Investigation of the functions of microfilaments and microtubules by cytomagnetometry, immunoflurescent microscopy and differential interference microscopy.

通过细胞磁力测定法、免疫荧光显微镜和微分干涉显微镜研究微丝和微管的功能。

基本信息

批准号：
12680846
负责人：
NEMOTO Iku
金额：
$ 1.86万
依托单位：
Tokyo Denki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

We investigated intracellular motility of living cells by cytomagnetometry and optical observations including immunoflurescent microscopy and differential interference microscopy. Cytomagnetometric method is our own invention and one of few methods able to investigate intracellular movement and the functions of the cytoskeletons within living cells. In the present study we concentrated on the measurement of the energy (E_r) of the intracellular movements responsible for the random movements of the phagosomes which causes the magnetic relaxation measured after magnetizing the magnetic particles engulfed by the cells. We used cytoskeletal drugs which affect the cytoskeletons such as microfilaments and microtubules, and measured E_r and observed the effects of those drugs on the filaments by immunoflurescent microscopy. One peculiar result was that MIA which inhibits ATP synthesis increased E_r which seems contradictory. MIA also inhibited microfilament synthesis, which may have to do with the increase in E_r although cytochalasin B which disrupts microfilaments decreased E_r. Colchicine, which has been known to break microtubules increased E_r also. This is probably due to its side effect of activation of cellular movements (which has been observed by other researchers) and of increasing microfilaments which has been clearly seen by immunoflurescent microscopy. Our measurement of E_r thus captured the phenomenon quantitatively. A different application of cytomagnetometry is measurement of the stiffness of the cell membrane. This was done by making magnetic particles coated with fibronectin adhere to the cell membrane and twisting them by an external magnetic field. This method was tentatively applied to investigate the relationship between the cytoskeletons and the cell membrane and seems promising to measure the mechanical properties of the cell membrane and their dependence on the cytoskeletons.

我们通过细胞磁法和光学观察结果（包括免疫膨胀显微镜和差异干扰显微镜）研究了活细胞的细胞内运动。细胞磁测量法是我们自己的发明，也是能够研究细胞内运动和活细胞内细胞骨架功能的少数方法之一。在本研究中，我们集中于负责吞噬体随机运动的细胞内运动的测量（E_R），这在磁化磁性颗粒被细胞吞没后导致磁性弛豫。我们使用了影响细胞骨架的细胞骨架药物，例如微丝和微管，并测量了E_R，并观察到这些药物对免疫泄漏显微镜的影响。一个特殊的结果是，抑制ATP合成的MIA增加了E_R，这似乎是矛盾的。 MIA还抑制了微丝合成，这可能与E_R的增加有关，尽管破坏微丝的细胞切拉蛋白B降低了E_R。已知会破坏微管的秋水仙碱也增加了E_R。这可能是由于其激活细胞运动的副作用（其他研究人员已经观察到了）以及增加的微丝，这已经通过免疫浮力显微镜显然可以看出。因此，我们对e_r的测量量捕获了现象。细胞磁法的不同应用是测量细胞膜的刚度。这是通过使覆盖在纤连蛋白上的磁性颗粒粘附到细胞膜上并通过外部磁场扭曲它们来完成的。该方法暂定地用于研究细胞骨架与细胞膜之间的关系，似乎有望测量细胞膜的机械性能及其对细胞骨架的依赖性。