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β-II tubulin isotype directs stiffness and differentiation of neuroblastoma SH-SY5Y cells

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Abstract

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.

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Abbreviations

MTs:

microtubules

AFM:

atomic force microscopy

MAPs:

microtubule-associated proteins

DMEM:

Dulbecco’s modified eagle medium

FBS:

fetal bovine serum

RA:

trans-retinoic acid

DM:

differentiation media

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Acknowledgements

We would like to thank the Central Instrumentation Facility of the Indian Institute of Technology Bombay for atomic force microscopy. We thank Prof. Abhijit Majumder for allowing us to use his phase-contrast microscope (EVOS-FL auto-inverted microscope, Life Technologies). We acknowledge the Indian Institute of Technology, Bombay for providing a postdoctoral fellowship to NM. We would like to acknowledge the funding provided by the JC Bose Fellowship (JCB/2019/000016) from the Department of Science and Technology Government of India to DP.

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Authors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India

    Nikita Mundhara & Dulal Panda

  2. National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Mohali, Punjab, 160062, India

    Dulal Panda

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  1. Nikita Mundhara
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NM and DP designed the experiments, analysed data, and wrote the manuscript. NM performed the experiments. All authors have approved the final version of the manuscript.

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Correspondence to Dulal Panda.

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Mundhara, N., Panda, D. β-II tubulin isotype directs stiffness and differentiation of neuroblastoma SH-SY5Y cells. Mol Cell Biochem 478, 1961–1971 (2023). https://doi.org/10.1007/s11010-022-04649-0

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