What is a lamin

Lamina Associated Polypeptides 2, Lamin B Receptor, and Lamine: Studies on Vertebrate Models

Lamina associated polypeptides 2, lamin B receptor and lamins: investigations on vertebrates

Please always quote using this URN: urn: nbn: de: bvb: 20-opus-11619

Kristina Prüfert

  • The nucleus is an important organelle of eukaryotic cells, which is separated from the cytoplasm by a double membrane. With the help of the zebrafish as a model organism and cultivated cells from different vertebrates, investigations on integral (lamina-associated polypeptide 2, lamin B receptor) and peripheral membrane proteins (lamines) of the inner nuclear membrane were carried out in several sub-projects. One of the most well-studied integral membrane proteins of the inner nuclear membrane is the lamina-associated polypeptide 2 The nucleus is an important organelle of eukaryotic cells that is separated from the cytoplasm by a double membrane. With the help of the zebrafish as a model organism and cultivated cells from different vertebrates, investigations on integral (lamina-associated polypeptide 2, lamin B receptor) and peripheral membrane proteins (lamines) of the inner nuclear membrane were carried out in several sub-projects. One of the best studied integral membrane proteins of the inner nuclear membrane is the lamina-associated polypeptide 2 (LAP2). Alternative splicing creates a number of different isoforms from a single gene, which are expressed in a developmentally and tissue-specific manner. In mammals, 6 different isoforms (LAP2 & # 945 ;, & # 946 ;, & # 948 ;, & # 949 ;, & # 947 ;, & # 950;) are known, all of which with the exception of LAP2 & # 945; and & # 950; are integrated into the inner nuclear membrane. The LAP2 & # 945; is located exclusively in the nucleoplasm and has so far only been described in mammals. Through the identification of a genomic clone ICRFc 7101293Q5 (RZPD) and comparative database analyzes, the structural structure of the zebrafish LAP2 gene could be determined. The gene encodes 15 exons within 19 kb (without regulatory sequences) from which 3 different isoforms result from alternative splicing (zLAP2,,). With the help of “Radiation Hybrid Mapping”, the LAP2 gene was localized on the “Linkage group” 4 of the zebrafish, between the EST markers fc01g04 (213.97) and fb49f01 (215.69cR). Due to the additional identification of the genomic sequence of the chicken LAP2 gene, it was possible to compare the genomic sequences of the LAP2 genes from lower vertebrates (zebrafish), through birds (chickens), to mammals (humans). On the one hand, it was shown that the & # 969; The zebrafish isoform is not present in the chicken and mammalian genome: On the other hand, the & # 945; Isoform of the mammals is also not found in any of the other species (chicken, zebrafish). Furthermore, in additional protein analyzes with monoclonal and polyclonal antibodies against the conserved amino terminal domain of the LAP2 proteins, only one protein with a molecular weight comparable to LAP2 & # 946; other species can be clearly detected. Based on these findings and the fact that the coding exon sequences show a greater similarity between humans and chickens than between humans and zebrafish, it is reasonable to assume that the isoform is a novelty in mammals. Another integral membrane protein that was first investigated in zebrafish is the Lamin B receptor (zLBR). With the help of radioactively labeled probes, two clones could be identified which contain both the entire cDNA sequence (MPMGp567K10194Q3 (RZPD) and the entire genomic sequence (ICRFc71M10137Q5 (RZPD)). Using comparative database analyzes, the structural structure of the zebrafish LBR- It codes within 12 kb (without regulatory sequences) with 13 exons for a 616 amino acid protein. Comparable to the LBR of other species, the zebrafish LBR also has 8 transmembrane domains in its carboxy-terminal domain with which it is anchored in the inner nuclear membrane The amino acid comparison with other species showed that the amino acid sequence is highly conserved both in the amino terminal region and in the region of the transmembrane domains. Furthermore, polyclonal antibodies against the first 210 amino acids of the zLBR were produced, which can be used for future immunological analyzes Like the integral membrane proteins, the lamines also play an important role in nuclear processes. The B-type laminates are all characterized by a preserved CxxM motif at the carboxy terminus. The CxxM motif is post-translationally modified, whereby it acquires lipophilic properties and is therefore responsible for the initial anchoring of the lamines in the inner nuclear membrane. In the case of A-type lamins, this motif is not contained in the primary sequence or it is proteolytically split off after being incorporated into the nuclear lamina. A specialty is the meiotic lamin C2, which is a splice product of the lamin A gene in mammals. Lamin C2 does not have a CxxM motif at its amino terminus, but a hexapeptide (GNAEGR) after the start methionine. The post-translational modification of this sequence gives the protein lipophilic properties. On the basis of functional analyzes, overexpression of GFP fusion proteins from various wild-type lamines and lamin mutants showed that the interaction of the lamines via the CxxM motif or the GNAEGR motif with the inner nuclear membrane induces the growth of the nuclear envelope. In a last project, specific “antisense” oligonucleotides called “morpholinos” were able to repress the expression of LAP2, LBR and the B-type lamines B1 and B2 in zebrafish embryos. The mRNA translation of the corresponding genes was blocked completely in all cases within the first 24 hours. Although the number of embryos observed was relatively small, all embryos showed a clear development delay and different degrees of developmental disorders. The morphological effects were less with the reduction of the LAP2 or the Lamine than with the reduction of the LBR, since in both cases maternal proteins such as the LA2 & # 969; or the B-type lamin LIII, could not be reduced. ...
