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A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in
Nadia Korfali1, Vlastimil Srsen, Martin Waterfall
1The Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
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.
Insights
Nuclear envelope proteins significantly impact cell cycle regulation. NET4/TMEM53 influences cell cycle progression by affecting key proteins like p53 and retinoblastoma protein, potentially leading to premature senescence.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Nuclear envelope protein mutations can disrupt cell cycle regulation, contributing to disease.
- Previous research identified limited nuclear envelope proteins affecting cell cycle progression.
Purpose of the Study:
- To screen novel nuclear envelope transmembrane proteins for their impact on cell cycle progression.
- To investigate the role of NET4/Tmem53 in cell cycle regulation.
Main Methods:
- Screening of 39 novel nuclear envelope transmembrane proteins using flow cytometry.
- Investigating NET4/Tmem53 function in p53(-/-) and retinoblastoma protein-deficient cells.
- Analyzing protein levels (p53, retinoblastoma protein, p21) and cell cycle markers (Ki-67, ß-galactosidase) after NET4/Tmem53 manipulation.
- Assessing the role of p38 MAP kinase pathway.
Main Results:
- Eight of 39 screened proteins altered cell cycle profiles; seven increased the 4N:2N ratio.
- NET4/Tmem53 knockdown mimicked overexpression effects and was dependent on p53 and retinoblastoma protein.
- NET4/Tmem53 loss decreased phosphorylated retinoblastoma protein, increased p53 and p21 levels, induced cell cycle withdrawal, and suggested premature senescence.
- NET4/Tmem53-mediated changes were dependent on p38 MAP kinase activity.
Conclusions:
- A significant proportion of nuclear envelope transmembrane proteins influence cell cycle progression.
- NET4/Tmem53 plays a critical role in cell cycle control, impacting key regulatory proteins and potentially inducing senescence.
- The nuclear envelope's role in cell cycle regulation is more substantial than previously recognized.
