Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Intermediate filaments,microtubules and microfilaments in epidermis of sea urchin tube foot

Summary Tube foot epidermal cells of the sea urchin Strongylocentrotus purpuratus were examined by transmission electron microscopy and fluorescence microscopy to identify the chemical nature of prominent bundles of cytoplasmic filaments. Cross sections revealed filaments of roughly 7–8 nm in diameter closely packed into dense bundles. These bundles, in turn, were each surrounded by a loose sheath of microtubules. The filament size and negative reaction with the fluorescent F-actin binding drug NBD-phallacidin indicated that they were not actin. Indirect immunofluorescence microscopy of whole tissues and frozen sections revealed a strong reaction of the filaments with a monoclonal antibody prepared against porcine stomach desmin. In SDS-polyacrylamide gels of whole tube foot protein, a band of apparent molecular weight around 50 000 daltons reacted with the anti-desmin monoclonal antibody. The combined data provide evidence that the epidermal filament bundles are related to vertebrate intermediate filaments, but further biochemical studies will be necessary to assign them to a particular class of filament proteins.     

Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Intermediate filament structure: 2. Molecular interactions in the filament

Molecules of intermediate filament (IF) proteins contain a central rod domain in which the two constituent chains have a predominantly α-helical conformation and are coiled around one another to form segments of two-strand rope. Possible interactions between the two long segments, termed 1B and 2 were investigated by a technique successfully employed in studies of the modes of association of collagen molecules by Miller and coworkers. Prominent maxima were found in all of the six possible modes of association between the rod domain segments in individual IF proteins and certain maxima were found to be common to all IF. The surface lattice of the IF from α-keratin has been determined and possible bonding arrangements between the rod-domain segments are catalogued. A systematic search was carried out for combinations of interaction maxima which were consistent with the dimensions of the surface lattice. By the further application of stereochemical constraints, models for the topological arrangement of the rod-domain segments on the surface lattice were derived and these are illustrated and discussed.     

Strand-specific nucleotide composition bias in echinoderm and vertebrate mitochondrial genomes

Summary The gene organization of starfish mitochondrial DNA is identical with that of the sea urchin counterpart except for a reported inversion of an approximately 4.6-kb segment containing two structural genes for NADH dehydrogenase subunits 1 and 2 (ND 1 and ND 2). When the codon usage of each structural gene in starfish, sea urchin, and vertebrate mitochondrial DNAs is examined, it is striking that codons ending in T and G are preferentially used more for heavy strand-encoded genes, including starfish ND 1 and ND 2, than for light strand-encoded genes, including sea urchin ND 1 and ND 2. On the contrary, codons ending in A and Care preferentially used for the light strand-encoded genes rather than for the heavy strand-encoded ones. Moreover, G-U base pairs are more frequently found in the possible secondary structures of heavy strandencoded tRNAs than in those of light strand-encoded tRNAs. These observations suggest the existence of a certain constraint operating on mitochondrial genomes from various animal phyla, which results in the accumulation of G and T on one strand, and A and C on the other.     

Intermediate filament typing of the human embryonic and fetal notochord

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: Implications for evolution of chordate IF proteins

We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I–IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences.     

Intermediate filament networks: in vitro and in vivo assembly models

We propose two systems of ordinary differential equations modeling the assembly of intermediate filament networks. The first one describes the in vitro intermediate filament assembly dynamics. The second one deals with the in vivo evolution of cytokeratin, which is the intermediate filament protein expressed by epithelial cells. The in vitro model is then briefly analyzed in a simplified case.     

"Laminopathies": a wide spectrum of human diseases

Mutations in genes encoding the intermediate filament nuclear lamins and associated proteins cause a wide spectrum of diseases sometimes called "laminopathies." Diseases caused by mutations in LMNA encoding A-type lamins include autosomal dominant Emery-Dreifuss muscular dystrophy and related myopathies, Dunnigan-type familial partial lipodystrophy, Charcot-Marie-Tooth disease type 2B1 and developmental and accelerated aging disorders. Duplication in LMNB1 encoding lamin B1 causes autosomal dominant leukodystrophy and mutations in LMNB2 encoding lamin B2 are associated with acquired partial lipodystrophy. Disorders caused by mutations in genes encoding lamin-associated integral inner nuclear membrane proteins include X-linked Emery-Dreifuss muscular dystrophy, sclerosing bone dysplasias, HEM/Greenberg skeletal dysplasia and Pelger-Huet anomaly. While mutations and clinical phenotypes of "laminopathies" have been carefully described, data explaining pathogenic mechanisms are only emerging. Future investigations will likely identify new "laminopathies" and a combination of basic and clinical research will lead to a better understanding of pathophysiology and the development of therapies.     

The sequence of a cytoplasmic intermediate filament (IF) protein from theannelid Lumbricus terrestris emphasizes a distinctive feature of protostomic IF proteins

The complete cDNA clone for a cytoplasmic intermidiatefilament (IF) protein from the annelid Lumbricus terrestris reported here, shows an extra 42 residues in the coil 1b subdomain of the central rod, as do the IF proteins from nematodes and moluscs. These extra six heptads are also present in all nuclear lamins but not in any known vertebrate cytoplasmic IF protein. Thus, it seems that protostomic metazoa conserve a lamin-like structural element in their cytoplasmic IF proteins, which was lost in the deuterostomic metazoan branch leading to the vertebrates.     

