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Actin dysfunction activates ERK1/2 and delays entry into mitosis in mammalian cells
Reports NRF is supported by Research Projects( Actin dysfunction activates ERK1/2 and delays entry into mitosis in mammalian cells | 2004 Year 신청요강 다운로드 PDF다운로드 | 이경희(연세대학교) ) data is submitted to the NRF Project Results
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Project Number C00025
Year(selected) 2004 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2007년 09월 07일
Year type 결과보고
Year(final report) 2007년
Research Summary
  • Korean
  • 세포분열 (mitosis) 과정에서 복제된 염색체의 물리적 분리가 일어나기 전 actin 세포골격계에 의해 조절되는 방추체의 방향성을 검증하는 actin checkpoint의 존재는 방추체의 방향성이 정확히 딸세포로 염색체를 분리하는데 매우 중요한 출아형 효모뿐 아니라 mitosis에서 방추체의 방향성에 의해 세포질분열 (cytokinesis)의 축이 결정되어 세포의 운명이 결정되는 다세포 동물 및 포유동물의 발생과정을 이해하기 위해서도 매우 중요하다. 그러나 다세포 동물 및 포유동물에서 방추체의 방향성을 검증하는 actin checkpoint의 존재 및 그 정확한 기전은 아직 잘 알려져 있지 않다.
    본 연구에서는 방추체의 역동적인 방향성에 의해 정확한 염색체 분리 및 세포질분열의 축이 조절되어 세포의 운명이 결정되는 다세포 동물 및 포유동물의 세포분열 및 발생과정을 이해하기 위하여 포유동물 세포에서 현재까지 잘 알려져 있지 않은 actin checkpoint의 존재 및 actin 세포골격계에 의한 세포분열 조절기전의 기능을 밝히고자 한다. Cytochalasin D와 BDM을 이용하여actin 세포골격계와 그 motor protein인 myosin II을 해체 시, 암 세포주는 유사분열 세포주기에 영향을 받지 않았다. 그러나, 세포의 방향성이 중요한 primary세포들인 IMR90와 MEF에서는 유사분열의 시작이 지연되는 것을 관찰할 수 있었으며, 이러한 세포들은 정상적인 분열 방추제를 형성하지 못하였으며, 결국 중심체의 분열과 이동이 정상적으로 이루어지지 못하는 것을 관찰할 수 있었다. 또한 actin 세포골격계 해체는 ERK1/2 MAP kinase의 지속적인 활성적 인산화를 야기하였고, 특이적 작용inhibitor인 U0126과 dominant negative mutants의 transfection을 이용한 지속적인 ERK1/2의 활성저해는 지연되었던 세포주기를 회복하게 하였다. 따라서, ERK1/2를 유사분열 초기에 잘못된 방추체의 방향성을 감지하여, 유사분열의 시작을 지연시킬 수 있는 새로운 형태의 mitotic checkpoint라고 할 수 있겠다.
    이것은 동물세포의 세포분열 과정에서 actin 세포골격계가 어떻게 미세소관과 상호작용 하여 유전체의 안정성을 유지하는가의 기전을 제시하는 것이며, 아직 포유동물 세포에서 보고되지 않은 actin checkpoint의 존재를 최초로 확인하는 주요한 연구라고 할 수 있을 것이다. 또한 궁극적으로 mitosis에서 actin 세포골격계에 의해 방추체의 방향성이 결정되는 기전의 이해는 발생과정에서 방추체의 방향 조절에 의한 세포질분열 축의 조절로 비대칭적으로 딸 세포의 운명이 결정되는 중요한 조절 기전에 대한 단서를 제공할 것이다.
  • English
  • The positioning of the cell division axis is a critical feature for ensuring normal cell growth. The proper spindle assembly and orientation are critical and must be coupled to cell polarity for correct positioning of daughter cells in symmetric or asymmetric cell division. Many studies have described the crucial functions of the microtubules and microtubule motors for spindle positioning. Lately, growing evidences suggest that spatial distribution of cortical signals seems to continuously guide spindle orientation and daughter cell positioning. However, it has been unclear whether actin is involved in cell division except cytokinesis in mammalian cells. In this study, we describe that cortical actin cyctoskeleton is also required for spindle assembly and orientation in mammalian cells. We mainly use cytochalasin D and butanedione-2-monoxime (BDM) to disrupt the actin cytoskeleton and myosin II. After being treated with each inhibitor, only primary cells not transformed cancer cells show delay at mitotic entry and mitotic spindle assembly and orientation are perturbed. Interestingly, the delay caused by disrupted actin cytoskeletons is accompanied with a strong and sustained activation of ERK1/2, suggesting that direct involvement of ERK1/2 is critical for this actin checkpoint pathway. Based on our finding, this ERK1/2 dependent actin checkpoint is considered as another G2 checkpoint or part of antephase checkpoint. Our work emphasizes the importance of cortical actin and myosin II exert for assembling a spindle and provides a new G2 checkpoint that accounts for how spindle is formed and becomes correctly positioned within the framework of a cell.
