title: A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.
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.
authors.affiliation: The Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Nadia
authors.initials: N
authors.lastname: Korfali
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Srsen
authors.forename: Martin
authors.initials: M
authors.lastname: Waterfall
authors.forename: Dzmitry G
authors.initials: DG
authors.lastname: Batrakou
authors.forename: Vanja
authors.initials: V
authors.lastname: Pekovic
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hutchison
authors.forename: Eric C
authors.initials: EC
authors.lastname: Schirmer
mesh_terms: D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D016922:Cellular Senescence; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D034622:RNA Interference
chemical_list: D018797:Cell Cycle Proteins; D008565:Membrane Proteins