NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.     

Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.     

Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus

AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.     

The transforming acidic coiled coil 3 protein is essential for spindle-dependent chromosome alignment and mitotic survival

Cancer-associated centrosomal transforming acidic coiled coil (TACC) proteins are involved in mitotic spindle function. By employing gene targeting, we have recently described a nonredundant and essential role of TACC3 in regulating cell proliferation. In this study, we used an inducible RNA interference approach to characterize the molecular function of TACC3 and its role in mitotic progression and cell survival. Our data demonstrate that a TACC3 knockdown arrests G(1) checkpoint-compromised HeLa cells prior to anaphase with aberrant spindle morphology and severely misaligned chromosomes. Interestingly, TACC3-depleted cells fail to accumulate the mitotic kinase Aurora B and the checkpoint protein BubR1 to normal levels at kinetochores. Moreover, localization of the structural protein Ndc80 at outer kinetochores is reduced, indicating a defective kinetochore-microtubule attachment in TACC3-deficient cells. As a consequence of prolonged TACC3 depletion, cells undergo caspase-dependent cell death that relies on a spindle checkpoint-dependent mitotic arrest. TACC3 knockdown cells that escape from this arrest by mitotic slippage become highly polyploid and accumulate supernumerary centrosomes. Similarly, deficiency of the post-mitotic cell cycle inhibitor p21(WAF) exacerbates the effects of TACC3 depletion. Our findings therefore point to an essential role of TACC3 in spindle assembly and cellular survival and identify TACC3 as a potential therapeutic target in cancer cells.     

The centrosomal, putative tumor suppressor protein TACC2 is dispensable for normal development, and deficiency does not lead to cancer

TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Studies of human breast cancer samples and cell lines suggest a putative role of TACC2 as a tumor suppressor protein. To analyze the physiological role of TACC2, we generated mice lacking TACC2. TACC2-deficient mice are viable, develop normally, are fertile, and lack phenotypic changes compared to wild-type mice. Furthermore, TACC2 deficiency does not lead to an increased incidence of tumor development. Finally, in TACC2-deficient embryonic fibroblasts, proliferation and cell cycle progression as well as centrosome numbers are comparable to those in wild-type cells. Therefore, TACC2 is not required, nonredundantly, for mouse development and normal cell proliferation and is not a tumor suppressor protein.     

胚胎干细胞体外分化为造血干细胞

造血干细胞移植已成为治疗白血病、再生障碍性贫血、重症免疫缺陷征、地中海贫血、急性放射病、某些恶性实体瘤和淋巴瘤等造血及免疫系统功能障碍性疾病的成熟技术和重要手段,另外这一技术还被尝试用于治疗艾滋病,已取得积极的效果。但是由于移植需要配型相同的供体,并且过程复杂,使得造血干细胞移植因缺少配型相同的供体来源以及费用昂贵而不能被广泛应用。胚胎干细胞是一种能够在体外保持未分化状态并且能进行无限增殖的细胞,在适合条件下能够分化为体内各种类型的细胞,研究胚胎干细胞分化为造血干细胞,不仅可作为研究动物的早期造血发生的模型,而且可以增加造血干细胞的来源,还可以通过基因剔除、治疗性克隆等方法来解决移植排斥的问题,从而为造血干细胞移植的发展扫除了障碍,因此有着重要的研究价值和应用前景。现对胚胎干细胞体外分化为造血干细胞的诱导方法,诱导过程中的调控机制,并对胚胎干细胞分化为造血干细胞的存在问题和发展前景进行讨论。     

The ATM/p53/p21 pathway influences cell fate decision between apoptosis and senescence in reoxygenated hematopoietic progenitor cells

