Evolution of the lamin protein family at the base of the vertebrate lineage.

Lamin proteins are major constituents of the nuclear lamina. They are required for fundamental nuclear activities, as evidenced by the large number of laminopathies. Mutations in the human lamin A/C gene exhibit a broad spectrum of clinical manifestations. Most non-vertebrates including the nearest relatives of the vertebrates have only a single lamin gene. In jawed vertebrates (Gnathostomata), four lamin subtypes (B1, B2, LIII, and A) are found. Lampreys and hagfish form the two orders of jawless vertebrates, Agnatha, which represent the sister group of the Gnathostomata at the base of the vertebrate lineage. Lamin sequence information of lampreys and hagfish sheds light on the evolution of the lamin protein family at the base of the vertebrate lineage. In the genomes of the lamprey (Petromyzon marinus) and the hagfish (Eptatretus burgeri), only three lamin genes are present, a lamin A gene is lacking. The presence of an LIII gene in both, lampreys and hagfish, proves that the distinguishing features of this gene had been established before the agnathan/gnathostome split. The other two agnathan lamins, LmnI and LmnII, deviate strongly in their sequences from those of the gnathostome lamins. For none of these two agnathan lamins can orthology be established to one of the gnathostome lamin types. In the direct chromosomal neighbourhood of all three hagfish lamin genes, a MARCH3 paralog is found. This can be interpreted as further evidence that the vertebrate lamin genes have arisen in the course of the two rounds of whole genome duplication that took place at the base of the vertebrate lineage.

Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm.

It is still controversial whether cranial placodes and neural crest cells arise from a common precursor at the neural plate border or whether placodes arise from non-neural ectoderm and neural crest from neural ectoderm. Using tissue grafting in embryos of Xenopus laevis, we show here that the competence for induction of neural plate, neural plate border and neural crest markers is confined to neural ectoderm, whereas competence for induction of panplacodal markers is confined to non-neural ectoderm. This differential distribution of competence is established during gastrulation paralleling the dorsal restriction of neural competence. We further show that Dlx3 and GATA2 are required cell-autonomously for panplacodal and epidermal marker expression in the non-neural ectoderm, while ectopic expression of Dlx3 or GATA2 in the neural plate suppresses neural plate, border and crest markers. Overexpression of Dlx3 (but not GATA2) in the neural plate is sufficient to induce different non-neural markers in a signaling-dependent manner, with epidermal markers being induced in the presence, and panplacodal markers in the absence, of BMP signaling. Taken together, these findings demonstrate a non-neural versus neural origin of placodes and neural crest, respectively, strongly implicate Dlx3 in the regulation of non-neural competence, and show that GATA2 contributes to non-neural competence but is not sufficient to promote it ectopically.

CaaX-less lamins: Lophotrochozoa provide a glance at the playground of evolution.

Nuclear lamins are the main components of the nuclear lamina in many eukaryotes. They are members of the intermediate filament (IF) protein family. Lamins differ from cytoplasmic IF proteins by the presence of a nuclear localisation sequence (NLS) and a C-terminal tetrapeptide, the CaaX motif. The CaaX motif is target of post-translational modifications including isoprenylation, proteolytic processing, and carboxyl-methylation. These modifications, in conjunction with the NLS, direct lamins to the inner nuclear membrane where they assemble into filaments. Lamins lacking a CaaX motif are unable to associate independently with nuclear membranes and remain in the nucleoplasm. So far, three species have been reported to exclusively express CaaX-less lamins. All three belong to the lophotrochozoan lineage. To find out whether they represent rare exceptions, we analysed lamins of representatives of 17 lophotrochozoan phyla. Here we report that all four clades of Rotifera as well as individual taxa of Mollusca and Annelida lack CaaX-lamins, but express lamins with alternative C-termini. Of note, the respective mollusc and annelid groups occupy very different phylogenetic ranks. Most of these alternative C-termini are rich in aromatic residues. A possible function of these residues in membrane association is discussed. Alternative splicing of terebellid lamin transcripts gives rise to two lamin variants, one with a CaaX motif and one with an alternative C-terminus. A similar situation is found in Arenicolidae, Opheliidae, Capitellidae, and Echiura. This points a way, how the switch from lamins carrying a CaaX motif to lamins with alternative C-termini may have occurred.

Targeted Feature Extraction in MALDI Mass Spectrometry Imaging to Discriminate Proteomic Profiles of Breast and Ovarian Cancer.

