Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein

Glia mediate a vast array of cellular processes and are critical for nervous system development and function. Despite their immense importance in neurobiology, glia remain understudied and the molecular mechanisms that direct their differentiation are poorly understood. Rap/Fzr is the Drosophila homolog of the mammalian Cdh1, a regulatory subunit of the anaphase-promoting complex/cyclosome (APC/C). APC/C is an E3 ubiquitin ligase complex well characterized for its role in cell cycle progression. In this study, we have uncovered a novel cellular role for Rap/Fzr. Loss of rap/fzr function leads to a marked increase in the number of glia in the nervous system of third instar larvae. Conversely, ectopic expression of UAS-rap/fzr, driven by repo-GAL4, results in the drastic reduction of glia. Data from clonal analyses using the MARCM technique show that Rap/Fzr regulates the differentiation of surface glia in the developing larval nervous system. Our genetic and biochemical data further indicate that Rap/Fzr regulates glial differentiation through its interaction with Loco, a regulator of G-protein signaling (RGS) protein and a known effector of glia specification. We propose that Rap/Fzr targets Loco for ubiquitination, thereby regulating glial differentiation in the developing nervous system.     

Mitosis in Neurons: Roughex and APC/C Maintain Cell Cycle Exit to Prevent Cytokinetic and Axonal Defects in Drosophila Photoreceptor Neurons

The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.     

Notch-dependent Fizzy-related/Hec1/Cdh1 expression is required for the mitotic-to-endocycle transition in Drosophila follicle cells

During Drosophila oogenesis, Notch function regulates the transition from mitotic cell cycle to endocycle in follicle cells at stage 6. Loss of either Notch function or its ligand Delta (Dl) disrupts the normal transition; this disruption causes mitotic cycling to continue and leads to an overproliferation phenotype. In this context, the only known cell cycle component that responds to the Notch pathway is String/Cdc25 (Stg), a G2/M cell cycle regulator. We found that prolonged expression of string is not sufficient to keep cells efficiently in mitotic cell cycle past stage 6, suggesting that Notch also regulates other cell cycle components in the transition. By using an expression screen, we found such a component: Fizzy-related/Hec1/Cdh1 (Fzr), a WD40 repeat protein. Fzr regulates the anaphase-promoting complex/cyclosome (APC/C) and is expressed at the mitotic-to-endocycle transition in a Notch-dependent manner. Mutant clones of Fzr revealed that Fzr is dispensable for mitosis but essential for endocycles. Unlike in Notch clones, in Fzr mutant cells mitotic markers are absent past stage 6. Only a combined reduction of Fzr and ectopic Stg expression prolongs mitotic cycles in follicle cells, suggesting that these two cell cycle regulators, Fzr and Stg, are important mediators of the Notch pathway in the mitotic-to-endocycle transition.     

Rap1a is a key regulator of fibroblast growth factor 2-induced angiogenesis and together with Rap1b controls human endothelial cell functions

Angiogenesis, the formation of new blood vessels from existing vasculature, is regulated primarily by endothelial cell activity. We show herein that the Ras family GTPase Rap1 has a key role in the regulation of angiogenesis by modulating endothelial cell functions. Blood vessel growth into fibroblast growth factor 2 (FGF2)-containing Matrigel plugs was absent from rap1a^−/− mice, and aortic rings derived from rap1a^−/− mice failed to sprout primitive tubes in response to FGF2, when the tissue was embedded in Matrigel. Knocking down either rap1a or rap1b, two closely related rap1 family members, in human microvascular endothelial cells (HMVECs) by utilizing siRNA confirmed that Rap1 plays key roles in endothelial cell function. The rap1a or rap1b knockdown resulted in decreased adhesion to extracellular matrices and impaired cell migration. HMVEC monolayers lacking Rap1 had increased permeability, and Rap1-deficient endothelial cells failed to form three-dimensional tubular structures when they were plated on Matrigel in vitro. Finally, the activation levels of extracellular signal-regulated kinase (ERK), p38, and Rac, which are important signaling molecules in angiogenesis, were all reduced in response to FGF2 when either of the Rap1 proteins was depleted. These observations place Rap1 centrally in the human angiogenic process and suggest that both the Rap1a and Rap1b proteins are required for angiogenesis and that Rap1 is a critical mediator of FGF-induced ERK activation.     

Cdk1 is essential for mammalian cyclosome/APC regulation

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome.     

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34^cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.     

