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1.
Trends Cell Biol ; 11(5): 196-202, 2001 May.
Article in English | MEDLINE | ID: mdl-11316608

ABSTRACT

The translocation of dynein along microtubules is the basis for a variety of essential cellular movements. Despite a general domain organization that is found in all the cytoskeletal motors, there are structural features of dynein that set it apart from the other motors. These include a track-binding site that is located at the tip of a long projection, and six nucleotide-binding modules that together form the globular head of dynein. These unique features suggest that dynein produces movement by a mechanism that is different from that used by the other motors.


Subject(s)
Dyneins/chemistry , Dyneins/metabolism , Molecular Motor Proteins/physiology , Protein Conformation , Amino Acid Sequence , Animals , Dyneins/ultrastructure , Evolution, Molecular , Humans , Microtubules/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding
4.
J Immunol ; 166(3): 1507-16, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11160190

ABSTRACT

The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated, protein tyrosine kinases of both the Src and Syk families. To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane. The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell. Syk is not demonstrably associated with the internalized receptor. Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of the clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation of the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating receptor internalization and down-regulation.


Subject(s)
Enzyme Precursors/metabolism , Immunologic Capping , Luminescent Proteins/metabolism , Nuclear Proteins , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, B-Cell/metabolism , src-Family Kinases/physiology , Animals , B-Lymphocytes/enzymology , B-Lymphocytes/metabolism , Catalysis , Cell Line , Chickens , DNA-Binding Proteins/metabolism , Enzyme Activation/immunology , Enzyme Precursors/deficiency , Enzyme Precursors/genetics , Enzyme Precursors/physiology , Genetic Vectors/chemical synthesis , Genetic Vectors/immunology , Genetic Vectors/metabolism , Green Fluorescent Proteins , Humans , Immunologic Capping/genetics , Intracellular Fluid/enzymology , Intracellular Fluid/immunology , Intracellular Fluid/metabolism , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice , NFATC Transcription Factors , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/physiology , Receptors, Antigen, B-Cell/immunology , Syk Kinase , Transcription Factors/metabolism , Transfection , src-Family Kinases/deficiency , src-Family Kinases/genetics
5.
J Cell Sci ; 113 ( Pt 14): 2557-65, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10862713

ABSTRACT

Syk is a protein-tyrosine kinase that is essential for B-lymphocyte development and B-cell signaling. Syk phosphorylates tubulin on tyrosine both in vitro and in intact lymphocytes. Here we show that (alpha)-tubulin present within the cytoskeletal microtubule network was phosphorylated in a Syk-dependent manner following the activation of B-cells by engagement of the B-cell antigen receptor or by treatment with the phosphotyrosine phosphatase inhibitor, pervanadate. Immunofluorescence staining of microtubule cytoskeletons and western blotting studies with antibodies to phosphotyrosine confirmed the phosphorylation of polymerized tubulin in Syk-expressing, but not Syk-deficient, cells. At low concentrations of pervanadate, centrosomes appeared to be preferentially tyrosine-phosphorylated. Tubulin phosphorylated to a high stoichiometry on tyrosine assembled into microtubules in vitro, and preassembled microtubules were also phosphorylated by Syk kinase in vitro. Thus, Syk has the capacity to interact with microtubule networks within the B-lymphocyte and catalyzes the phosphorylation of the (alpha)-tubulin subunit. Syk-dependent phosphorylation of microtubules may affect the ability of the microtubule cytoskeleton to serve as a platform upon which signaling complexes are assembled.


