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First published online 6 May 2003
doi: 10.1242/jcs.00450

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The lamina-associated polypeptide 2 (LAP2) isoforms ß, and of zebrafish: developmental expression and behavior during the cell cycle

Vera K. Schoft^*,1, Ariane J. Beauvais^*,2, Carmen Lang¹, Andreas Gajewski^1,3, Kristina Prüfert¹, Christoph Winkler⁴, Marie-Andrée Akimenko⁵, Micheline Paulin-Levasseur² and Georg Krohne^1,

¹ Division of Electron Microscopy, Biocenter of the University of Wü rzburg, Am Hubland, D-97074 Wü rzburg, Germany
² Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5
³ Department of Biochemistry and Molecular Cell Biology, Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria
⁴ Department of Physiological Chemistry I, Biocenter of the University of Wü rzburg, Am Hubland, D-97074 Wü rzburg, Germany
⁵ Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9

View larger version (52K): [in a new window]   Fig. 1. Isoforms of lamina-associated polypeptide 2 in the zebrafish (ZLAP2). (A) ZLAP2, ZLAP2ß and ZLAP2. The position of the LEM motif (LEM), lamin binding domain (lamin binding), transmembrane domain (TM) and the position of individual amino acids is marked for each isoform. The insertion sites of the ß- and - specific sequences are marked by lines. (B) Amino acid sequence comparison (single letter code) of ZLAP2, ß and . Bold printed letters represent amino acids identical in the three proteins. Proteins contain 369 (), 515 (ß) and 657 () amino acids. Gaps have been introduced to allow an optimal alignment of the three sequences. The sequences have been published under Accession Nos AJ320192 (), AJ320191 (ß) and AJ320190 () in the EMBL Nucleotide Sequence Database.

View larger version (46K): [in a new window]   Fig. 2. Characterization of ZLAP2-specific antibodies and proteins encoded by ZLAP2 cDNAs. (A) Total proteins from zebrafish testicle (lane 1), liver (lane 2), blastula (lane 3) and ovary (lane 4) were separated by SDS-PAGE (11% acrylamide) and immunoblotted with the ZLAP2-serum1. A typical radiogram is shown. (B) Coupled in vitro transcription and translation of the cDNAs coding for ZLAP2 (lane 1), ZLAP2ß (lane 2) and ZLAP2 (lane 3). [35S]-Methionine-labeled polypetides were separated by SDS-PAGE (11% acrylamide) and visualized by fluorography. The additional smaller polypeptides (B, lane 3) with mobilities of ZLAP2 and ZLAP2ß probably result from the internal start of translation at triplets coding for methionine at positions 241 and 447/448 of ZLAP2. Molecular masses of reference proteins (in kDa) are marked in (A).

View larger version (38K): [in a new window]   Fig. 7. The behaviour of LAP2 during mitosis in zebrafish AB9 cells. Immunofluorescence microscopy of an AB9 cell at metaphase (A; A', phase contrast) and anaphase (B; B', phase contrast), after staining with ZLAP2-specific antibodies. Note that metaphase chromosomes are not stained by LAP2 antibodies, and that anaphase chromosomes are stained only in regions where the nuclear envelope has begun to reassemble. Bars, 10 µm. (C) LAP2 isoforms of zebrafish AB9 cells. Total proteins of AB9 cells were separated by SDS-PAGE (11% acrylamide) and immunoblotted with ZLAP2-serum1. ZLAP2 is the major isoform in AB9 cells, and only minor amounts of ZLAP2ß were detectable. Identical results were obtained with zebrafish ZF4 cells (data not shown). Molecular masses of reference proteins (in kDa) are marked.

View larger version (25K): [in a new window]   Fig. 3. Biochemical properties of a GFP-ZLAP2 fusion protein, and of full-length ZLAP2. A GFP-ZLAP2 fusion protein (amino acids 503-657 of ZLAP2; GFP503-657) expressed in Xenopus A6 cells (A), and ZLAP2 from zebrafish ovaries (B) was analyzed. The GFP was fused to the aminoterminus of this LAP2 deletion mutant. Proportional amounts of proteins of pellet fractions (P) and supernatants (S) were separated by SDS-PAGE and immunoblotted with GFP antibodies (A) or ZLAP2-specific antibodies (ZLAP2-serum1). The quality of the fractionation was controlled by immunoblotting with antibodies against lamin B2 (A', B'). Lamins should be recovered in the supernatant after urea extraction. (A, A') GFP-ZLAP2 fusion protein; Xenopus A6 cells extracted with 6 M and 8 M urea (A, GFP antibody; B', lamin B2 antibody). (B,B') Membranes of zebrafish ovaries purified by sucrose step gradient centrifugation were extracted with 4 M and 6 M urea. (B, ZLAP2-serum1; B', lamin B2 antibody). The positions of ZLAP2 (o) and ZLAP2 (g) are marked in (B) as well as molecular masses of reference proteins (in kDa).

