TY - JOUR
T1 - Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET analyses in living HeLa cells
AU - Shimi, Takeshi
AU - Koujin, Takako
AU - Segura-Totten, Miriam
AU - Wilson, Katherine L.
AU - Haraguchi, Tokuko
AU - Hiraoka, Yasushi
N1 - Funding Information:
We thank Dr. R. Craigie for BAF plasmids, Dr. R. Foisner for LAP2β plasmids, Dr. H. Worman for the MAN1 plasmid, Dr. K. Furukawa for the BAF antibody, Dr. H. Kimura for histone H2B–GFP and helpful discussion on the FRAP experiments, and the Riken Cell Bank for HeLa cells. This work was supported by grants from the Human Frontier Science Program (to K. L. W. and Y. H.), the Japan Science and Technology Corporation (to Y. H. and T. H.), Grant-in-Aid for Scientific Research B (to Y. H. and T. H.), and National Institutes of Health RO1 grant GM48646 (to K. L. W.).
PY - 2004/7
Y1 - 2004/7
N2 - 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.
AB - 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.
KW - Barrier-to-autointegration factor
KW - Emerin
KW - Emery-Dreifuss muscular dystrophy
KW - LAP2
KW - Laminopathy
KW - MAN1
KW - Nuclear membrane
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U2 - 10.1016/j.jsb.2003.11.013
DO - 10.1016/j.jsb.2003.11.013
M3 - Article
C2 - 15109603
AN - SCOPUS:1842578717
SN - 1047-8477
VL - 147
SP - 31
EP - 41
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 1
ER -