利用流式细胞仪研究拟南芥叶发育过程中细胞周期的调控

叶的形态建成依赖于细胞不断地分裂增殖和不同类型细胞的特化。在叶发育早期,叶细胞主要通过旺盛的有丝分裂来增加原基中细胞的数目。随着叶片的生长,叶细胞自顶部向基部逐渐退出有丝分裂进入内复制来增加细胞的倍性,同时伴随细胞的扩展和分化。本文介绍利用流式细胞仪研究双子叶模式植物拟南芥叶发育过程中细胞周期调控的方法和具体研究实例。我们发现至少存在3种类型的细胞周期异常的拟南芥叶发育突变体。此外,我们还介绍利用流式细胞仪测定DNA复制效率的方法。     

Perturbation Analysis of Heterochromatin-Mediated Gene Silencing and Somatic Inheritance

Repetitive sequences in eukaryotic genomes induce chromatin-mediated gene-silencing of juxtaposed genes. Many components that promote or antagonize silencing have been identified, but how heterochromatin causes variegated and heritable changes in gene expression remains mysterious. We have used inducible mis-expression in the Drosophila eye to recover new factors that alter silencing caused by the bw^D allele, an insertion of repetitive satellite DNA that silences a bw⁺ allele on the homologous chromosome. Inducible modifiers allow perturbation of silencing at different times in development, and distinguish factors that affect establishment or maintenance of silencing. We find that diverse chromatin and RNA processing factors can de-repress silencing. Most factors are effective even in differentiated cells, implying that silent chromatin remains plastic. However, over-expression of the bantam microRNA or the crooked-legs (crol) zinc-finger protein only de-repress silencing when expressed in cycling cells. Over-expression of crol accelerates the cell cycle, and this is required for de-repression of silencing. Strikingly, continual over-expression of crol converts the speckled variegation pattern of bw^D into sectored variegation, where de-repression is stably inherited through mitotic divisions. Over-expression of crol establishes an open chromatin state, but the factor is not needed to maintain this state. Our analysis reveals that active chromatin states can be efficiently inherited through cell divisions, with implications for the stable maintenance of gene expression patterns through development.     

Suppression of Rap1 Impairs Cardiac Myofibrils and Conduction System in Zebrafish

Numerous studies have revealed that Rap1 (Ras-proximate-1 or Ras-related protein 1), a small GTPase protein, plays a crucial role in mediating cAMP signaling in isolated cardiac tissues and cell lines. However, the involvement of Rap1 in the cardiac development in vivo is largely unknown. By injecting anti-sense morpholino oligonucleotides to knock down Rap1a and Rap1b in zebrafish embryos, and in combination with time-lapsed imaging, in situ hybridization, immunohistochemistry and transmission electron microscope techniques, we seek to understand the role of Rap1 in cardiac development and functions. At an optimized low dose of mixed rap1a and rap1b morpholino oligonucleotides, the heart developed essentially normally until cardiac contraction occurred. Morphant hearts showed the myocardium defect phenotypes, most likely due to disrupted myofibril assembly and alignment. In vivo heart electrocardiography revealed prolonged P-R interval and QRS duration, consistent with an adherens junction defect and reduced Connexons in cardiac myocytes of morphants. We conclude that a proper level of Rap1 is crucial for heart morphogenesis and function, and suggest that Rap1 and/or their downstream factor genes are potential candidates for genetic screening for human heart diseases.     

Regulation of the Anaphase-promoting Complex/Cyclosome by bimAAPC3 and Proteolysis of NIMA

Surprisingly, although highly temperature-sensitive, the bimA1^APC3 anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1^APC3 is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34^cdc2 kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1^APC3-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1^APC3 double mutant arrests in a mitotic state with very high p34^cdc2 H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1^APC3 mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimA^APC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1^APC3. The bimA1^APC3 mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimA^APC3 regulates the APC/C in a NIMA-dependent manner.     

Rap1 maintains adhesion between cells to affect Egfr signaling and planar cell polarity in Drosophila

The small GTPase Rap1 affects cell adhesion and cell motility in numerous developmental contexts. Loss of Rap1 in the Drosophila wing epithelium disrupts adherens junction localization, causing mutant cells to disperse, and dramatically alters epithelial cell shape. While the adhesive consequences of Rap1 inactivation have been well described in this system, the effects on cell signaling, cell fate specification, and tissue differentiation are not known. Here we demonstrate that Egfr-dependent cell types are lost from Rap1 mutant tissue as an indirect consequence of DE-cadherin mislocalization. Cells lacking Rap1 in the developing wing and eye are capable of responding to an Egfr signal, indicating that Rap1 is not required for Egfr/Ras/MAPK signal transduction. Instead, Rap1 regulates adhesive contacts necessary for maintenance of Egfr signaling between cells, and differentiation of wing veins and photoreceptors. Rap1 is also necessary for planar cell polarity in these tissues. Wing hair alignment and ommatidial rotation, functional readouts of planar cell polarity in the wing and eye respectively, are both affected in Rap1 mutant tissue. Finally, we show that Rap1 acts through the effector Canoe to regulate these developmental processes.     