PURPOSE: To develop a mass spectrometry imaging (MSI) based workflow for extracting m/z values related to putative protein biomarkers and using these for reliable tumor classification. EXPERIMENTAL DESIGN: Given a list of putative breast and ovarian cancer biomarker proteins, a set of related m/z values are extracted from heterogeneous MSI datasets derived from formalin-fixed paraffin-embedded tissue material. Based on these features, a linear discriminant analysis classification model is trained to discriminate the two tumor types. RESULTS: It is shown that the discriminative power of classification models based on the extracted features is increased compared to the automatic training approach, especially when classifiers are applied to spectral data acquired under different conditions (instrument, preparation, laboratory). CONCLUSIONS AND CLINICAL RELEVANCE: Robust classification models not confounded by technical variation between MSI measurements are obtained. This supports the assumption that the classification of the respective tumor types is based on biological rather than technical differences, and that the selected features are related to the proteomic profiles of the tumor types under consideration.

Ectopic expression of prelamin A in early Xenopus embryos induces apoptosis.

Lamin proteins are components of metazoan cell nuclei. During evolution, two classes of lamin proteins evolved, A- and B-type lamins. B-type lamins are expressed in nearly all cell types and in all developmental stages and are thought to be indispensable for cellular survival. In contrast, A-type lamins have a more restricted expression pattern. They are expressed in differentiated cells and appear late in embryogenesis. In the earliest steps of mammalian development, A-type lamins are present in oocytes, pronuclei and during the first cleavage stages of the developing embryo. But latest after the 16-cell stage, A-type lamin proteins are not any longer detectable in embryonic cells. Amphibian oocytes and early embryos do not express lamin A. Moreover, extracts of Xenopus oocytes and eggs have the ability to selectively remove A-type lamins from somatic nuclei. This observation and the restricted expression pattern suggest that the presence of lamin A might interfere with developmental processes in the early phase of embryogenesis. To test this, we ectopically expressed lamin A during early embryonic development of Xenopus laevis by microinjection of synthetic mRNA. Here, we show that introducing mature lamin A does not interfere with normal development. However, expression of prelamin A or lamin A variants that cannot be fully processed cause severe disturbances and lead to apoptosis during gastrulation. The toxic effect is due to lack of the conversion of prenylated prelamin A to its mature form. Remarkably, even a cytoplasmic prelamin A variant that is excluded from the nucleus drives embryos into apoptosis.

Evolutionary changes in lamin expression in the vertebrate lineage.

The nuclear lamina is involved in fundamental nuclear functions and provides mechanical stability to the nucleus. Lamin filaments form a meshwork closely apposed to the inner nuclear membrane and a small fraction of lamins exist in the nuclear interior. Mutations in lamin genes cause severe hereditary diseases, the laminopathies. During vertebrate evolution the lamin protein family has expanded. While most vertebrate genomes contain 4 lamin genes, encoding the lamins A, B1, B2, and LIII, the majority of non-vertebrate genomes harbor only a single lamin gene. We have collected lamin gene and cDNA sequence information for representatives of the major vertebrate lineages. With the help of RNA-seq data we have determined relative lamin expression levels for representative tissues for species of 9 different gnathostome lineages. Here we report that the level of lamin A expression is low in cartilaginous fishes and ancient fishes and increases toward the mammals. Lamin B1 expression shows an inverse tendency to that of lamin A. Possible implications for the change in the lamin A to B ratio is discussed in the light of its role in nuclear mechanics.

Evolutionary aspects in intermediate filament proteins.

Intermediate filament (IF) proteins, together with tubulins and actins, constitute the majority of cytoskeletal proteins in metazoans. Proteins of the IF family fulfil increasingly diverse functions but share common structural features. Phylogenetic analysis within the metazoan lineage traces back their origin to a common lamin-like ancestor. Major steps in lamin evolution occurred at the base of the vertebrate radiation, while cytoplasmic IF protein subfamilies evolved independently in the major metazoan lineages.

A peculiar lamin in a peculiar mammal: Expression of lamin LIII in platypus (Ornithorhynchus anatinus).

Platypus (Ornithorhynchus anatinus) holds a unique phylogenetic position at the base of the mammalian lineage due to an amalgamation of mammalian and sauropsid-like features. Here we describe the set of four lamin genes for platypus. Lamins are major components of the nuclear lamina, which constitutes a main component of the nucleoskeleton and is involved in a wide range of nuclear functions. Vertebrate evolution was accompanied by an increase in the number of lamin genes from a single gene in their closest relatives, the tunicates and cephalochordates, to four genes in the vertebrate lineage. Of the four genes the LIII gene is characterized by the presence of two alternatively spliced CaaX-encoding exons. In amphibians and fish LIII is the major lamin protein in oocytes and early embryos. The LIII gene is conserved throughout the vertebrate lineage, with the notable exception of marsupials and placental mammals, which have lost the LIII gene. Here we show that platypus has retained an LIII gene, albeit with a significantly altered structure and with a radically different expression pattern. The platypus LIII gene contains only a single CaaX-encoding exon and the head domain together with coil 1a and part of coil1b of the platypus LIII protein is replaced by a novel short non-helical N-terminus. It is expressed exclusively in the testis. These features resemble those of male germ cell-specific lamins in placental mammals, in particular those of lamin C2. Our data suggest (i) that the specific functions of LIII, which it fulfills in all other vertebrates, is no longer required in mammals and (ii) once it had been freed from these functions has undergone structural alterations and has adopted a new functionality in monotremes.

