Purification, characterization and molecular cloning of muscle paranemin

Paranemin is an incompletely characterized ~280 kilodalton protein previously identified and immunolocalized in embryonic chick skeletal muscle. Paranemin has been purified from the same tissue source, has the same molecular weight by SDS-PAGE, and has the same antibody localization at the Z-lines of adult avian cardiac muscle. The method developed for preparation of purified paranemin from embryonic (chick) skeletal muscle includes homogenization, centrifugation and gel filtration, hydroxyapatite, and DEAE-cellulose chromatography. By using this method, ~2 mg of purified paranemin was routinely obtained. Amino acid analysis revealed that paranemin has a high acidic to basic amino acid ratio, which agrees with the measured pI range of 4.1-4.5. When the purified protein was stained with a cationic carbocyanine dye, Stains-all, paranemin stained an intense blue, indicating it is a phosphoprotein and/or a glycoprotein. Further testing determined that paranemin is a glycoprotein. A monoclonal antibody (4D3) was made to use in one-and two-dimensional Western blots, which were used to identify paranemin throughout the purification procedure, and for immunofluorescence studies. Double-label confocal immunofluorescence showed colocalization of paranemin with desmin at the Z-lines of adult cardiac and skeletal muscle cells and at cardiac muscle intercalated disks;I determined the full-length cDNA sequence of paranemin by immunoscreening a [lambda]gt22 cDNA library from embryonic chick skeletal muscle with a monoclonal antibody specific for paranemin (4D3) and by hybridization screening. Northern blot analysis reveals a single transcript of 5.3 kb, which is much smaller than predicted from the size of paranemin (~280 kDa) by SDS-PAGE. The pI and molecular weight, predicted from the deduced amino acid sequence of paranemin, are 4.17 and 178,161 Daltons, respectively. I found that paranemin is a novel intermediate filament (IF) protein, which may be classified as a type VI IF protein. Paranemin contains the conserved IF rod domain (308 amino acids), which is 63.3% identical in amino acid sequence to the rod domain of tanabin and 45.5% identical to the rod domain of nestin. The partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, which overlap each other by 402 nucleotides, are almost identical to parts of the cDNA sequence of paranemin

The expression of a "Xenopus borealis" cardiac actin gene in normal and transformed frog embryos

The major aim of this project has been to ascertain if the expression of a cloned Xenopus gene, which is normally expressed in a tissue - specific fashion during early development, is regulated in the same way as its chromosomal counterpart in micro- injected Xenopus embryos. A number of clones, which contain genes encoding different actin isoforms, have been isolated from Xenopus borealis genomic libraries. One of these contains an entire cardiac actin gene, on the basis of the isotype - specific sequence of its encoded product. Indeed, in the adult frog, the chromosomal gene is only expressed in the heart. However, in the embryo, transcripts are also detected in the myotomes, which contain skeletal muscle cells. Two transcriptional assays have been developed, so that transcripts from the unmodified, cloned X. borealis cardiac actin gene can be detected separately from endogenous transcripts in micro-injected X. laevis embryos. In such transformed animals, injected linear DNA forms high-molecular-weight extrachromosomal concatenates, which replicate and become relatively evenly distributed throughout all tissues. Properly initiated transcripts from the cloned gene are correctly localised to the myotomes in both neurulae and tadpoles. The temporal regulation of expression also shares strong similarities with that of the endogenous, chromosomal actin gene. In a preliminary investigation of the sequences responsible for this regulation, a fusion construct between the first two actin exons and the last exon of a mouse β-globin gene has been injected. The same wide distribution of DNA, but spatially restricted pattern of expression, as the actin gene is found, whereas transcripts from a histone-globin fusion gene are formed in all tissues. This is the first report of correct spatial control of expression from an injected, cloned gene in Xenopus. I discuss the wider significance of these results for future studies on the early developmental regulation of gene expression

Characterization of mammalian WDR1 during dynamic actin rearrangement events.

The actin cytoskeleton functions within processes such as cell extension, migration, and neurogenesis; yet the mechanisms of actin regulation are not completely understood. WD repeat proteins (WDR1) have recently been shown to interact with actin and regulate cortical actin dynamics through interactions with cofilin in a number of species. However studies on mammalian WDR1 have not been reported. Further studies in chick systems have also indicated several cofilin-independent functions of WDR1 within cytokinesis and cell migration. We investigated the human homologue of yeast WDR1 and identified the expression of two isoforms, a full length 60 kDa protein and an N-terminal truncated 50 kDa protein. Analysis of WDR1 expression in transformed and non-transformed cell lines indicated that the two isoforms were differentially expressed. Sequence analysis revealed the WD motifs were homologous to kelch motifs found within Drosophila kelch. Kelch containing proteins are believed to mediate protein-protein interactions. Protein interaction experiments demonstrated WDR1 bound actin and formed hetero-multimeric complexes; however no interaction with cofilin was observed. Localization studies showed WDR1 localized to actin filaments (similar to vinculin) and to areas undergoing actin rearrangement with cofilin and CAP1. Interestingly, WDR1 was shown to remain attached to glass coverslips as part of a WDR1 aggregated complex (WAC) after trypsin mediated cell detachment. Latrunculin A and cytochalasin D treatments indicated WDR1 may stabilize actin filaments during depolymerizing events. Therefore, these results have provided an initial characterization of the important role of hWDR1 within the critical cellular processes of attachment and migration, and have provided experimental avenues for future pursuit.Dept. of Biological Sciences. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2004 .N66. Source: Masters Abstracts International, Volume: 43-05, page: 1656. Adviser: D. Hubberstey. Thesis (M.Sc.)--University of Windsor (Canada), 2004

