A τ Fragment Containing a Repetitive Sequence Induces Bundling of Actin Filaments

Abstract: Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule-associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin-binding domain on τ with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence of τ protein, increased turbidity at 320 nm in a dose-dependent fashion. A salient feature of the τ peptide-induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the τ peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6–4.7 resulted in a higher effect of τ peptide in promoting bundles of actin filaments. A similar pH-dependent effect was observed for τ protein-induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of ɛ-aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a τ repetitive sequence (also found in MAP-2 and MAP-4) containing a common tubulin binding motif may constitute a functional domain on τ for the dynamics of the interconnections between actin filaments and microtu-bules.     

Half-lives of oat mRNAs in vivo and in a polysome-based in-vitro system

We have used a cell-free polysome-based in-vitro mRNA-degradation system to investigate the halflives of plant cell mRNAs. In order to establish the fidelity of the in-vitro system, we used cordycepin to determine the in-vivo half-lives of -tubulin and actin mRNAs in the primary leaves of 4-d-old etiolated oat (Avena sativa L.) seedlings. The in-vitro rank order of half-lives for phytochrome A (45 min), -tubulin (105 min), and actin (220 min) mRNAs mimicked the in-vivo rank order. A pulse of red light given to excised etiolated primary leaves caused an in-vivo reduction in the half-life of -tubulin mRNA. The selectivity of the polysome-based system was further demonstrated by the decrease in the half-life of -tubulin mRNA (from 105 min to 60 min) induced by a pulse of red light given to the etiolated oat seedlings prior to isolation of polysomes. Red light did not affect the apparent half-lives of phytochrome A or actin mRNAs.Abbreviations cab gene for chlorophyll-a/b-binding protein - kb(p) kilobase (pair) - phyA gene for type-I phytochrome protein - rbcS gene for ribulose-1,5-bisphosphate-carboxylase small-subunit We thank Dr. Richard B. Meagher for the pSAc3 actin clone. We thank Dr. Cecil Stewart for the use of his density-gradient fractionator, and Dr. Virginia Crane for instruction in using the fractionator. We also appreciate the helpful comments provided by the other members of the laboratory during the course of this research: Dr. Isaac John, Dr. Iffat Rahim, Linda Barnes, Bruce Held, David Higgs, and Theresa Tirimanne. This work was supported by USDA grants CRGO 88-37261-4196 and 91-37304-6397, and the Iowa State University Biotechnology Program.     

Novel changes in the plasma membrane and cortical cytoplasm during wound-induced contraction in a giant-celled green alga

Summary Changes in the plasma membrane surface and in the cortical cytoplasm during wound healing in giant green algal cells ofErnodesmis verticillata (Kützing) Brgesen were followed using scanning and transmission electron microscopy. Microvillus-like structures that contain cytoplasmic and cytoskeletal constituents were observed emanating from the surface of the plasma membrane at the retracting/cut end of wounded cells. These delicate structures seem to be remnants of cell wall-plasmalemma connections that draw out the plasma membrane and cortical components from the contracting cytoplasm as it pulls away from the cell wall. Most of these connections break during wound healing and, when contraction stops, the microvillus-like protrusions become progressively shorter. In cells treated with a calmodulin antagonist (W-7), a number of distinctive bodies accumulate that are of unknown composition, are oblong in shape, and have a diameter slightly smaller than the protoplasmic protrusions. Ultrastructural and other data indicate that these bodies result from retrieved constituents of the plasma-membrane protrusions, as they do not accumulate in unwounded drugtreated cells or in cells treated in W-5. These findings suggest that the protoplasmic protrusions accumulate membrane and cytoplasmic components that are retrieved and recycled during wound healing inErnodesmis by a novel mechanism. The combined plasma membrane surfaces of the microvillus-like protrusions may help to account for the drastic decrease in surface area that occurs during wound healing.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy - W-7 N-[6-aminohexyl]-5-chloro-1-naph-thalenesulfonamide - W-5 N-[6-aminohexyl]-1-naphthalenesulfonamide     

