We analyzed the influence of lamins on nuclear envelope growth in cultured Xenopus A6 cells by the overexpression of human lamin A, Xenopus and zebrafish lamins B2 and Drosophila lamins Dm0 and C as GFP fusion proteins. Lamins containing a CxxM motif in their primary sequence (lamins A, B2, Dm0) induced the formation of lobulated nuclei with multi-membrane-layered, highly folded nuclear membranes and intranuclear membrane assemblies, as observed by electron microscopy. Such morphological alterations were not observed with Drosophila lamin C, a lamin without this motif or with a lamin B2 mutant (B2-SxxM) where the cysteine of the CxxM motif is replaced by a serine. Drosophila lamin C mutants containing a CxxM motif behaved like B-type lamins thus confirming that this tetrapeptide is directly involved in the morphological changes we observed. Nuclear membrane proliferation could also be induced by lamin B2 in COS-7 cells and in zebrafish embryos but not by human lamin A in COS-7 cells. We speculate that the human lamin A is incompletely processed in Xenopus A6 cells and therefore behaves in this cell line like a B-type lamin. Our results indicate that the CxxM motif of B-type lamins has a dual function: it mediates lamin targeting to the inner nuclear membrane thereby promoting nuclear membrane growth.
Cysteine string proteins (CSPs) are conserved secretory vesicle proteins involved in regulating neurotransmitter and peptide release. While the function of the J-domain has been studied in detail, little is known about other conserved regions. We have constructed mutant genes coding for proteins with modified cysteine string, linker region or C terminus and transformed them into Csp nullmutant Drosophila. In the living animal, mutated CSP lacking all cysteines fails to associate with membranes, does not concentrate in synaptic terminals, and cannot rescue adult temperature-sensitive paralysis and short life span, both prominent null mutant phenotypes. A mutant protein with 5 instead of 11 string cysteines appears to be normally targeted but cannot rescue paralysis at 37°C. We propose that the cysteine string, in addition to its role in targeting, may be essential for a function of CSP that is dependent on the number of cysteines in the string. A deletion in the linker region or the C terminus does not affect CSP targeting, and function in adults is only marginally impaired.
Zebrafish lamina-associated polypeptides 2 (ZLAP2) β, γ and ω have in common an N-terminal region with a LEM domain, and in the C-terminal half of the molecule a lamina binding domain and a membrane spanning sequence. The maternally synthesized ω is the largest isoform and the only LAP2 present in the rapidly dividing embryonic cells up to the gastrula stage. ZLAP2ω levels decrease during development, concomitant with the increase of the somatic isoforms ZLAP2β and γ. In somatic zebrafish cells ZLAP2γ is the predominant isoform, whereas only small amounts of ZLAP2β are present.During early embryonic development, ZLAP2ω becomes associated with mitotic chromosomes before anaphase. The surface of these chromosomes is decorated with vesicles, and each chromosome assembles its own nuclear envelope at the end of mitosis (karyomere formation). Ectopically expressed ZLAP2ω-green fluorescent protein (GFP) fusion protein targets vesicles to mitotic chromosomes in Xenopus A6 cells, suggesting that ZLAP2ω is involved in karyomere formation during early zebrafish development.When ZLAP2β and γ were expressed as GFP fusion proteins in Xenopus A6 cells, the β-but not the γ-isoform was found in association with mitotic chromosomes, and ZLAP2β-containing chromosomes were decorated with vesicles. Further analysis of ZLAP2-GFP fusion proteins containing only distinct domains of the ZLAP2 isoforms revealed that the common N-terminal region in conjunction with β-or ω-specific sequences mediate binding to mitotic chromosomes in vivo.Key words: Zebrafish LAP2, Karyomere, Chromatin binding, Lamina, Mitosis
SummaryThe lamina-associated polypeptide 2 (LAP2) isoforms β, γ and ω of zebrafish: developmental expression and behavior during the cell cycle
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