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First published online 6 February 2007
doi: 10.1242/jcs.03390

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LAP2-binding protein LINT-25 is a novel chromatin-associated protein involved in cell cycle exit

¹ Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, Dr. Bohr-Gasse 9, Vienna Biocenter, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
² Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria

View larger version (33K): [in this window] [in a new window]   Fig. 1. LINT-25 is a novel interaction partner of LAP2. (A) Diagram of the domain organization of LAP2, full-length human OIP5/LINT-25 (229 residues, TIGR THC2238209) and the yeast two-hybrid fragment identified using LAP2-specific C-terminus as bait. The light gray box in human LINT-25 (residues 75-219) delineates conserved regions found also in protein sequences in Mus musculus (TIGR TC1500192), Rattus norvegicus (GB XM_575214), Gallus gallus (GB XM_421136), Xenopus laevis (TIGR TC267260), Danio rerio (TIGR TC296675) and Homo sapiens (TIGR THC2246127) with dotted lines showing the predicted full-length proteins. The black rectangle represents a predicted coiled-coil domain. Accession numbers given are from the Institute for Genomic Research (TIGR) and from GenBank (GB). The two numbers below each bar indicate the percentage identity and similarity, respectively, to human LINT-25 protein sequence. (B) RT-PCR analyses of LINT-25, LAP2, and actin mRNA as control, using mRNAs from the indicated human tissues or cell line: bone marrow (Bm), brain (B), colon (C), heart (H), kidney (K), skeletal muscle (Sk), thymus (Th), testis (T) and uterus (U). An ethidium bromide-stained gel of PCR fragments is shown.

View larger version (38K): [in this window] [in a new window]   Fig. 2. LINT-25 and LAP2 interact in vitro. (A) His-tagged, recombinant LINT-25 and LAP2 were incubated alone or together and mixed with immobilized LAP2 antibody. Immunoprecipitates (P) and supernatants (SN) were analyzed by immunoblotting detecting LAP2 (upper panel) and the His-Tag (lower panel). Input represents 10% of protein used for IPs. (B) In vitro translated, [35S]methionine-labeled LINT-25 was incubated alone or together with in vitro translated and labeled LAP2 or LAP21-408 and mixed with immobilized LAP2- and LAP2 antibodies (IP) or hybridoma medium (DMEM) as a control. Immunoprecipitates (P) and supernatants (SN) were analyzed by SDS-PAGE and autoradiography. Input represents 2% of protein used for IP. Note, that the signals of the different protein bands do not reflect the relative protein levels due to different numbers of methionines in the proteins' primary sequences. (C) Recombinant LINT-25 was transblotted to nitrocellulose, stained with Ponceau S (Ponc), or detected with His-tag reagent (His) or overlaid with recombinant LAP2 or buffer. Bound LAP2 was detected using LAP2 antibodies (LAP2). Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25.

View larger version (38K): [in this window] [in a new window]   Fig. 3. LINT-25 localizes to the nucleus and associates with chromatin during mitosis. (A) HeLa cells were lysed in hypotonic buffer with iodoacetamide (IAA) (left panel), and total lysates (Pre), nuclear (N) and cytoplasmic (C) fractions were analyzed by immunoblotting using antibodies against LINT-25, lamin A/C and LAP2 (right panel). HeLa cells were lysed in culture dishes in buffer with the indicated concentrations of Triton X-100 and salt and total lysates (Pre), insoluble pellet fractions (P) and supernatants (S) were analyzed by immunoblotting. Note that the LINT-25 double band is caused by additional alkyl-groups resulting from the IAA treatment (see also supplement). Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25; La, lamin. (B,C) HeLa cells were fixed with formaldehyde and double-stained for immunofluorescence microscopy using LINT-25 antiserum and antibodies against the indicated antigens. DNA was stained with Hoechst dye. Confocal images of interphase cells (B) or various stages of mitosis (C) are shown. Arrowheads indicate outer core regions. Bars, 10 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 4. LINT-25 is upregulated and translocated to chromocenters in non-proliferating fibroblasts. BJ1 human foreskin fibroblasts (A,B), human primary skin fibroblasts (C) and mouse primary skin fibroblasts (D) were cultivated in normal medium (P), under low serum conditions for 6-9 days (Q6, Q7, Q9) followed by serum re-stimulation for 38 hours (Q9P), or passaged in culture until reaching replicative senescence (S). Cells were processed for immunofluorescence microscopy (confocal images are shown. Bars, 10 µm) or immunoblotting of cell lysates (B, left panel). Samples were analyzed using antibodies against the indicated antigens or stained for senescence-associated -galactosidase activity (SA -gal). For testing mRNA levels (B, upper right panel), semiquantitative RT-PCR was performed using primers for indicated cDNAs. Ethidium bromide-stained agarose gels of PCR fragments are shown. In order to test proteasomal degradation of LAP2 (B, lower right panel), cells were starved in low serum medium for 4 days and incubated with or without proteasome inhibitor MG-132 during the last 12 hours. Proteins were precipitated from lysates using LAP2 antibody or hybridoma medium (DMEM) as a control, and immunoprecipitates (P) and supernatants (SN) were analyzed by immunoblotting using antibodies against LAP2 (upper panel) and ubiquitin (lower panel). Input (Inp) represents 1.5% of protein used for IPs. Black and white arrowheads point to ubiquitylated LAP2. Note, that the ubiquitin blot is weak because of the large background detected in the region >110 kDa. Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25; La, lamin; Ubi-LAP2, ubiquitylated LAP2; pRb, retinoblastoma protein.

View larger version (63K): [in this window] [in a new window]   Fig. 5. LINT-25 is upregulated during differentiation. (A) E14.5 fetal liver-derived proliferating erythroid progenitors were switched to erythroid differentiation medium and processed for immunoblotting or immunofluorescence microscopy at the indicated time points, using antibodies as indicated. Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25; La, lamin; pRb, retinoblastoma protein. (B) C2C12 myoblasts or myotubes differentiated in 1% FCS for 10 days were processed for immunofluorescence microscopy using antibodies against the indicated antigens. Confocal images are shown. Bars, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 6. LINT-25 protein levels change during the cell cycle of proliferating cells. (A) BJ1 cells at different phases of the cell cycle were prepared by centrifugal elutriation and cell fractions were analyzed by DNA flow cytometry. Cell lysates were processed for immunoblotting using antibodies against LINT-25 and LAP2. FACS profiles from representative fractions and from an asynchronous culture (log) are shown. (B) WI-38 human lung fibroblasts were synchronized by an overnight aphidicolin block and released from the block for indicated time periods. Samples were processed for immunoblotting after the indicated time points and analyzed using antibodies against LINT-25, LAP2 and actin. The first lane (–) is a sample from untreated control cells; numbers on the right are molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25.

View larger version (71K): [in this window] [in a new window]   Fig. 7. LINT-25 acts upstream of LAP2. (A) Primary skin fibroblasts from LAP2 knock-out mice were passaged in culture until senescence (S) and processed for immunofluorescence microscopy together with proliferating control cells (P) using antibodies against LINT-25, LAP2 or Ki67. (B) GFP-tagged LINT-25 (upper panel) or eGFP alone (lower panel) were transiently expressed in BJ1 cells and samples were processed for immunofluorescence microscopy 15 hours after transfection using antibodies against Ki67, LAP2 and Hoechst dye (blue). Confocal images are shown. Bars, 10 µm.