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Reconstituting the Interaction Between Purified Nuclei and Microtubule Network
Gökçe Agsu1,2, Jérémie Gaillard1,2, Bruno Cadot3
1INSERM, CEA, U976 - HIPI, Institut de Recherche Saint Louis, Université de Paris, Paris, France.
Methods in Molecular Biology (Clifton, N.J.)
|April 27, 2022
Summary
This study presents a new protocol for purifying cell nuclei from nonadherent cells. This method enables in-depth in vitro studies of nucleus-microtubule interactions and their role in cell mechanics.
Area of Science:
- Cell Biology
- Biophysics
- Cytoskeleton Dynamics
Background:
- The cell nucleus, the stiffest organelle, undergoes deformation crucial for processes like cell migration and differentiation.
- Nucleus stiffness and shape are regulated by chromatin, lamin density, and cytoskeletal elements (actin and microtubules).
- While actin dominates in adherent cells, microtubules are key in nonadherent cells, necessitating further investigation.
Purpose of the Study:
- To develop a nucleus purification protocol for nonadherent cells.
- To investigate nucleus-microtubule interactions in vitro.
- To establish a system for studying the mechanical balance regulating nucleus deformation.
Main Methods:
- Developed a protocol for purifying nuclei from nonadherent cells.
- Utilized reconstitution assays combining purified nuclei with microtubules.
- Integrated nucleus-microtubule interaction studies with microtubule gliding assays.
Main Results:
- Successfully purified nuclei from nonadherent cells.
- Demonstrated that purified nuclei interact with microtubules.
- Observed differential wrapping of nuclei from various cell types within microtubule arrays.
- Showcased the ability to apply motor-based forces to microtubules to study nucleus-membrane interactions.
Conclusions:
- The developed protocol facilitates in-depth in vitro studies of microtubule-nucleus interactions.
- This method allows for the dissection of the mechanical contributions of microtubules to nucleus deformation.
- The findings provide a foundation for understanding nucleus mechanics in nonadherent cell contexts.
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