Subject(s)
B-Lymphocytes/metabolism , Enzyme Precursors/metabolism , Lymphocyte Activation , Microtubules/metabolism , Protein-Tyrosine Kinases/metabolism , Tubulin/metabolism , Animals , Antibodies, Anti-Idiotypic/pharmacology , B-Lymphocytes/drug effects , B-Lymphocytes/ultrastructure , Biopolymers/metabolism , Cell Line , Centrosome/drug effects , Centrosome/metabolism , Chickens , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Enzyme Inhibitors/pharmacology , Enzyme Precursors/drug effects , Enzyme Precursors/physiology , Gene Deletion , Intracellular Signaling Peptides and Proteins , Microtubules/drug effects , Phosphorylation , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein-Tyrosine Kinases/drug effects , Protein-Tyrosine Kinases/physiology , Signal Transduction/physiology , Syk Kinase , Tubulin/drug effects , Vanadates/pharmacology
6.
Methods Cell Biol ; 62: 433-40, 2000.
Article in English | MEDLINE | ID: mdl-10503208

ABSTRACT

As illustrated elsewhere in this volume, Tetrahymena is an extraordinary experimental system in which molecular genetics, biochemistry, and cytology can be applied to the study of a cell biological problem. Our long-term goal is to understand how the different structural domains of dynein contribute to its function. Our strategy is to create targeted modifications in an axonemal dynein heavy chain gene, recover the dynein protein from cilia, and evaluate the in vitro activity of the isolated dynein. In this chapter, we have summarized our procedures for the isolation and characterization of the ciliary outer arm dynein.


Subject(s)
Dyneins/analysis , Tetrahymena thermophila/chemistry , Animals , Cilia/chemistry
7.
Mol Biol Cell ; 10(3): 771-84, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10069817

ABSTRACT

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.


Subject(s)
Cytoplasm/metabolism , Dyneins/genetics , Protozoan Proteins/genetics , Tetrahymena thermophila/genetics , Amino Acid Sequence , Animals , Chromosome Segregation , Cytoplasm/chemistry , Cytoskeleton/genetics , Cytoskeleton/metabolism , Dyneins/metabolism , Genetic Techniques , Mitosis/genetics , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phagocytosis/genetics , Protozoan Proteins/metabolism , Sequence Homology, Amino Acid
8.
Mol Biol Cell ; 9(2): 237-47, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9450951

ABSTRACT

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.


Subject(s)
Cytoplasm/chemistry , Dyneins/analysis , Isoenzymes/analysis , Testis/chemistry , Animals , Antibody Specificity , Brain Chemistry , Centrifugation, Density Gradient , Dyneins/chemistry , Dyneins/genetics , Isoenzymes/chemistry , Isoenzymes/genetics , Male , Molecular Weight , Osmolar Concentration , Photolysis , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Sodium Chloride
9.
J Cell Sci ; 109 ( Pt 7): 1891-8, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8832411

ABSTRACT

Organisms that have cilia or flagella express over a dozen dynein heavy chain genes. Of these heavy chain genes, most appear to encode axonemal dyneins, one encodes conventional cytoplasmic dynein (MAP1C or DHC1a), and one, here referred to as DHC1b, encodes an unclassified heavy chain. Previous analysis of sea urchin DHC1b (Gibbons et al. (1994) Mol. Biol. Cell 5, 57-70) indicated that this isoform is either an axonemal dynein with an unusual protein sequence or a cytoplasmic dynein whose expression increases during ciliogenesis. In the present study, we examined the expression of DHC1b in rat tissues. The DHC1b gene is expressed in all tissues examined, including unciliated liver and heart cells. In contrast, rat axonemal dyneins are only expressed in tissues that produce cilia or flagella. In cultured rat tracheal epithelial (RTE) cells, DHC1b is expressed in undifferentiated cells and increases in expression during ciliogenesis. In contrast, the expression of conventional cytoplasmic dynein, DHC1a, does not change during RTE differentiation and axonemal dynein is not expressed until after differentiation commences. In order to examine the expression of DHC1b protein, we produced an isoform-specific antibody to a synthetic peptide derived from the rat DHC1b sequence. The antibody demonstrated that DHC1b is a relatively minor component of partially purified cytoplasmic dynein. Indirect immunofluorescence microscopy revealed that DHC1b is not detected in cilia and remains in the cytoplasm of ciliated RTE cells, often accumulating at the apical ends of the cells. These results suggest that DHC1b is a cytoplasmic dynein that may participate in intracellular trafficking in polarized cells.


