Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET analyses in living HeLa cells

Takeshi Shimi, Takako Koujin, Miriam Segura-Totten, Katherine L. Wilson, Tokuko Haraguchi, Yasushi Hiraoka*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

106 Scopus citations

Abstract

Barrier-to-autointegration factor (BAF) is a conserved 10kDa DNA-binding protein. BAF interacts with LEM-domain proteins including emerin, LAP2β, and MAN1 in the inner nuclear membrane. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we compared the mobility of BAF to its partners emerin, LAP2β, and MAN1 in living HeLa cells. Like endogenous BAF, GFP-BAF was enriched at the nuclear envelope, and found inside the nucleus and in the cytoplasm during interphase. At every location, FRAP and FLIP analysis showed that GFP-BAF diffused rapidly; the halftimes for recovery in a 0.8μm square area were 260ms at the nuclear envelope, and even faster inside the nucleus and in the cytoplasm. GFP-fused emerin, LAP2β, and MAN1 were all relatively immobile, with recovery halftimes of about 1min, for a 2μm square area. Thus, BAF is dynamic and mobile during interphase, in stark contrast to its nuclear envelope partners. FLIP results further showed that rapidly diffusing cytoplasmic and nuclear pools of GFP-BAF were distinctly regulated, with nuclear GFP-BAF unable to replenish cytoplasmic BAF. Fluorescence resonance energy transfer (FRET) results showed that CFP-BAF binds directly to YFP-emerin at the inner nuclear membrane of living cells. We propose a "touch-and-go" model in which BAF binds emerin frequently but transiently during interphase. These findings contrast with the slow mobility of both GFP-BAF and GFP-emerin during telophase, when they colocalized at the 'core' region of telophase chromosomes at early stages of nuclear assembly.

Original languageEnglish (US)
Pages (from-to)31-41
Number of pages11
JournalJournal of Structural Biology
Volume147
Issue number1
DOIs
StatePublished - Jul 2004

Keywords

  • Barrier-to-autointegration factor
  • Emerin
  • Emery-Dreifuss muscular dystrophy
  • LAP2
  • Laminopathy
  • MAN1
  • Nuclear membrane

ASJC Scopus subject areas

  • Structural Biology

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