Lang C et al. (1999), Molecular characterization and developmentally ...

XB-ART-13539

J Cell Sci 1999 Mar 01;112 ( Pt 5):749-59. doi: 10.1242/jcs.112.5.749.

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Molecular characterization and developmentally regulated expression of Xenopus lamina-associated polypeptide 2 (XLAP2).

Lang C , Paulin-Levasseur M , Gajewski A , Alsheimer M , Benavente R , Krohne G .

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Lamina-associated polypeptides 2 (LAP2alpha, beta, gamma)/thymopoietins (TPalpha, beta, gamma) are a family of proteins that are generated by alternative splicing from a single gene. These proteins have been primarily characterized in mammals. One member of this protein family, the integral membrane protein LAP2beta/TPbeta, has been localized to the inner nuclear membrane of somatic cells where it binds to chromatin and B-type lamins. By cDNA cloning we have characterized XLAP2, a Xenopus homologue of the mammalian LAP2beta. Using LAP2-specific antibodies, the Mr 68,000 XLAP2 was found to be the only member of the LAP2/TP family expressed in somatic cells and adult tissues. XLAP2 was not detected in oocytes, eggs and in early embryos up to the gastrula stage at the mRNA and protein level demonstrating that it is not synthesized from maternal mRNA. In counterpart oocytes, eggs, and embryos contained one LAP2-related integral membrane proteins of Mr 84,000. Northern blot analysis with the XLAP2 cDNA showed that a single hybridizing mRNA band of 1.8-2.0 kb was present in Xenopus somatic cells whereas two other hybridizing mRNA species of 2.8-3.0 and 0. 9-1.1 kb were present in oocytes, eggs and early embryos. All together, these results indicated that at least three distinct LAP2-related proteins might be expressed in Xenopus. The LAP2/TP protein of Mr 84,000 is present in the early embryos but its amount decreases during embryogenesis concomitant with the increase of XLAP2 in the embryo. Our results are the first description of the developmentally regulated expression of integral nuclear envelope proteins during early embryogenesis.

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Species referenced: Xenopus
Genes referenced: hsp90aa1.1 lmnb1 lmnb2 lmnb3 prim2 tmpo
???displayArticle.antibodies??? Lmna Ab2 Lmnb2/3 Ab2 Lmnb2/3 Ab3 Tmpo Ab1