Hematopoietic cells are often exposed to transient hypoxia as they develop and migrate between blood and tissues. We tested the hypothesis that hypoxia-then-reoxygenation represent a stress for hematopoietic progenitor cells. Here we report that reoxygenation-generated oxidative stress induced senescence, tested as staining for SA-beta-galactosidase (SA-beta-gal), of bone marrow progenitor cells. Reoxygenation induced significant DNA damage and inhibited colony formation in lineage-depleted bone marrow cells enriched for progenitor cells. These reoxygenated cells exhibited a prolonged G(0)/G(1) accumulation without significant apoptosis after 24 h of treatments. Reoxygenated bone marrow progenitor cells expressed SA-beta-gal and senescence-associated proteins p53 and p21(WAF1). Reoxygenated Fancc-/- progenitor cells, which underwent significant apoptosis and senescence, tested as staining for SA-beta-gal, also expressed p16(INK4A). Suppression of apoptosis by the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone dramatically increased senescent Fancc-/- progenitor cells. Senescence induction, tested as staining for SA-beta-gal, in reoxygenated progenitor cells was closely correlated with extent of DNA damage and phosphorylation of ATM at Ser-1981 and p53 at Ser-15. Moreover, inhibition of ATM signaling reduced SA-beta-gal positivity but increased apoptosis of reoxygenated progenitor cells. Thus, these results suggest that the ATM/p53/p21 pathway influences cell fate decision between apoptosis and senescence in reoxygenated hematopoietic progenitor cells.     

miR-33-mediated downregulation of p53 controls hematopoietic stem cell self-renewal

Hematopoietic stem cells (HSCs) are defined by their exclusive capacity to both self-renew and to give rise to multipotent progenitors (MPPs) that in turn differentiate into the mature blood cell lineages. The tumor suppressor p53, in addition to its role in the regulation of the cell cycle, plays an importatn role in HSC self-renewal, although it has not fully resolved. Here we report that in super-p53 mice (sp53), which carry one extra gene dose of p53, the miR-33 is down-regulated in HSCs and highly expressed in MPPs. Transplantation assays of miR-33-transduced sp53 HSC results in a significant acquisition of repopulating capacity and a decrease of recipients survival. Moreover, high levels of miR-33 represses the endogenous level of p53 protein in murine embryonic fibroblasts (MEFs), leads both to neoplastic transformation and anchorage independent growth of MEFs, and displays a decrease of apoptotic response using tumor-derived cell lines. Accordingly, we demonstrate that miR-33-mediated down-regulation of p53 is dependent on the binding of miR-33 to two conserved motifs in the 3′UTR of p53. Together, these data show that the miR-33 modifies HSC repopulating efficiency of sp53 mice by impairing the p53 function. Defining the role of miR-33 in controlling the HSC self-renewal through p53 may lead to the prevention and treatment of hematopoietic disorders.     

The centrosome and mitotic spindle apparatus in cancer and senescence

Altered cell division is associated with overproliferation and tumorigenesis, however, mitotic aberrations can also trigger antiproliferative responses leading to postmitotic cell cycle exit. Here, we focus on the role of the centrosome and in particular of centrosomal TACC (transforming acidic coiled coil) proteins in tumorigenesis and cellular senescence. We have compiled recent evidence that inhibition or depletion of various mitotic proteins which take over key roles in centrosome and kinetochore integrity and mitotic checkpoint function is sufficient to activate a p53-p21^WAF driven premature senescence phenotype. These findings have direct implications for proliferative tissue homeostasis as well as for cellular and organismal aging.     

Deficiency of Trp53 rescues the male fertility defects of Kit(W-v) mice but has no effect on the survival of melanocytes and mast cells.

Mutations of the receptor tyrosine kinase, Kit, or its ligand, mast growth factor (Mgf), affect three unrelated cell populations: melanocytes, germ cells, and mast cells. Kit signaling is required initially to prevent cell death in these lineages both in vitro and in vivo. Mgf appears to play a role in the survival of some hematopoietic cells in vitro by modulating the activity of p53. Signaling by Mgf inhibits p53-induced apoptosis of erythroleukemia cell lines and suppresses p53-dependent radiation-induced apoptosis of bone marrow cells. We tested the hypothesis that cell survival in Kit mutant mice would be enhanced by p53 deficiency in vivo. Double-mutant mice, which have greatly reduced Kit receptor tyrosine kinase activity and also lack Trp53, were generated and the affected cell lineages examined. Mast cell, melanoblast, and melanocyte survival in the double Kit(W-v/W-v):Trp53(-/-) mutants was not increased compared to the single Kit(W-v/W-v):Trp53(+/+) mutants. However, double-mutant males showed an increase in sperm viability and could father litters, in contrast to their homozygous Kit mutant, wild-type p53 littermates. This germ cell rescue appears to be male specific, as female ovaries were similar in mice homozygous for the Kit mutant allele with or without p53. We conclude that defective Kit signaling in vivo results in apoptosis by a p53-independent pathway in melanocyte and mast cell lineages but that in male germ cells apoptosis in the absence of Kit is p53-dependent.     

