Distinct functions of the unique C terminus of LAP2alpha in cell proliferation and nuclear assembly.
The non-membrane-bound lamina-associated polypeptide 2 isoform, LAP2alpha, forms nucleoskeletal structures with A-type lamins and interacts with chromosomes in a cell cycle-dependent manner. LAP2alpha contains a LEM (LAP2, emerin, and MAN1) domain in the constant N terminus that binds to chromosomal barrier-to-autointegration factor, and a C-terminal unique region that is essential for chromosome binding. Here we show that C-terminal LAP2alpha fragment efficiently bound to mitotic chromosomes and inhibited assembly of endogenous LAP2alpha, nuclear membranes, and lamins A/C in in vitro nuclear assembly assays. Full-length recombinant LAP2alpha, which bound to chromosomes, and N-terminal fragment, which did not bind, had no effect on assembly. This suggested an essential role for the LAP2alpha C terminus in chromosome association and for the N-terminal LEM domain in subsequent assembly stages. In vivo analysis upon transient expression of GFP-tagged LAP2alpha fragments confirmed that, unlike the N-terminal fragment, the C-terminal fragment was able to bind to chromosomes during mitosis, if expressed weakly. At higher expression levels, C-terminal LAP2alpha fragment and full-length protein led to cell cycle arrest in interphase and apoptosis, as shown by fluorescence-activated cell sorter analysis, time lapse microscopy, and BrdUrd incorporation assays. These data indicated distinct functions of LAP2alpha in cell cycle progression during interphase and in nuclear reassembly during mitosis.
Vlcek
Sylvia
S
Department of Biochemistry and Molecular Cell Biology, Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria.
Korbei
Barbara
B
Foisner
Roland
R
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002455:Cell Division / Q000502:physiology*; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology*; D002875:Chromosomes; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C093413:lamina-associated polypeptide 2; D049452:Green Fluorescent Proteins