Analysis of Different Methodological Approaches to Measuring Microtubule Length in the Cytoplasm of Cultured Cells
Analysis of Different Methodological Approaches to Measuring Microtubule Length in the Cytoplasm of Cultured Cells
复制标题
测量培养细胞细胞质中微管长度的不同方法的分析
DOI：
10.1023/a:1009413211185
复制
发表时间：
2004
影响因子：
0.7
通讯作者：
I. Vorobjev
中科院分区：
文献类型：
--
作者：
O. A. Chernobelskaya;I. Grigoriev;I. Alieva;I. Vorobjev
一般认为组织培养细胞中的微管从中心体延伸至细胞外周，单个微管的长度平均为几十微米。然而，直接电子显微镜测量对这一假设提出了一些质疑。在这项研究中，使用组合方法估计了培养的 Vero 细胞中微管的平均长度。通过电子显微镜连续切片测定游离细胞质和中心体微管的长度；同时，通过追踪活细胞中沿微管的跳跃颗粒运动，在微管蛋白抗体染色的制剂中测量片层中游离微管的长度（间接免疫荧光方法）。根据免疫荧光显微镜数据，片层中的微管长度平均为4.57±3.69μm。然而，由于两个或多个微管可以重叠，因此这种方法可能会稍微高估它们的长度。另一方面，跳跃运动很容易相当准确地监测和测量，但由于马达（驱动蛋白和动力蛋白）的加工能力有限，它们的范围可能比实际微管长度短。平均而言，活细胞的跳跃运动轨迹为 3.85 ± 0.72 μm 长。在电子显微镜水平上，使用中心体周围和片层中的微管系统的伪三维重建来分析微管长度。片层中游离微管的长度达到18μm，平均3.33±2.43μm；中心体微管的平均长度为1.49±0.82μm。通过不同方法获得的微管长度和排列数据之间良好的对应性，可以得出这样的结论：Vero细胞中大多数游离微管的长度实际上为2-5μm；即，它们比细胞半径（约 25 μm）短得多。从中心体延伸的微管更短并且不会到达细胞外围。因此，Vero 细胞片层中的大多数微管是自由的，它们的有序排列与它们与中心体的附着无关。
It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 ± 3.69 μm. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 ± 0.72 μm long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 μm, averaging 3.33 ± 2.43 μm; the average length of centrosomal microtubules was 1.49 ± 0.82 μm. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of the free microtubules in Vero cells actually have a length of 2–5 μm; i.e., they are much shorter than the cell radius (about 25 μm). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.
DOI：
10.1073/pnas.96.1.115
发表时间：
1999-01-05
影响因子：
11.1
作者：
Rodionov, V;Nadezhdina, E;Borisy, G
通讯作者：
Borisy, G
DOI：
10.1073/pnas.94.10.5078
发表时间：
1997-05-13
影响因子：
11.1
作者：
Keating, TJ;Peloquin, JG;Borisy, GG
通讯作者：
Borisy, GG
培养细胞中微管的细胞质组装。
影响因子：
4
作者：
Vorobjev,IA;Svitkina,TM;Borisy,GG
通讯作者：
Borisy,GG