A Flow Cytometry-Based Screen of Nuclear Envelope Transmembrane Proteins Identifies NET4/Tmem53 as Involved in Stress-Dependent Cell Cycle Withdrawal

Korfali, Nadia; Srsen, Vlastimil; Waterfall, Martin; Batrakou, Dzmitry G.; Pekovic, Vanja; Hutchison, Christopher J.; Schirmer, Eric C.

Abstract

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N∶2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53−/− cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

Citation

Korfali, N., Srsen, V., Waterfall, M., Batrakou, D. G., Pekovic, V., Hutchison, C. J., & Schirmer, E. C. (2011). A Flow Cytometry-Based Screen of Nuclear Envelope Transmembrane Proteins Identifies NET4/Tmem53 as Involved in Stress-Dependent Cell Cycle Withdrawal. PLoS ONE, 6(4), Article e18762. https://doi.org/10.1371/journal.pone.0018762

• Journal Article Type: Article
• Acceptance Date: Mar 17, 2011
• Online Publication Date: Apr 14, 2011
• Publication Date: Apr 14, 2011
• Deposit Date: May 14, 2018
• Publicly Available Date: May 14, 2018
• Journal: PLoS ONE
• Publisher: Public Library of Science
• Peer Reviewed: Peer Reviewed
• Volume: 6
• Issue: 4
• Article Number: e18762
• DOI: https://doi.org/10.1371/journal.pone.0018762