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Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells
Abstract:
Receptor-mediated endocytosis is inhibited during mitosis in mammalian cells and earlier work on A431 cells suggested that one of the sites inhibited was the invagination of coated pits (Pypaert, M., J. M. Lucocq, and G. Warren. 1987. Eur. J. Cell Biol. 45: 23-29). To explore this inhibition further, we have reproduced it in broken HeLa cells. Mitotic or interphase cells were broken by freeze-thawing in liquid nitrogen and warmed in the presence of mitotic or interphase cytosol. Using a morphological assay, we found invagination to be inhibited only when mitotic cells were incubated in mitotic cytosol. This inhibition was reversed by diluting the cytosol during the incubation. Reversal was sensitive to okadaic acid, a potent phosphatase inhibitor, showing that phosphorylation was involved in the inhibition of invagination. This was confirmed using purified cdc2 kinase which alone could partially substitute for mitotic cytosol.
Insights
Receptor-mediated endocytosis invagination is blocked during mitosis. This inhibition in HeLa cells is dependent on mitotic cytosol and reversed by dilution, indicating phosphorylation
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Receptor-mediated endocytosis is crucial for cellular processes.
- This process is known to be inhibited during mitosis in mammalian cells.
- Previous studies suggested inhibition of coated pit invagination.
Purpose of the Study:
- To investigate the mechanism of endocytosis inhibition during mitosis.
- To identify factors responsible for inhibiting coated pit invagination in mitotic cells.
- To explore the role of phosphorylation in this process.
Main Methods:
- Utilized a broken cell system with HeLa cells.
- Incubated mitotic or interphase cells with corresponding cytosol.
- Employed a morphological assay to assess coated pit invagination.
- Investigated the effect of cytosol dilution and okadaic acid.
- Tested the role of purified cdc2 kinase.
Main Results:
- Coated pit invagination was inhibited specifically when mitotic cells were incubated with mitotic cytosol.
- This inhibition was reversible upon dilution of the mitotic cytosol.
- Sensitivity to okadaic acid indicated the involvement of phosphorylation.
- Purified cdc2 kinase partially mimicked the effect of mitotic cytosol.
Conclusions:
- Mitotic cytosol contains factors that inhibit coated pit invagination.
- Phosphorylation, likely mediated by cdc2 kinase, plays a key role in this mitotic inhibition.
- The findings provide insights into the regulation of endocytosis during cell division.