  • The nucleus is an essential organelle of eucaryotic cells and separated by a double membrane, the nuclear envelope, from the cytoplasm. Using the zebrafish as one model organism and also cultured cells of various vertebrates, analysis of integral (lamina associated polypeptide 2, lamin B receptor) and peripheral membrane proteins (lamins) of the inner nuclear membrane were performed. One of the best investigated protein of the inner nuclear membrane is the lamina associated polypeptide 2. By alternative splicing the LAP2 gene encodes a number The nucleus is an essential organelle of eucaryotic cells and separated by a double membrane, the nuclear envelope, from the cytoplasm. Using the zebrafish as one model organism and also cultured cells of various vertebrates, analysis of integral (lamina associated polypeptide 2, lamin B receptor) and peripheral membrane proteins (lamins) of the inner nuclear membrane were performed. One of the best investigated protein of the inner nuclear membrane is the lamina associated polypeptide 2. By alternative splicing the LAP2 gene encodes a number of isoforms that are expressed in a developmental and tissue specific manner. In mammals 6 different isoforms (LAP2 & # 945 ;, & # 946 ;, & # 948 ;, & # 949 ;, & # 947 ;, & # 950;) are known, and all of them except for LAP2 & # 945; and LAP2 & # 950; get integrated into the inner nuclear membrane. LAP2? is predominantly found in the nucleoplasm and has so far only been described in mammals. Due to the identification of the genomic clone ICRFc 7101293Q5 (RZPD) and an additional contig-sequence published by a database of the Sanger Institute, I was able to analyze the genomic structure of the zebrafish lamina associated polypeptide 2 (zLAP2) gene. It spans approximately 19 kb (with no regulatory sequences) and contains 15 exons. By alternative splicing the 15 exons encode 3 isoforms (zLAP2 & # 946 ;, & # 947 ;, & # 969;). Using the method of “radiation hybrid mapping” the zLAP2 gene could be localized onto the linkage group 4 between the EST markers fc01g04 (213.97) and fb49f01 (215.69cR). The additional identification of the genomic LAP2 sequence of chicken, made it possible to compare the genomic sequences of lower vertebrate LAP2 (zebrafish), avian LAP2 (chicken) and mammal LAP2 (human). This comparison revealed that the zebrafish LAP2 & # 969; was not found in the chicken and the mammalian genome. On the other hand sequences encoding a LAP2 & # 945; isoform were not detected in the chicken and zebrafish genome. The analysis of total proteins of 10 day old chicken embryos and fibroblasts using monoclonal and polyclonal antibodies raised against the evolutionary conserved aminoterminal domain confirmed the abundance of a LAP2 & # 945; isoform. The predominant isoform detected was comparable to LAP2 & # 946; of other species. These results suggest, in addition to the fact that the exons of human LAP2 show a higher identity to the exons of the chicken LAP2 than to the exons of the zebrafish LAP2, that the & # 945; Isoform is a novelty of mammals. A second integral membrane protein, which I focused on, is the lamin B receptor of zebrafish (zLBR). By using radioactive labeled probes of a previously identified LBR fragment, I was able to identify to clones that were coding for the cDNA- (MPMGp567K10194Q3 (RZPD) and the genomic sequence (ICRFc71M10137Q5 (RZPD). By further computer analysis a genomic sequence was determined and revealed that the LBR gene spans over 12 kb nucleotides (no regulatory sequences included). The gene contains 13 exons and encodes a protein of 616 amino acids. Comparable to other species the zLBR contains 8 transmembrane domains in its carboxyterminal domain. Comparison of LBRs of different species on the amino acid level demonstrated that the aminoterminal region and the carboxyterminal domain are highly conserved. Especially the carboxyterminal domain coding for the membrane spanning domains exhibited a high conservation. Furthermore, polyclonal antibodies, specific for the amino acids 1-210 were raised and can be used for immulological analysis in the future. Comparable to integral membrane proteins of The inner nuclear membrane lamins are also essential for nuclear processes. A main feature of B type laminates is the carboxy terminal CxxM-motif. This motif undergoes a posttranslational isoprenylation and is responsible for the correct targeting and initial association of the lamins to the inner nuclear membrane. A type lamins on the other and do not possess a CxxM motif in the primary sequence (mammalian lamin C, C2). In the lamin A isoform the CxxM-motif is encoded in the primary structure but gets proteolytically removed after the protein was targeted to the lamina. The lamin C2, a meiotic splice variant of lamin A, demonstrates a peculiarity. It does not encode a CxxM-motif but possesses the hexapeptide GNAEGR at its aminoterminal end. The GNEAGR sequence also undergoes a posttranslational modification and becomes myristoylated. Functional studies using overexpressed GFP-fusion proteins of different wildtype lamins and lamin mutants demonstrated that both the CxxM motif and the GNAEGR motif promote nuclear membrane proliferation. With the help of specific antisense oligonucleotides, known as "Morpholinos", I was able to block the expression of LAP2, LBR and B-type lamins B1 and B2 in zebrafish embryos. The mRNA translation of each gene was block completely for at least 24 hours after fertilization. Although only few embryos were investigated I realized an obvious delay in development and developmental defects in different degrees in all analyzed embryos. The degree of morphological deformations was more obvious in knock downs of LBR, than after the reduction of LAP2 or the B type lamins, where maternal proteins, like LAP2 & # 969; and the B Type lamin LIII could not get reduced. ...