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Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions
Published on: June 16, 2018
Electron microscopic examination of podosomes induced by phorbol 12, 13 dibutyrate on the surface of A7r5 cells
Hideyuki Tanaka1, Hong-Hui Wang2, Sean E Thatcher3
1Department of Anatomy, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
Abstract:
The role of myosin light chain kinase (MLCK) in inducing podosomes was examined by confocal and electron microscopy. Removal of myosin from the actin core of podosomes using blebbistatin, a myosin inhibitor, resulted in the formation of smaller podosomes. Downregulation of MLCK by the transfection of MLCK small interfering RNA (siRNA) led to the failure of podosome formation. However, ML-7, an inhibitor of the kinase activity of MLCK, failed to inhibit podosome formation. Based on our previous report (Thatcher et al. J.Pharm.Sci. 116 116-127, 2011), we outlined the important role of the actin-binding activity of MLCK in producing smaller podosomes.
Insights
Myosin light chain kinase (MLCK) is crucial for podosome formation. Its actin-binding activity, not kinase function, drives smaller podosome production, as shown by blebbistatin and siRNA experiments.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Podosomes are dynamic actin-rich structures involved in cell adhesion and matrix degradation.
- The precise molecular mechanisms regulating podosome formation and stability are not fully understood.
Purpose of the Study:
- To investigate the role of myosin light chain kinase (MLCK) in podosome formation and regulation.
- To differentiate between the kinase activity and actin-binding activity of MLCK in podosome dynamics.
Main Methods:
- Confocal and electron microscopy were used to visualize podosome structures.
- Blebbistatin, a myosin inhibitor, was employed to assess the role of myosin.
- Small interfering RNA (siRNA) was used to downregulate MLCK expression.
- ML-7, a specific inhibitor of MLCK kinase activity, was utilized.
Main Results:
- Inhibition of myosin activity with blebbistatin led to the formation of smaller podosomes.
- Downregulation of MLCK via siRNA resulted in the complete failure of podosome formation.
- Inhibition of MLCK's kinase activity with ML-7 did not prevent podosome formation.
- These findings suggest MLCK's actin-binding activity is essential for podosome formation.
Conclusions:
- MLCK plays a critical role in initiating podosome formation.
- The actin-binding property of MLCK, rather than its kinase activity, is essential for producing smaller podosomes.
- Further research into MLCK's structural role in podosomes is warranted.