Effect of cytochalasin A on actin distribution in the fission yeastSchizosaccharomyces pombe studied by fluorescent and electron microscopy

Summary Actin distribution and ultrastructure of the fission yeastSchizosaccharomyces pombe treated with cytochalasin A (CA) were investigated by fluorescence microscopy using rhodamine-conjugated phalloidin (rh-ph) and freeze substitution electron microscopy. Among the cytochalasins tested, CA was most effective and at 5 g/ ml inhibited the appearance of the actin ring at the cell equator at the stage prior to septum formation and the accumulation of actin dots at the septum-forming site both in wild-type cells and the mutantcdc 11, which is defective in septum formation at restrictive temperature. Freeze substitution electron microscopy of CA-treated cells revealed the displacement and morphological alteration of cytoplasmic vesicles and dictyosomes within 30 min and the appearance of dense bodies in the cytoplasm. A sub-population of cytoplasmic vesicles and dictyosomes were insensitive to CA and maintained their original structure. An electron less dense layer containing filamentous material was noted beneath the plasma membrane and thought to be the area of heavy actin patches stained with rh-ph at the cells ends. These results suggest that CA disrupted an actin network that normally maintains the organization of the secretory pathway involving dictyosomes and vesicles.     

Sequence analysis of an actin isoform in the flatworm Diphyllobothrium dendriticum (Cestoda)

We have examined actin cDNA of the flatworm Diphyllobothrium dendriticum (Cestoda). Actin is a contractile protein that has been implicated in a variety of developmental and cellular processes. It is highly conserved and present in all eukaryotic cells. It is of particular interest to analyze evolutionary preserved genes in flatworms, because ancestral flatworms are regarded to play a central role in the evolution of the metazoans (Barnes et al., 1998). Screening a cDNA library of D. dendriticum (UniZap XR, Stratagene) with a human -actin probe resulted in several positive clones. One of the cDNA inserts, Didactl, consisting of 1392 bp was completely sequenced. The established nucleotide sequence revealed a 5 untranslated region of 33 bp, the entire open reading frame of 1128 bp and a 3 untranslated region of 231 bp which ends in a stretch of 21 A residues. The potential polyadenylation signal (AATAAA) is located 14 bp upstream of the poly (A) tail. The deduced amino acid sequence of Didactl is 376 amino acids long. It is a typical invertebrate actin (Fyrberg et al., 1981) resembling more the cytoplasmic than the muscular isoforms of vertebrate actins. Didactl is for example 96% homologous to human cytoplasmic -actin but only 92.6% identical with human smooth muscle -actin. The actin proteins are generally encoded by a multigene family which differs in size from species to species. Most organisms have four to eight genes coding for actin in their genome, but the number of actin genes can also be over 20 (Hamelin et al., 1988). Sequence comparisons of Didactl and the partly sequenced cDNA clones indicate that D. dendriticum has at least four different genes coding for actin in its genome.     

Persistent Enhancement of Sustained Calcium-Dependent Glutamate Release by Phorbol Esters: Role of Calmodulin-Independent Serine/Threonine Phosphorylation and Actin Disassembly

Abstract: The phorbol ester 4β-phorbol 12,13-dibutyrate increases the final extent of Ca²⁺-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca²⁺. Previous work from our laboratory demonstrated that this 4β-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4β-phorbol 12,13-dibutyrate, requires localized Ca²⁺ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca²⁺/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca²⁺/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca²⁺-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the effect of 4β-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.     