The mouse synemin gene encodes three intermediate filament proteins generated by alternative exon usage and different open reading frames

We have previously cloned and characterized the human synemin gene, which encodes two intermediate filament proteins (IFPs). We now show that the mouse synemin gene encodes three different synemin isoforms through an alternative splicing mechanism. Two of them, synemin H and M are similar to human alpha and beta synemin, and the third isoform, L synemin, constitutes a new form of IFP. It has a typical rod domain and a short tail (49 residues) with a novel sequence that is produced by a different open reading frame. The synthesis of H/M synemins starts in the embryo, whereas the synemin L isoform is present in adult muscles. The H/M isoforms are bound to desmin or vimentin in the muscle cells of wild-type mice. Using desmin- and vimentin-deficient mice, we have obtained direct evidence that synemin is associated with muscle intermediate filaments in vivo. The organization of the synemin fibril is disrupted in skeletal and cardiac muscle when desmin is absent and in smooth muscle when vimentin is absent. The fact that the three synemin isoforms differ in the sequences of their tail domains as well as in their developmental patterns suggests that they fulfill different functions.     

Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates

Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.     

Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus

The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes within physiological timescales, the only major exception being the reversible destiffening of the mammalian uterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (sea urchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions in timescales of around a second to minutes. Elucidation of the molecular mechanism underlying such mutability has implications for the zoological, ecological and evolutionary field. Important information could also arise for veterinary and biomedical sciences, particularly regarding the pathological plasticization or stiffening of connective tissue structures. In the present investigation we analyzed aspects of the ultrastructure and biochemistry in two representative models, the compass depressor ligament and the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three different mechanical states. The results provide further evidence that the mechanical adaptability of echinoderm connective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher glycosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compass depressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues. The possible involvement of GAG in the mutability phenomenon will need further clarification. During the shift from a compliant to a standard condition, significant changes in GAG content were detected only in the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling) and biochemistry (two alpha chains) were found between the two models and mammalian collagen. Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLAST alignment highlighted the uniqueness of sea urchin collagen with respect to mammalian collagen.     

Simultaneous labelling of microtubules and fibrillar bundles in tobacco BY-2 cells by the anti-intermediate filament antibody,ME 101

Summary In previous studies on plant cells, antibodies directed against intermediate filaments (IFs) have shown that IF antigens are distributed in one of two quite distinct forms. The first co-distributes with each of the four microtubule arrays (cortical, preprophase band, spindle and phragmoplast), while the second form is associated with cytoplasmic paracrystalline fibrillar bundles (FBs) of 10 nm filaments. Conditions allowing one form to be labelled with antibody have generally proved unsuitable for labelling of the other; this has prevented the simultaneous visualization of the two forms of IF antigen in plants and the study of any possible physical relationships between them at the electron microscopic level. In this paper, we show that ME 101, which recognizes an epitope in the N-terminal portion of all classes of intermediate filaments, stains both forms of plant IF antigen simultaneously in tobacco suspension cells using immunofluorescence or immunogold labelling techniques. These cells contain in their cortex short (ca. l m) fibrillar bundles which stain with ME 101. These bundles appear to be independent of the microtubule-associated epitope which stains in a continuous linear manner with ME 101. When protoplasts are either cleaved open on grids or sequentially extracted with detergents prior to critical point drying, the short fibrillar bundles are specifically labelled by ME 101 tagged with colloidal gold. ME 101 also co-distributed with underlying linear filaments, which appeared to be microtubules. In addition to these structures, the cortex also contains a meshwork of variably-sized fine filaments but these are not labelled with ME 101 nor with an antibody raised against the plant cytoskeleton, which recognizes cytokeratin 8. These results confirm that the fibrillar bundles and the microtubule-associated form of plant IF antigens are present simultaneously rather than experimentally-interconvertible, and that they appear to be physically unconnected.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FB fibrillar bundle - FITC fluorescein isothiocyanate - IF intermediate filaments - MTSB microtubule stabilizing buffer - TBS Tris-buffered-saline     

The potency of the first two cleavage cells in echinoderm development: the experiments of Driesch revisited

A hundred years have passed since Driesch performed the classical experiment of separating sea urchin blastomeres from a two-cell-stage embryo, finding that each developed into a complete though smaller larva. The earlier studies of Roux using frogs showed that inactivating one of the two blastomeres by a heated needle resulted, during the early stages of development, in the formation of a half embryo. In this type of experiment, in which the two blastomeres are not separated, the live blastomere continues its development while it is still attached to an inactivated neighbour. In the work reported here, Roux's experimental design was used on two-cell-stage embryos of sea urchins. In contrast to the findings of Roux using amphibians, it was found (as claimed by Driesch) that the living blastomere developed as in the case of separated blastomeres.     

Immunohistochemical distribution of simple-epithelial-type keratins and other intermediate filament proteins in the developing human pituitary gland

Summary An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation.     

Testing claims of gene conversion between multigene family members: examples from echinoderm actin genes

Molecular genetics studies often infer the occurrence of gene conversion events based on simple sequence similarity observations that do not include any statistical analyses. I show that the statistical significance of two previously proposed gene conversion events can easily be tested and point out that a variety of methods are available to perform gene conversion analyses. Received: 6 June 2001 / Accepted: 22 June 2001     

Formation of the sea urchin male pronucleus in vitro: Membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca²⁺ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.