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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.

我们通过细胞磁测量和包括免疫荧光显微镜和差示干涉显微镜在内的光学观察来研究活细胞的细胞内运动性。细胞磁性计量学方法是我们自己发明的，也是少数几种能够研究活细胞内细胞运动和细胞骨架功能的方法之一。在本研究中，我们主要测量吞噬小体的随机运动引起的细胞内运动的能量(E_R)，吞噬小体的随机运动导致被细胞吞噬的磁性颗粒磁化后测量到的磁弛豫。我们使用影响细胞骨架的药物如微丝和微管，测量E_r，并用免疫荧光显微镜观察这些药物对微丝的影响。一个奇怪的结果是，抑制ATP合成的MIA增加了E_r，这似乎是矛盾的。MIA也抑制微丝合成，这可能与破坏微丝的细胞松弛素B降低E_r有关，而已知的破坏微管的秋水仙素也增加了E_r。这可能是由于它的副作用激活细胞运动(其他研究人员已经观察到)和增加微丝，这已经在免疫荧光显微镜下清楚地看到。因此，我们对E_r的测量定量地捕捉到了这一现象。细胞磁学的另一种应用是测量细胞膜的硬度。这是通过使包裹着纤维连接蛋白的磁性颗粒附着在细胞膜上，并在外部磁场的作用下扭曲它们来实现的。该方法被初步应用于研究细胞骨架与细胞膜之间的关系，有望用于测量细胞膜的力学性能及其对细胞骨架的依赖关系。