Association of Hsp90 with p53 and Fizzy related homolog (Fzr) synchronizing Anaphase Promoting Complex (APC/C): An unexplored ally towards oncogenic pathway

The intricate molecular interactions leading to the oncogenic pathway are the consequence of cell cycle modification controlled by a bunch of cell cycle regulatory proteins. The tumor suppressor and cell cycle regulatory proteins work in coordination to maintain a healthy cellular environment. The integrity of this cellular protein pool is perpetuated by heat shock proteins/chaperones, which assist in proper protein folding during normal and cellular stress conditions. Among these versatile groups of chaperone proteins, Hsp90 is one of the significant ATP-dependent chaperones that aid in stabilizing many tumor suppressors and cell cycle regulator protein targets. Recently, studies have revealed that in cancerous cell lines, Hsp90 stabilizes mutant p53, ‘the guardian of the genome.’ Hsp90 also has a significant impact on Fzr, an essential regulator of the cell cycle having an important role in the developmental process of various organisms, including Drosophila, yeast, Caenorhabditis elegans, and plants. During cell cycle progression, p53 and Fzr coordinately regulate the Anaphase Promoting Complex (APC/C) from metaphase to anaphase transition up to cell cycle exit. APC/C mediates proper centrosome function in the dividing cell. The centrosome acts as the microtubule organizing center for the correct segregation of the sister chromatids to ensure perfect cell division. This review examines the structure of Hsp90 and its co-chaperones, which work in synergy to stabilize proteins such as p53 and Fizzy-related homolog (Fzr) to synchronize the Anaphase Promoting Complex (APC/C). Dysfunction of this process activates the oncogenic pathway leading to the development of cancer. Additionally, an overview of current drugs targeting Hsp90 at various phases of clinical trials has been included.     

The plant cell inhibitor KRP6 is involved in multinucleation and cytokinesis disruption in giant-feeding cells induced by root-knot nematodes

The plant cell cycle inhibitor gene KRP6 has been investigated in roots infected by plant-parasitic root-knot nematodes (Meloidogyne spp.). Unexpectedly, KRP6 overexpressing lines revealed a distinct role for this specific KRP as an activator of the mitotic cell cycle. This function was confirmed in Arabidopsis thaliana suspension cultures ectopically expressing KRP6. A blockage in the mitotic exit was observed in cell suspensions and in giant cells resulted in the appearance of multi-nucleated cells. KRP6 expression during nematode infection and the similarity in phenotypes among KRP6 overexpressing cell cultures and giant-cell morphology strongly suggest that KRP6 is involved in multinucleation and acytokinesis occurring in giant-cells. Once again nematodes have been shown to manipulate the plant cell cycle machinery in order to promote gall establishment.     

Primordial germ cells and early stages of oogenesis in Ophryotrocha puerilis (Polychaeta,Dorvillediae)

Summary Each setigerous segment of the protandric polychaete Ophryotrocha puerilis contains two primordial germ cells. A ventral furrow in the gut wall together with the peritoneal lining of the gut forms a genital blood vessel. The gonocytes are located within the peritoneum of this genital blood vessel. At sexual maturity the gonocytes undergo a proliferation cycle, the first division of which gives rise to a cell which is extruded into a forming outpocketing of the coelomic lining. The stem cell remains within the peritoneum. Inside the forming gonad the detached cell goes through a series of four mitotic divisions. The resulting 16 cells are interconnected by cytoplasmic bridges. These bridges are arranged in a very regular pattern which allows the mitotic cycles to be followed. While remaining still within the gonad the 16 cells begin to synthesize yolk and to take up exogenous yolk precursors. At this stage a differentiation into oocytes and nurse cells becomes visible. The oocytes deposit yolk platelets of the definitive size whereas the polyploid nurse cells produce only small yolk bodies that are passed to the adjacent oocytes. In a later stage the cell bridges between adjacent nurse cells are cut and pairs of one oocyte and one nurse cell are released to the coelomic cavity during breakdown of the gonadal sac. Oocyte-nurse cell-complexes then freely float in the coelomic fluid. The proliferation of gonadal cells is well synchronized within one segment. In anterior segments, however, gonadal proliferation usually begins earlier than in posterior segments but smaller oocytes in posterior segments catch up within a few days. Finally a batch of oocytes is produced in which all the oocytes are of the same size (120 m). The origin of the primordial germ cells remains unknown.