Compression-induced changes in the shape and volume of the chondrocyte nucleus

Changes in cell shape and volume are believed to play a role in the process of mechanical signal transduction by chondrocytes in articular cartilage. One proposed pathway through which chondrocyte deformation may be transduced to an intracellular signal is through cytoskeletally mediated deformation of intracellular organelles, and more specifically, of the cell nucleus. In this study, confocal scanning laser microscopy was used to perform in situ three-dimensional morphometric analyses of the nuclei of viable condrocytes during controlled compression of articular cartilage explants from the canine patellofemoral groove. Unconfined compression of the tissue to a 15% surface-to-surface strain resulted in a significant decrease of chondrocyte height and volume by 14.7 ± 6.4 and 11.4 ± 8.4%, respectively, and of nuclear height and volume by 8.8 ± 6.2% and 9.8 ± 8.8%, respectively. Disruption of the actin cytoskeleton using cytochalasin D altered the relationship between matrix deformation and changes in nuclear height and shape, but not volume. The morphology and deformation behavior of the chondrocytes were not affected by cytochalasin treatment. These results suggest that the actin cytoskeleton plays an important role in the link between compression of the extracellular matrix and deformation of the chondrocyte nuclei and imply that chondrocytes and their nuclei undergo significant changes in shape and volume in vivo.     

Deuterostomic Actin Genes and the Definition of the Chordates: cDNA Cloning and Gene Organization for Cephalochordates and Hemichordates

The evolutionary relationship of muscle and nonmuscle actin isoforms in deuterostomia was studied by the isolation and characterization of two actin genes from the cephalochordate Branchiostoma lanceolatum and two from the hemichordate Saccoglossus kowalevskii The Branchiostoma genes specify a muscle and a nonmuscle actin type, respectively. Together with earlier results on muscle actins from vertebrates and urochordates, a N-terminal sequence signature is defined for chordate muscle actins. These diagnostic amino acid residues separate the chordates from the echinoderms and other metazoa. Although the two Saccoglossus actins characterized so far lack the diagnostic residues, in line with the presumptive phylogenetic position of hemichordates outside the chordates, a definitive conclusion can only be expected once the full complement of actin genes of Saccoglossus is established. Comparison of the intron patterns of the various deuterostomic actin genes shows that intron 330-3, which is present in all vertebrate genes, is conspicuously absent from nonvertebrate genes. The possible origin of this intron is discussed. Received: 4 July 1997 / Accepted: 29 August 1997     

Isolation and Characterization of Five Actin cDNAs from the Cestode Diphyllobothrium dendriticum: A Phylogenetic Study of the Multigene Family

Five cDNAs (pDidact2–pDidact6), representing different actin genes, were isolated from a Diphyllobothrium dendriticum cDNA library, and the DNA as well as the putative amino acid sequences were determined. The corresponding Didact2 and Didact4 genes code for peptides 376 amino acids long, with molecular weights 41,772 and 41,744 Da, respectively, while the deduced Didact3 protein is 377 amino acids long and weighs 41,912 Da. The pDidact5 and -6 cDNAs lack nucleotides corresponding to three to six amino acids at the amino-terminus. Two of the five cDNAs contain the conventional AATAAA as the putative polyadenylation signal, one has the common variant ATTAAA, whereas the hexanucleotide AATAGA is found 15 and 18 nucleotides, respectively, upstream of the poly(A) site in two of the cDNAs. Phylogenetic studies including 102 actin protein sequences revealed that there are at least four different types of cestode actins. In this study three of these types were found to be expressed in the adult D. dendriticum tapeworm. Structurally the cestode actin groupings differ from each other to an extent seen only among the metazoan actins between the vertebrate muscle and cytoplasmic isoforms. In the phylogenetic trees constructed, cestode actins were seen to map to two different regions, one on the border of the metazoan actins and the other within this group. It is, however, difficult to say whether the cestode actins branched off early in the metazoan evolution or if this position in the phylogenetic tree only reflects upon differences in evolutionary rate. Received: 19 June 1996 / Accepted: 20 August 1996     

Evidence for Gene Conversion Between Tandemly Duplicated Cytoplasmic Actin Genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly eclosed adults. A region 5′ of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs. Received: 10 January 1996 / Accepted: 12 March 1996