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

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

Research output: Contribution to journalArticlepeer-review

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 LAP2 alpha. 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 beta-galactosidase. The beta-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.

Original languageEnglish
Article numbere18762
Number of pages12
JournalPLoS ONE
Volume6
Issue number4
DOIs
Publication statusPublished - 14 Apr 2011

Keywords

  • Cell Aging
  • Cell Cycle
  • Cell Cycle Proteins
  • Cell Line
  • Flow Cytometry
  • Gene Knockdown Techniques
  • Humans
  • Membrane Proteins
  • Nuclear Envelope
  • RNA Interference

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