxic effects against four human tumor cell lines (HeLa, A-549, HCT-8 and HepG2) and less toxic to normal human fetal lung fibroblast WI-38 cells than etoposide. In particular, N-(morpholinosulfonyl)-4-podophyllotoxin carbamate (9) exhibited the most potent activity towards these four tumor cells with IC₅₀ values in the range of 0.5–16.5 μM. Furthermore, immunofluorescence analysis revealed that 9 induced cell apoptosis by up-regulating the expression of p53 and ROS. Meanwhile, 9 effectively inhibited tubulin polymerization and microtubule assembly at cellular levels in HeLa cells. In addition, 9 could induce cell cycle arrest in the G2/M phase in HeLa cells by up-regulating levels of cyclinB1 and cdc2 and decreasing the expression of p-cdc2. These results indicated that 9 had potential for further development as anticancer agents.     

Expression and Localization of Brain Ankyrin Isoforms and Related Proteins During Early Developmental Stages of Rat Nervous System

Abstract: Expression and localization of two isoforms of brain ankyrin, 440- and 220-kDa ankyrin_B, were studied in the developing nervous system of the rat fetus. The 440-kDa ankyrin_B appeared on as early as embryonic day 13, and its level increased progressively toward the day of birth, which was similar to the expression pattern of growth-associated protein (GAP)-43, a well-established axonal protein. On the other hand, 220-kDa ankyrin_B was expressed at a low level but constitutively throughout the latter prenatal period and was a major isoform even before embryonic day 14. Whereas the localization of 440-kDa ankyrin_B was essentially confined to the axons, judging from the similarity with that of GAP-43, 220-kDa ankyrin_B showed a rather general distribution in neural tissue. The localization of L1, known as an ankyrin_B-binding protein, was similar to that of 440-kDa ankyrin_B in the brain tissue, whereas it was similar to that of 220-kDa ankyrin_B in cultured neurons, suggesting that the interaction of L1 with brain ankyrins in neurons is affected by their environment.     

Bidirectional motility of the fission yeast kinesin-5, Cut7

Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules, but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.