University of Paris, INSERM, CEA, UMRS1160, Institut de Recherche Saint Louis, CytoMorpho Lab, Hôpital Saint Louis, 10 Avenue Claude Vellefaux, 75010 Paris, France.
Aix Marseille Université, CNRS, INP UMR7051, NeuroCyto Lab, 27 Boulevard Jean Moulin, 13385 Marseille, France.
Curr Biol. 2023 Jan 9;33(1):122-133.e4. doi: 10.1016/j.cub.2022.11.060. Epub 2022 Dec 23.
Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 μm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.
微管自修复已在体外和体内作为微管稳定性的潜在机制进行了研究。微管中微管蛋白二聚体的周转率挑战了仅生长末端具有动态性的先存教条。然而，尽管有明确的证据表明微管蛋白在体外掺入聚合微管的轴中，但这种事件在活细胞中发生的可能性仍然不确定。在这项研究中，我们通过将用红色荧光染料标记的纯化微管蛋白二聚体微注射到表达 GFP-微管蛋白的细胞的细胞质中，研究了这种可能性。我们在几分钟内观察到红色斑点沿着预先存在的绿色微管出现。我们发现这些红点的荧光强度与绿色信号呈负相关，表明红色二聚体被掺入微管中并取代了预先存在的绿色二聚体。与微管中心的横向距离与掺入位点和生长末端的距离相似。掺入的大小和空间频率随注入微管蛋白浓度和注射后延迟的饱和表明，注入的二聚体掺入到有限数量的受损位点中。根据我们的低估计，在注射后的几分钟内，游离二聚体每 70μm 的微管就会掺入到主要的修复位点。最后，我们在微图案化细胞中定位了这些位点，并发现它们在肌动蛋白丝网络密度较低且微管表现出更大的横向波动的区域更加集中。