Structure and Expression of the Actin Gene Family of Drosophila melanogaster

We have isolated the six actin genes of Drosophila melanogaster from a Drosophila genomic DNA library and have compared structural features of the genes by restriction mapping, electron microscopy and DNA sequencing. We found that at least two of the actin genes contain intervening sequences which interrupt the genes at different positions. Several of the genes were shown to be lacking intervening sequences in the analogous positions. This nonconservation of intron position is in striking contrast to the strong conservation of intron positions seen in other gene families. The DNA sequences of the protein coding regions of the genes are highly conserved while the intron and untranslated sequences are not. The primary sequences of all the Drosophila actins resemble mammalian cytoplasmic actins more than mammalian muscle actins. We studied the distribution of actin mRNAs in different developmental stages and in different dissected body parts with the use of gene specific hybridization probes which we isolated from the 3' untranslated portions of the genes. We found that the genes fall into three main categories with respect to their patterns of expression in Drosophila. Trancripts from two of the genes are found throughout Drosophila development. They are expressed at higher levels in ovaries and embryonic cultured cells than in muscle containing tissue and are thought to be cytoplasmic actins. Two others encode thoracic muscle actins. Their transcripts accumulate predominantly in the thoracic regions of the adult where the flight and jump muscles are found. The other two genes are most active in larvae and in adult abdomens. They are thought to encode actins used in the larval, pupal, and adult intersegmental muscles. We studied the structure of the cytoplasmic actin gene, act5C, in detail and found that it encodes at least six different mRNAs. At the 5' end there are two nonhomologous leader exons which are alternately spliced to the remainder of the gene. At the 3' end of the gene, three sites of polyadenylation are used. The 3' variation is the principal cause of the transcript length heterogeneity observed in the transcripts. In whole animal RNA, the two leader exons are expressed with the same pattern through development and with all three polyadenylation sites. There is some developmental variability in the use of the three polyadenylation sites. In order to determine if each exon is preceded by a functional promoter and to identify sequences important for transcription initiation from each exon, we made fusions between act5C promoter fragments and the bacterial chloramphenicol acetyltransferase (CAT) gene and tested these for promoter activity in transient assays in Kc cells. We found that each exon is preceded by a separate, functional promoter. At least two regions of DNA sequences are necessary for optimal expression from exon 1. One of these lies greater than 1.9 kb upstream from the exon 1 cap site. All of the sequences required for exon 2 transcription lie within 450 bases of its cap site. There is evidence from some constructions that transcription initiation from exon 1 may inhibit transcription initiation from ex on 2.</p

Structural and Functional Studies on the Subunits of the Nicotinic Acetylcholine Receptor