    Although Mitogen-activated protein kinase (MAPK) pathways are required for cell cycle progression into S phase and through G2 and M phases, an involvement in mitotic cell cycle control of the JNKs has significantly less well characterized. Here, we demonstrate that the c-Jun N-terminal kinase 1 and 2 (JNK1/2) has a role in mitosis. Phosphorylation of histone H3 at serine 10 (Ser10) is considered to be essential event for onset of mitosis. In this study, we show that c-Jun N-terminal kinase (JNK) is required for histone H3 Ser10 phosphorylation at mitosis. Inhibition of JNK using specific pharmacological inhibitors or dominant negative JNK1 and 2 mutants prevent histone H3 Ser10 phosphorylation in vivo and JNK can phosphorylate histone H3 in vitro. In cells treated with SP600125, neither the activating dephosphorylation of CDK1 nor the mitotic cyclin A degradation at the beginning of mitosis was observed, verifying the arrest in mitotic entry. During G2/M transition, basal JNK phosphorylation is increased and enriched on nucleus and subsequently localized in centrosomes. The nuclear localization of active JNK only at G2 is tightly correlated with histone H3 phophirylation. Finally, we suggest that JNK might be a mitotic H3 kinase and appears to be distinct from mitogen-induced H3 phosphorylations, maybe helping to explain how reduced JNK activity inhibits G2/M cell cycle progression and proliferation.
Research result report
  • Abstract
  • Investigations of actin function during the cell cycle have focused primarily on cytokinesis. Here, we describe the role of actin at the entry into mitosis in primary mammalian cells. Depolymerization of actin with cytochalasin D or inhibition of myosin ATPase with butanedione-2-monoxime (BDM) at G2 blocked the mitotic spindle formation and central positioning of the nucleus in synchronized MEF and IMR90 cells. Time-lapse microscopy confirmed that these treatments inhibit both spindle formation and separation of duplicated centrosomes to the opposite poles. Concurrent with actin dysfunction, activation of Cdc2 and nuclear localization of cyclin B1 were delayed. Furthermore, cyclin A degradation that is necessary for nuclear envelope breakdown (NEBD) in early mitosis was deferred, supporting the conclusion that mitotic onset was delayed. The activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was sustained in these cells, and the use of a specific ERK inhibitor or a dominant negative form of ERK2 abrogated this delay of entry into mitosis. This delay of mitotic entry and the sustained ERK1/2 activity by actin dysfunction was observed only in primary cells and not in transformed cancer cell lines. These observations demonstrate that an intact actin cytoskeleton is necessary for entry into mitosis and that ERK1/2 is involved in monitoring actin dysfunction to control the onset of mitosis, suggesting the presence of an actin checkpoint at the G2/M transition in primary mammalian cells.
  • Research result and Utilization method
  • 1. Disruption of actin cytoskeleton during G2/M transition delays mitotic cell cycle progression
    2. Disruption of actin cytoskeleton blocks mitotic spindle assembly and proper positioning of the nucleus
    3. Disruption of actin cytoskeleton leads to strong and prolonged activation of ERK1/2 in mitosis
    4. Actin dysfunction at G2/M inhibits Cdc25C phosphorylation
    5. Sustained ERK1/2 activation is not directly involved in the block of centrosome separation caused by actin defects

    이러한 연구결과는 궁극적으로 유사분열에서 actin 세포골격계에 의해 방추체의 방향성이 결정되는 기전의 이해에 접근하여 발생과정에서 방추체의 방향 조절에 의한 세포질분열 축의 조절로 비대칭적으로 딸세포의 운명이 결정되는 다세포 동물 및 포유동물의 세포분열 및 발생과정에 대한 이해에 대한 단서를 제공할 것이라 사료됨
  • Index terms
  • actin cytoskeleton, centrosome, astral microtubule, MEF, IMR90, ERK1/2, mitotic entry, actin checkpoint, G2/M
  • List of digital content of this reports
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