View larger version (45K): [in a new window]   Fig. 4. Expression of ZLAP2 isoforms during zebrafish development. (A,B) Northern blot analysis of the ZLAP2 expression. Total RNA of D. rerio embryos at the eight-cell stage (lane 1), early blastula (3 hpf, lane 2), late blastula (4.5 hpf, lane 3), late gastrula (7.5 hpf, lane 4), one-somite stage (10 hpf, lane 5), at the age of 24 hours (24 h embryo, lane 6) and 48 hours (48 h embryo, lane 7) were hybridized with in vitro synthesized [32P]-labeled RNA complementary to ZLAP2 mRNAs. (A) A sequence exclusively present in the ZLAP2 mRNA (nucleotides 642-942 of ZLAP2) or (B) a probe complementary to the complete ZLAP2 mRNA were used for hybridization. The hybridized mRNAs have estimated sizes of 3600 (3.6), 2800 (2.8), 2600 (2.6), 1900 (1.9) and 1600 (1.6) nucleotides. (C) Total proteins of nine developmental stages were separated by SDS-PAGE (11% acrylamide) and immunoblotted with ZLAP2-serum1. In each lane, total proteins of 2.5 embryos were loaded. The developmental stage is specified on top of each lane. Expression of ZLAP2ß and was first seen in the late gastrula (lane 4), and ZLAP2 was only detectable in minor amounts in embryos aged 36 hours (36 h embryo; lane 9). The apparent different mobilities of ZLAP2 in some lanes is caused by abundant yolk proteins that have slightly lower mobilities than ZLAP2. (D) Total proteins of seven developmental stages identical to those in lanes 1-7 of (A) were separated by SDS-PAGE (11% acrylamide) and immunoblotted with lamin antibody X155 reacting in zebrafish with lamin B2. In each lane, total proteins of 2.5 embryos were loaded. Lamin B2 is first detectable in the late gastrula (lane 4). Molecular masses of reference proteins (in kDa) are marked (C,D).

View larger version (68K): [in a new window]   Fig. 5. Distribution of LAP2 during the cell cycle in zebrafish blastomeres. Localization of ZLAP2 in whole-mount preparations of embryos by indirect immunofluorescence microscopy with polyclonal antibodies specific for ZLAP2 (A-F; A'-F', DNA staining by Hoechst). Embryos at the cleavage period (A,B; 1.5 hpf), early blastula stage (C,D; 2.5 hpf) and late blastula stage (E, F; 4 hpf) have been analyzed. Cells at metaphase (A-A',E-E'), anaphase (B-B',F-F'), the karyomere stage (C-C') and interphase (D-D') are shown. (D) The nuclear envelope of the interphase nucleus (D) is highly invaginated, resulting in an apparent intranuclear staining with LAP2 antibodies. This morphology is characteristic for nuclei that have been formed by the fusion of karyomeres. Digital images were taken with a CLSM (C-C',D-D',E-E',F) and a Zeiss Axiophot (A-A',B-B',F'). Bars, 10 µm.

View larger version (94K): [in a new window]   Fig. 6. Morphology of chromosomes during cell division of zebrafish blastomeres at the early blastula stage (2.5 hpf). Electron micrographs showing longitudinal sectioned mitotic chromosomes (A, compare with Fig. 4B) and chromosomes at an early karyomere stage (B, earlier than that shown in Fig. 4C). Numerous flattened vesicles are attached to the surface of mitotic chromosomes (A), and nuclear envelope fragments containing pore complexes are attached to the surface of each chromosome at the early karyomere stage (B). Bars, 0.5 µm.

View larger version (69K): [in a new window]   Fig. 8. Intracellular distribution of ZLAP2-GFP fusion proteins in transfected Xenopus A6 cells during interphase and cell division. In all constructs GFP was fused to the C-terminus of full-length LAP2 isoforms. Transfected cells expressing ZLAP2- GFP (A,B), ZLAP2ß-GFP (C) and ZLAP2-GFP (D) are shown during interphase (A) and mitosis (B-D). The GFP fluorescence (A-D) and the same cells after staining with lamin B2 antibodies (A') or the DNA dye Hoechst (B'-D') are shown. ZLAP2-GFP is distributed in mitotic cells throughout the cytoplasm and forms local aggregates (D), whereas ZLAP2- GFP (B) and ZLAP2ß-GFP (C) predominantly colocalized with chromatin. Digital images were taken with a Leica CLSM (A,A') and a Zeiss Axiophot (B-D'). Bars, 10 µm.

View larger version (65K): [in a new window]   Fig. 9. Intracellular distribution of deletion mutants of ZLAP2 expressed as GFP fusion proteins in transfected Xenopus A6 cells during cell division. In all constructs, GFP was fused to the carboxyterminus of LAP2 deletion mutants. Transfected cells expressing mutant 1-214GFP (A; amino acids 1-214 common to all ZLAP2 isoforms), mutant 1-360GFP (B, amino acids 1-360 of ZLAP2ß) and mutant 214-502GFP (C; amino acids 214-502 of ZLAP2) are shown during mitosis. The GFP fluorescence (A-C) and the same cells after staining with the DNA dye Hoechst (A'-C') are shown. Note the association of mutant 1-360GFP with mitotic chromosomes and the exclusion of 214-502GFP from regions containing chromosomes. Digital images were taken with a Leica CLSM (A-C) and a Zeiss Axiophot (A'-C'). Bars, 10 µm.

View larger version (160K): [in a new window]   Fig. 10. Mitotic chromosomes of transfected Xenopus A6 cells expressing full-length ZLAP2- GFP (A,B,D) or ZLAP2-GFP (C,E) analyzed by light (A) and electron microscopy (B-E). (A,B,D) The same cell at metaphase is shown by fluorescence microscopy (A) and on electron micrographs of ultrathin sections through this cell (B,D). The metaphase chromosomes are clearly visible at low magnification (B). (D) Higher magnification of the boxed area in (B) shows that chromosomes are associated with vesicles. (C,E) Electron micrographs of an ultrathin section through a transfected cell at metaphase expressing ZLAP2-GFP. The chromosomes are shown in an overview (C) and higher magnification (E, central region of C). A membrane stack (arrowheads in C and E) containing ZLAP2-GFP, chromosomes (single arrows) and spindle microtubules (double arrows) are marked. Metaphase chromosomes of cells expressing ZLAP2-GFP are not associated with vesicles. Bars, 0.5 µm (D,E), 2 µm (C) and 5 µm (A,B).

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