An introduction to the work in the study of the nicotinic acetylcholine receptor is presented. The author reviews the field to place the work of the present volume in its proper context The major developments in studying the protein biochemistry of the receptor are reviewed, including the subunit makeup, ligand binding, and protein sequences. These studies led to the cloning and sequencing of many of the subunits as cDNA or genomic DNA constructions. This wealth of sequence information has allowed the formulation of detailed models of receptor structure. Current work centers on testing various aspects of these models and expanding the scope of the field into different species and tissues that utilize this receptor. Partial cDNA clones specific for the β and δ subunits of the acetylcholine receptor of Torpedo californica were isolated by the following method. A cDNA library was constructed from electric organ poly( A)+ RNA and enriched by screening for clones more abundantly represented in electric organ than in brain or-liver mRNA preparations. These clones were tested by hybridization selection of clone specific mRNA which was then translated in vitro. Protein products were immunoprecipitated and analyzed by gel electrophoresis. The isolated clones were used to screen a library of Torpedo genomic DNA which resulted in the isolation of the gene for the Torpedo δ subunit. The δ gene was found to be single copy in Torpedo, and it contains at least four introns. A cDNA library was constructed in λgt10 from membrane bound poly(A)+ RNA from mouse BC3H-1 cells. This library was screened with cDNA encoding the complete protein region of the Torpedo γ and δ subunits. Positively hybridizing clones isolated with the Torpedo γ subunit were sequenced and compared with published data. The deduced amino acid sequence was more highly homologous to the Torpedo δ than to the Torpedo γ and on this basis the mouse clone was tentatively identified as a δ subunit of the acetylcholine receptor. The mouse nucleotide sequence has several stretches of strong homology with the Torpedo γ subunit cDNA, but no such homology with the Torpedo δ subunit . A genomic blotting experiment indicated that there is probably one, but at most two chromosomal genes encoding this or closely related sequences. In order to test the assignment of the mouse δ cDNA by a more functional criterion than simple amino acid homology, the following experiment was done. The phage SP6 transcription system was used to transcribe mRNA from the four individual Torpedo subunits and from the mouse δ. When the four Torpedo subunit specific mRNAs were injected into Xenopus oocytes, functional receptors appeared in the oocyte membrane. If the β or γ subunit RNA was omitted, no response to acetylcholine was detected, while a small response was detected if the δ subunit RNA was omitted. When mouse δ specific RNA was injected in place of the Torpedo δ, a 3-4 fold larger response was measured in response to acetylcholine under voltage clamp conditions. The replacement of Torpedo γ RNA with mouse δ RNA gave no detectable response. Surface binding of α-bungarotoxin was not significantly altered by exchanging the δ subunits, which indicates that the difference is intrinsic to the channel rather than a matter of stability or synthesis rates. Examination of the amino acid sequences of the two δ subunits and the Torpedo γ did not identify an obvious region of subunit specific homology. The amino acid features necessary to determine a specific subunit are not obvious from simple homology comparisons. We have constructed a series of chimeric subunits to try to localize subunit determining regions of the acetylcholine receptor polypeptides. Each chimera was tested in the oocyte system by replacing its RNA for each of the parent RNAs in turn. None of the chimeras we have constructed retained enough of either parental subunit characteristics to function fully in place of that parent subunit to form an acetylcholine receptor that is responsive to acetylcholine. We conclude that a minimum of two subunit-specific regions are widely dispersed over the subunit length. These data are also consistent with the conclusion that there are no discrete regions that determine subunit identity, but instead that this information is rather evenly distributed along subunit length. In some combinations, the chimeras were incorporated into surface AchRs, although these complexes were only weakly responsive to Ach. We further conclude that there are regions needed for efficient function of these subunits that are not necessary for the formation of surface complexes. We have demonstrated that the α subunits of both mouse and chick form functional receptors in the Xenopus oocyte system in combination with the β and γ subunits from Torpedo and a δ from either Torpedo or mouse. The responses of these hybrid AchRs are smaller than the response from the Torpedo AchR. In contrast, the mouse γ subunit did not form functional AchRs in any combination of the subunits mentioned above. The present author spent the early part of her career studying the molecular biology of the actin genes of Drosophila melanogaster. Portions of each of the six actin genes were sequenced. These sequences revealed that the amino acid sequence of actin is highly conserved but that the positions of introns in these genes are strikingly nonconserved. Further, each of the Drosophila actins resembles the cytoplasmic isoforms from vertebrates, while none resemble the muscle isoforms.</p

Functional analysis of RAC1B during neuronal development

Neurite extension requires the concerted action of a number of events including actin polymerization at the growth cone, formation of new adhesive sites to the substrate, and membrane addition, to extend the surface of the elongating neurite. The small GTPase Rac IB, identified from developing chicken retina, is specifically expressed during neural development, and overexpression of Rac IB in embryonic retinal neurons specifically stimulates neurite extension and branching (Albertinazzi et al., 1998). Very little is known about the molecular mechanisms which coordinate Rac IB-mediated neurite extension. In order to study in detail this mechanism in an in vivo system, recombinant embryonic stem cells (ES) for the deletion of mouse Rac IB gene were produced. ES cells from four independent clones that resulted positive for the deletion of mouse Rac IB gene were microinjected in blastocysts, and chimeric mice were generated to be used for the generation of Rac IB-null mice. Recently, p95-APPl was identified as a protein able to interact with Rac IB in a GTP-dependent manner (Di Cesare et al. 2000). P95-APP1 is an ArfGAP of the GIT family, highly expressed in the developing nervous system. Expression of p95- APPl with a mutated or deleted ArfGAP domain in retinal neurons prevented Rac IB-induced neuritogenesis, leading to PIX-mediated accumulation of mutant p95-APPl and associated proteins at large Rab 11-positive endocytic vesicles. Analysis of neurons expressing different p95-derived constructs together with wild-type or mutant Arf6 GTPases revealed the requirement of both p95-APPl and a cycling Arf6 for a normal Rac IB-mediated neuritogenesis. The data obtained in this thesis show a functional connection between the localization of the p95-APPl complex at recycling endosomes and Rac IB-dependent neuritogenesis, and suggest a role of this complex in the regulation of membrane recycling to/from the neuronal surface during neuritogenesis