TACC3-TSC2 maintains nuclear envelope structure and controls cell division

Studies of the role of tuberous sclerosis complex (TSC) proteins (TSC1/TSC2) in pathology have focused mainly on their capacity to regulate translation and cell growth, but their relationship with alterations of cellular structures and the cell cycle is not yet fully understood. The transforming acidic coiled-coil (TACC) domain-containing proteins are central players in structures and processes connected to the centrosome. Here, TACC3 interactome mapping identified TSC2 and 15 other physical interactors, including the evolutionary conserved interactions with ch-TOG/CKAP5 and FAM161B. TACC3 and TSC2 co-localize and co-purify with components of the nuclear envelope, and their deficiency causes morphological alterations of this structure. During cell division, TACC3 is necessary for the proper localization of phospho-Ser939 TSC2 at spindle poles and cytokinetic bridges. Accordingly, abscission alterations and increased frequency of binucleated cells were observed in Tacc3- and Tsc2-deficient cells relative to controls. In regulating cell division, TSC2 acts epistatically to TACC3 and, in addition to canonical TSC/mTOR signaling and cytokinetic associations, converges to the early mitotic checkpoint mediated by CHFR, consistently with nuclear envelope associations. Our findings link TACC3 to novel structural and cell division functions of TSC2, which may provide additional explanations for the clinical and pathological manifestations of lymphangioleiomyomatosis (LAM) disease and TSC syndrome, including the greater clinical severity of TSC2 mutations compared to TSC1 mutations.     

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform.     

vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubule and the kinetochore. Our recent ultrastructural studies demonstrated a dynamic distribution of TTK, from the kinetochore to the centrosome, as cell enters into anaphase. Here, we show that a centrosomal protein TACC2 is phosphorylated in mitosis by TTK signaling pathway. TACC2 was pulled down by wild type TTK but not kinase death mutant, suggesting the potential phosphorylation-mediated interaction between these two proteins. Our immunofluorescence studies revealed that both TTK and TACC2 are located to the centrosome. Interestingly, expression of kinase death mutant of TTK eliminated the centrosomal localization of TACC2 but not other centrosomal proteins such as gamma-tubulin and NuMA, a phenotype seen in TTK-depleted cells. In these centrosomal TACC2-liberated cells, chromosomes were lagging and mis-aligned. In addition, the distance between two centrosomes was markedly reduced, suggesting that centrosomal TACC2 is required for mitotic spindle maintenance. The inter-relationship between TTK and TACC2 established here provides new avenue to study centrosome and spindle dynamics underlying cell divisional control.     

The ups and downs of p53 regulation in hematopoietic stem cells

Hematopoietic stem cells provide an indispensible source for replenishing the blood with all its constituents throughout the organism's lifetime. Mice with a compromised hematopoietic stem cell compartment cannot survive. p53, a major tumor suppressor gene, has been implicated in regulation of hematopoiesis. In particular, p53 plays a role in homeostasis by regulating HSC quiescence and self renewal. We recently utilized a hypomorphic p53^515C allele in conjunction with Mdm2, a negative regulator of p53 to gain insights into the role of p53 in hematopoietic regulation. Our analyses revealed that p53^515C/515CMdm2^-/- double mutant mice die soon after birth due to hematopoietic failure. Further mechanistic studies revealed that in the absence of Mdm2, ROS induced postnatal p53 activity depletes hematopoietic stem cells, progenitors and differentiated cells.     

NF-kappaB family proteins participate in multiple steps of hematopoiesis through elimination of reactive oxygen species

To examine the roles for NF-kappaB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-kappaB(IkappaBSR) in a factor-dependent cell line, Ba/F3. Although IkappaBSR neither affected thrombopoietin-dependent nor gp130-mediated growth, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IkappaBSR enhanced factor-deprived apoptosis through the accumulation of reactive oxygen species (ROS). When expressed in normal hematopoietic stem/progenitor cells, IkappaBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS. We also expressed IkappaBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IkappaBSR was expressed at the stage of Flk-1(+) cells (putative hemangioblasts), IkappaBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IkappaBSR was expressed after the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-kappaB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS.     

Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.     

Mitotic catastrophe occurs in the absence of apoptosis in p53-null cells with a defective G1 checkpoint

Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD) that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.