Lamins of the sea lamprey (Petromyzon marinus) and the evolution of the vertebrate lamin protein family.

Lamin proteins are found in all metazoans. Most non-vertebrate genomes including those of the closest relatives of vertebrates, the cephalochordates and tunicates, encode only a single lamin. In teleosts and tetrapods the number of lamin genes has quadrupled. They can be divided into four sub-types, lmnb1, lmnb2, LIII, and lmna, each characterized by particular features and functional differentiations. Little is known when during vertebrate evolution these features have emerged. Lampreys belong to the Agnatha, the sister group of the Gnathostomata. They split off first within the vertebrate lineage. Analysis of the sea lamprey (Petromyzon marinus) lamin complement presented here, identified three functional lamin genes, one encoding a lamin LIII, indicating that the characteristic gene structure of this subtype had been established prior to the agnathan/gnathostome split. Two other genes encode lamins for which orthology to gnathostome lamins cannot be designated. Search for lamin gene sequences in all vertebrate taxa for which sufficient sequence data are available reveals the evolutionary time frame in which specific features of the vertebrate lamins were established. Structural features characteristic for A-type lamins are not found in the lamprey genome. In contrast, lmna genes are present in all gnathostome lineages suggesting that this gene evolved with the emergence of the gnathostomes. The analysis of lamin gene neighborhoods reveals noticeable similarities between the different vertebrate lamin genes supporting the hypothesis that they emerged due to two rounds of whole genome duplication and makes clear that an orthologous relationship between a particular vertebrate paralog and lamins outside the vertebrate lineage cannot be established.

Evolution of the lamin protein family: what introns can tell.

Lamins are the major components of the nuclear lamina, a filamentous layer found at the interphase between chromatin and the inner nuclear membrane. The lamina supports the nuclear envelope and provides anchorage sites for chromatin. Lamins and their associated proteins are required for most nuclear activities, mitosis, and for linking the nucleoskeleton to the network of cytoskeletal filaments. Mutations in lamins and their associated proteins give rise to a wide range of diseases, collectively called laminopathies. This review focuses on the evolution of the lamin protein family. Evolution from basal metazoans to man will be described on the basis of protein sequence comparisons and analyses of their gene structure. Lamins are the founding members of the family of intermediate filament proteins. How genes encoding cytoplasmic IF proteins could have arisen from the archetypal lamin gene progenitor, can be inferred from a comparison of the respective gene structures. The lamin/IF protein family seems to be restricted to the metazoans. In general, invertebrate genomes harbor only a single lamin gene encoding a B-type lamin. The archetypal lamin gene structure found in basal metazoans is conserved up to the vertebrate lineage. The completely different structure of lamin genes in Caenorhabditis and Drosophila are exceptions rather than the rule within their systematic groups. However, variation in the length of the coiled-coil forming central domain might be more common than previously anticipated. The increase in the number of lamin genes in vertebrates can be explained by two rounds of genome duplication. The origin of lamin A by exon shuffling might explain the processing of prelamin A to the mature non-isoprenylated form of lamin A. By alternative splicing the number of vertebrate lamin proteins has increased even further. Lamin C, an alternative splice form of the LMNA gene, is restricted to mammals. Amphibians and mammals express germline-specific lamins that differ in their protein structure from that of somatic lamins. Evidence is provided that there exist lamin-like proteins outside the metazoan lineage.

Follicle separation during Drosophila oogenesis requires the activity of the kinesin II-associated polypeptide Kap in germline cells.

Cellular localization of organelles, protein complexes and single mRNAs depends on the directed transport along microtubule tracks, a process mediated by ATP-driven molecular motor proteins of the dynein and kinesin superfamilies. Kinesin II is a heterotrimeric protein complex composed of two motor subunits and a unique nonmotor Kinesin-associated protein (Kap). Kap was shown to transport both particulate cargo, as axoneme components in rafts, and membrane-bounded organelles such as melanosomes. Drosophila Kinesin II was shown to be essential for the axonal transport of choline acetyltransferase in a specific set of neurons. We have generated Kap mutants and show that gene activity is not only required for neuronal function but also for separation of follicles during early oogenesis. The data suggest that Kap participates in the transport of signalling components required for instructive interactions between germline and soma cells.

Mapping and identification of essential gene functions on the X chromosome of Drosophila.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.