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	Fig. 1. (A) Amino acid sequence comparison (single letter code) of the Xenopus laevis LAP2 (X) and LAP2b of Rattus norvegicus (R). The membrane spanning domain is underlined in the rat sequence. Bold printed letters represent amino acids identical in both proteins. Proteins contain 518 (X) and 452 (R) amino acids. Gaps (marked by dots) have been introduced in some areas of the sequences to allow their optimal alignement. The XLAP2 sequence has been published under accession no Y17861 in the EMBL Nucleotide Sequence Database. (B) Schematic drawings of rat LAP2b and Xenopus LAP2. Domains of XLAP2 that show a high degree of identiy with the chromatin/chromosome binding domain, the lamina/lamin B1/B2 binding domain, and the transmembrane domain (TM) of the rat LAP2b have been marked with the same patterns. The degree of identity between both molecules in these regions is given as a percentage (%) below XLAP2. Two XLAP2 domains not contained in the rat protein are marked in black. The positions of individual amino acids has been marked on top of each molecule.
	Fig. 2. Antibodies of the human serum designated MAN are specific for the mammalian LAP2/TP proteins and XLAP2. (A) SDS-PAGE (12% acrylamide) of total bacterial proteins of a culture expressing the full-length rat LAP2b (lane 1) or the amino-terminal 187 amino acids of rat LAP2b (lane 2). Both rat LAP2b proteins specifically reacted on the immunblot shown with antibodies of the MAN-serum. (B) Coupled in vitro transcription and translation of the cDNAs coding for XLAP2 (lane 1) or Xenopus p58 (lane 2), and immunoprecipitation of the [35S]methionine labeled polypetides (XLAP2, lane 3; p58, lane 4) with antibodies of the MAN-serum bound to Protein A-Sepharose. XLAP2 in vitro translation products remaining after immunoprecipitation in the supernatant are shown in lane 5 (compare lanes 1 and 5). Note that the in vitro translation product of Mr 42,000 (marked by arrows in lanes 1 and 5) is not recognized by the MAN-serum. Proteins were separated by SDSPAGE (11% acrylamide) and visualized by fluorography. Molecular masses of reference proteins (in kDa) are marked in A and B.
	Fig. 3. (A) Total cellular proteins of HeLa cells (HeLa, lane 1), Xenopus cultured A6 cells (A6, lane 2), and Xenopus tissues (lane 3, follicle cells [Fol]; lane 4, spleen; lane 5, liver) after separation by SDS-PAGE (12% acrylamide) and immunoblotting with affinity purified antibodies of the MAN-serum specific for LAP2/TP proteins. Antibodies had been affinity purified on the 187 aminoterminal amino acids of the rat LAP2b. (B) Comparison of the electrophoretic mobility of in vitro translated XLAP2 (lane 1) with the major polypeptide detected by immunoblotting with the whole MAN-serum in Xenopus follicle cells (lane 2). The in vitro translation products were synthesized by coupled in vitro transcription and translation of the cDNA coding for XLAP2. Polypeptides of the two samples shown in B were separated in neighbouring lanes in the same SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose strip containing lane 1 was directly processed for fluorography, whereas the second strip (lane 2) was incubated with the whole MAN-serum. Molecular masses of reference proteins (in kDa) are marked in A and B. (C,C¢) Indirect immunofluorescence microscopy (C) on Xenopus A6 cells with affinity purified LAP2/TP antibodies of the MAN-serum. Antibodies had been affinity purified on the full-length rat LAP2. (C¢) Staining with the DNA dye Hoechst. Bar, 10 mm.
	Fig. 4. Extraction properties of Xenopus antigens recognized by the antibodies of the MAN-serum in somatic cells (A,A¢) and egg membranes (B,C). Proteins were separated by SDS-PAGE and immunoblotted with the whole MAN-serum (A,B,C). Molecular masses of reference proteins (in kDa) are marked (A-C).(A) Xenopus A6 cells were sequentially extracted with 1% Triton, DNase, and 250 mM NaCl (lanes 3, 4) or with 8 M urea (lanes 5, 6), and fractionated by centrifugation into supernatants (S) and pellets (P). For comparison total proteins of non-extracted A6 cells (A6, lane 2) and HeLa cells (HeLa, lane 1) were analyzed. (A¢) The same nitrocellulose shown in A was incubated with lamin antibody X155 after removal of bound MAN-antibodies. Antibody X155 does not react with mammalian lamins. (B) Aliquots of the membrane fraction (P200, lane 3; non-extracted membranes) of unfertilized Xenopus eggs were sequentially extracted with 1% Triton and 250 mM NaCl (lanes 4, 5) or with 8 M urea (lanes 6, 7), and fractionated by centrifugation into supernatants (S) and pellets (P). For comparison total proteins of A6 cells (A6, lane 2) and HeLa cells (HeLa, lane 1) were analyzed. (C) Aliquots of the membrane fraction (P200, lane 1; non-extracted membranes) of unfertilized Xenopus eggs (P200) were incubated with 1 M KCl (lanes 2, 3), 2 (lanes 4, 5), 4 (lanes 6, 7) or 6 M urea (lanes 8, 9) and fractionated by centrifugation into supernatants (S) and pellets (P). Note that XLAP2 (Mr 68,000) is not detectable in egg membranes (A-C).
	Fig. 5. Xenopus oocyte polypeptides detected by MAN-antibodies. Proteins were separated by SDS-PAGE (12% acrylamide) and immunoblotted with the whole MAN-serum. Molecular masses of reference proteins (in kDa) are marked. (A) Total proteins of oocyte follicle cells (lane 1), of the oocyte cytoplasmic pellet (lane 2; protein of 5 defolliculated and enucleated oocytes), and manually isolated oocyte nuclei (lane 3; 40 nuclei) after separation by SDSPAGE. (B) Manually subfractionation of oocyte nuclei into nuclear envelopes (lane 1; 25 nuclear envelopes) and nuclear contents (lane 2; 25 nuclear contents). Note that XLAP2 (Mr 68,000) is absent from cytoplasmic and nuclear membranes of oocytes.
	Fig. 6. Xenopus oocyte and egg membrane polypeptides reacting with affinity purified antibodies of the MAN-serum. Molecular masses of reference proteins (in kDa) are marked. (A) Total cellular proteins of oocyte follicle cells (Fol, lane 1), and defolliculated oocytes (Oocyte, lane 2) after separation by SDS-PAGE (12% acrylamide) and immunoblotting with affinity purified antibodies of the MAN-serum specific for the 187 amino-terminal amino acids of rat LAP2. Total proteins of 1/2 oocyte had been loaded (lane 2). (B) Total cellular proteins of HeLa cells (HeLa, lane 1), Xenopus A6 cells (A6, lane 2), and total membrane proteins of Xenopus egg membranes (P200, lane 3) after separation by SDS-PAGE (12% acrylamide) and immunoblotting with antibodies of the MAN-serum affinity purified on XLAP2 of A6 cells.
	Fig. 7. Northern blot analysis of the XLAP2 expression. Poly(A+) RNA of Xenopus unfertilized eggs (Egg, lane 1), embryos at the gastrula stage (Gastrula, lane 2; initial gastrula of stage 10), the neurula stage (Neurula, lane 3; neurula stage 18), and of A6 cells (lane 4) was hybridized with in vitro synthesized 32P-labelled RNA complementary to the XLAP2 mRNA. The positions of the 28S (28) and 18S rRNA (18), and the three hybridizing mRNA bands (dots) are marked.
	Fig. 8. Expression of XLAP2 during Xenopus development. (A) Total proteins of 8 developmental stages were separated by SDS-PAGE (11% acrylamide) and immunoblotted with the whole MAN-serum. In each case the total protein of 1/2 embryo was analyzed. (lane 1, Fertilized egg), eggs 45 minutes after fertilization; (lane 2, Blastula), medium cell blastula of stage 8; (lane 3, Gastrula), initial gastrula of stage 10; (lane 4, Neurula St. 18), neurula groove stage; (lane 5, St. 23), elongated embryo of 2.2-2.4 mm length with eye vesicles; (lane 6, St. 33/34), elongated embryo of 4.7-5.3 mm length; (lane 7, St. 40), tadpole with mouth broken through; (lane 8, St. 44), feeding tadpole. XLAP2 expression was first seen in the early gastrula (lane 3), and the Mr 84,000 polypeptide was only detectable in very minor amounts in embryos older than stage 33/34 when the X-ray film was exposed 10 times longer to the nitrocellulose as shown in A. (A¢) The same nitrocellulose shown in A was incubated with antibodies against Xenopus lamin A (antibody X94) after removal of bound MANantibodies. Lamin A is first detectable in swimming tadpoles of stage 40 (lane 7). Molecular masses of reference proteins (in kDa) are marked (A,A¢). (B,B¢) Indirect immunofluorescence microscopy (B) of a squashed embryo at the 128 cell stage after incubation with the whole MAN-serum (B¢, staining with the DNA dye Hoechst). Note that XLAP2 is yet not expressed (see A) whereas the Mr 84,000/35,000 polypeptides are present in this developmental stage. Bar, 20 mm.