Subject(s)
Dyneins/isolation & purification , Trachea/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Cilia , Cytoplasm/metabolism , Dyneins/genetics , Dyneins/metabolism , Epithelium/metabolism , Male , Molecular Sequence Data , Rats , Rats, Inbred F344
10.
J Biol Chem ; 271(9): 4755-62, 1996 Mar 01.
Article in English | MEDLINE | ID: mdl-8617742

ABSTRACT

Syk (p72syk) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha-tubulin.


Subject(s)
B-Lymphocytes/metabolism , Enzyme Precursors/metabolism , Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, B-Cell/metabolism , Tubulin/metabolism , Amino Acid Sequence , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Cytosol/enzymology , Enzyme Activation , Enzyme Precursors/isolation & purification , Glutathione Transferase/metabolism , Humans , Immunoglobulin M/pharmacology , Intracellular Signaling Peptides and Proteins , Kinetics , Mice , Molecular Sequence Data , Phosphorylation , Protein-Tyrosine Kinases/isolation & purification , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Syk Kinase
11.
Mol Biol Cell ; 7(1): 71-9, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8741840

ABSTRACT

Axonemal dyneins are molecular motors that drive the beating of cilia and flagella. We report here the identification and partial cloning of seven unique axonemal dynein heavy chains from rat tracheal epithelial (RTE) cells. Combinations of axonemal-specific and degenerate primers to conserved regions around the catalytic site of dynein heavy chains were used to obtain cDNA fragments of rat dynein heavy chains. Southern analysis indicates that these are single copy genes, with one possible exception, and Northern analysis of RNA from RTE cells shows a transcript of approximately 15 kb for each gene. Expression of these genes was restricted to tissues containing axonemes (trachea, testis, and brain). A time course analysis during ciliated cell differentiation of RTE cells in culture demonstrated that the expression of axonemal dynein heavy chains correlated with the development of ciliated cells, while cytoplasmic dynein heavy chain expression remained constant. In addition, factors that regulate the development of ciliated cells in culture regulated the expression of axonemal dynein heavy chains in a parallel fashion. These are the first mammalian dynein heavy chain genes shown to be expressed specifically in axonemal tissues. Identification of the mechanisms that regulate the cell-specific expression of these axonemal dynein heavy chains will further our understanding of the process of ciliated cell differentiation.


Subject(s)
Cell Differentiation , Cilia/chemistry , Dyneins/genetics , Gene Expression Regulation , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cells, Cultured , Cloning, Molecular , Epithelial Cells , Molecular Sequence Data , Polymerase Chain Reaction , Rats , Trachea
12.
Mol Biol Cell ; 6(11): 1549-62, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8589455

ABSTRACT

The genes encoding two Paramecium dynein heavy chains, DHC-6 and DHC-8, have been cloned and sequenced. Sequence-specific antibodies demonstrate that DHC-6 encodes ciliary outer arm beta-chain and DHC-8 encodes a cytoplasmic dynein heavy chain. Therefore, this study is the first opportunity to compare the primary structures and expression of two heavy chains representing the two functional classes of dynein expressed in the same cell. Deciliation of paramecia results in the accumulation of mRNA from DHC-6, but not DHC-8. Nuclear run-on transcription experiments demonstrate that this increase in the steady state concentration of DHC-6 mRNA is a consequence of a rapid induction of transcription in response to deciliation. This is the first demonstration that dynein, like other axonemal components, is transcriptionally regulated during reciliation. Analyses of the sequences of the two Paramecium dyneins and the dynein heavy chains from other organisms indicate that the heavy chain can be divided into three regions: 1) the sequence of the central catalytic domain is conserved among all dyneins; 2) the tail domain sequence, consisting of the N-terminal 1200 residues, differentiates between axonemal and cytoplasmic dyneins; and 3) the N-terminal 200 residues are the most divergent and appear to classify the isoforms. The organization of the heavy chain predicts that the variable tail domain may be sufficient to target the dynein to the appropriate place in the cell.


