Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.
The linker of nucleoskeleton and cytoskeleton (LINC) complex is situated in the nuclear envelope and forms a connection between the lamina and cytoskeletal elements. Sun1, Sun2 and nesprin-2 are important components of the LINC complex. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts and studied their diffusional mobilities using fluorescence recovery after photobleaching. We show that they all are more mobile in embryonic fibroblasts from mice lacking A-type lamins than in cells from wild-type mice. Knockdown of Sun2 also increased the mobility of a short, chimeric form of nesprin-2 giant (mini-nesprin-2G), whereas the lack of emerin did not affect the mobility of Sun1, Sun2 or mini-nesprin-2G. Fluorescence resonance energy transfer experiments showed Sun1 to be more closely associated with lamin A than is Sun2. Sun1 and Sun2 had similar affinity for the nesprin-2 KASH domain in plasmon surface resonance (Biacore) experiments. This affinity was ten times higher than that previously reported between nesprin-2 and actin. Deletion of the actin-binding domain had no effect on mini-nesprin-2G mobility. Our data support a model in which A-type lamins and Sun2 anchor nesprin-2 in the outer nuclear membrane, whereas emerin, Sun1 and actin are dispensable for this anchoring.
Department of Medicine, Columbia University, New York, NY 10032, USA.
Cecilia
C
Ostlund
Eric S
ES
Folker
Jason C
JC
Choi
Edgar R
ER
Gomes
Gregg G
GG
Gundersen
Howard J
HJ
Worman
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D055785:Gene Knockdown Techniques; D034904:Lamin Type A / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D034881:Nuclear Lamina / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D034904:Lamin Type A; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C464932:SUN1 protein, mouse; C520338:Sun2 protein, mouse; C485903:Syne2 protein, mouse; D034501:Telomere-Binding Proteins; C092710:emerin