Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India.
National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Mohali, Punjab, 160062, India.
Mol Cell Biochem. 2023 Sep;478(9):1961-1971. doi: 10.1007/s11010-022-04649-0. Epub 2022 Dec 30.
β-tubulin isotypes regulate the structure and bundling of microtubule (MT) lattice, its dynamics, and resulting functions. They exhibit differential tissue expression, varying due to physical and biochemical cues. In this work, we investigated the effect of transient heat shock at 42 °C on the nuclear and cytoplasmic stiffness of SH-SY5Y neuroblastoma cells through atomic force microscopy. Moreover, the variations in the expression of β-tubulin isotypes as a heat shock response were also monitored. The heat-exposed cells endured a recovery at 37 °C for 24 h and they manifested an increase of cytoplasmic stiffness by 130 ± 25% with respect to untreated controls. The expression of β-II tubulin isotype in heat-recovered cells is augmented by 51 ± 5% whereas the levels of total tubulin and β-III tubulin isotype remain unaltered. Upon depletion of β-II tubulin isotype using shRNA, the increase in cytoplasmic stiffness was dampened. However, it remained unaffected upon depletion with β-III tubulin isotype shRNA. This features the role of the β-II tubulin isotype in regulating cellular stiffness. In addition, neuroblastoma SH-SY5Y cells undergo differentiation by initiating neuritogenesis and prior evidence suggests the indispensable role of β-II tubulin isotype in this process. The heat-recovered cells which expressed higher levels of β-II tubulin isotype expedited the differentiation process in 3-day which was around 5-day for control cells, however, upon depletion of β-II tubulin isotype, the cells almost lost their differentiation potential. Altogether, this work highlights the role of β-II tubulin isotype as a biomarker for cellular stiffness.
β-微管蛋白异构体调节微管（MT）晶格的结构和束集、其动态变化以及由此产生的功能。它们表现出不同的组织表达，其变化是由于物理和生化线索。在这项工作中，我们通过原子力显微镜研究了 42°C 短暂热休克对 SH-SY5Y 神经母细胞瘤细胞核和细胞质硬度的影响。此外，还监测了β-微管蛋白异构体作为热休克反应的表达变化。暴露于热的细胞在 37°C 下恢复 24 小时，与未经处理的对照相比，细胞质硬度增加了 130±25%。热恢复细胞中β-II 微管蛋白异构体的表达增加了 51±5%，而总微管蛋白和β-III 微管蛋白异构体的水平保持不变。使用 shRNA 耗尽β-II 微管蛋白异构体后，细胞质硬度的增加减弱。然而，在用β-III 微管蛋白异构体 shRNA 耗尽时，它仍然不受影响。这突出了β-II 微管蛋白异构体在调节细胞硬度中的作用。此外，神经母细胞瘤 SH-SY5Y 细胞通过启动神经突发生分化来进行分化，先前的证据表明β-II 微管蛋白异构体在这个过程中不可或缺。表达更高水平β-II 微管蛋白异构体的热恢复细胞在 3 天内加速了分化过程，而对照细胞则需要大约 5 天，然而，当耗尽β-II 微管蛋白异构体时，细胞几乎失去了分化能力。总的来说，这项工作强调了β-II 微管蛋白异构体作为细胞硬度生物标志物的作用。