Subject(s)
Dyneins/biosynthesis , Dyneins/genetics , Genes, Protozoan , Paramecium tetraurelia/genetics , Amino Acid Sequence , Animals , Cilia/physiology , Conserved Sequence , Dyneins/chemistry , Genomic Library , Macromolecular Substances , Molecular Sequence Data , Paramecium tetraurelia/enzymology , Paramecium tetraurelia/physiology , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Transcription, Genetic
13.
J Neurobiol ; 27(2): 216-26, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7544825

ABSTRACT

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo.


Subject(s)
Axonal Transport/physiology , Dyneins/physiology , Lampreys/physiology , Animals , Axonal Transport/drug effects , Axonal Transport/radiation effects , Colchicine/pharmacology , Fluorescence , Image Processing, Computer-Assisted , Larva/physiology , Microinjections , Microscopy, Fluorescence , Microscopy, Video , Microspheres , Microtubules/physiology , Spinal Cord/cytology , Spinal Cord/drug effects , Spinal Cord/radiation effects , Ultraviolet Rays , Vanadates/pharmacology
14.
Cell Motil Cytoskeleton ; 32(2): 129-32, 1995.
Article in English | MEDLINE | ID: mdl-8681394

ABSTRACT

Axonemal dyneins and cytoplasmic dynein have evolved separate strategies to perform their tasks. The multi-dynein hypothesis accurately describes the highly specialized axonemal isoforms; each isoform is encoded by a separate gene, is located in a precise place, produces specific forces which contribute to the overall generation of propagated bending, and is not functionally interchangeable with other isoforms. In contrast, cytoplasmic dynein, although carrying many different cargoes, appears to be one isoform. An intriguing question is to determine whether there are additional cytoplasmic dyneins, heretofore uncharacterized, which, like their axonemal counterparts, are customized to perform specific tasks.


Subject(s)
Dyneins/physiology , Isoenzymes/physiology , Models, Biological , Animals , Cilia/chemistry , Cytoplasm/chemistry , Dyneins/chemistry , Dyneins/genetics , Flagella/chemistry , Genes , Isoenzymes/chemistry , Isoenzymes/genetics , Mammals/metabolism , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/physiology , Microtubules/physiology , Multigene Family , Rats
16.
J Cell Sci ; 107 ( Pt 4): 839-47, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8056840

ABSTRACT

Paramecium tetraurelia is a unicellular organism that utilizes both axonemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-directed polymerase chain reaction. Eight different P-loop-containing cDNA fragments were cloned. Southern hybridization analysis indicated that each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Seven of the eight cloned contain sequence motif A, which is found in axonemal dyneins, and one contains sequence motif B, which is found in the dyneins from cell types that do not have cilia or flagella. Two of the Paramecium dynein genes were further investigated: DHC-6 which contains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary beta heavy chain and DHC-8 is similar to the cytoplasmic dynein from Dictyostelium. Deciliation of the cells resulted in a substantial increase in the steady state concentration of DHC-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Competitive solid-phase binding assays demonstrated that each antiserum was peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bovine brain but did not react with ciliary dynein.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Cilia/chemistry , Cytoplasm/chemistry , Dyneins/genetics , Genes, Protozoan , Paramecium tetraurelia/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Antibody Specificity , Base Sequence , Cell Compartmentation , Consensus Sequence , Dyneins/chemistry , Dyneins/immunology , Molecular Sequence Data , Paramecium tetraurelia/immunology , Paramecium tetraurelia/ultrastructure , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Protein Structure, Secondary , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Sea Urchins/chemistry , Sequence Alignment , Sequence Homology, Amino Acid
17.
Dev Biol ; 162(1): 143-53, 1994 Mar.
Article in English | MEDLINE | ID: mdl-7510253

ABSTRACT

The functional significance of microtubule-associated protein 1B (MAP1B) phosphorylation during neuronal differentiation is unknown. In the present study we examined the hypothesis that the phosphorylation of MAP1B is required for neurite outgrowth. We reasoned that if MAP1B phosphorylation was important for neurite outgrowth then the intracellular distribution of phosphorylated MAP1B might exist as a discrete subset of the pattern for total MAP1B. We utilized a monoclonal antibody (mAb 7-1.1) that specifically recognizes a phosphorylated epitope on MAP1B and a polyclonal antiserum that recognizes all MAP1B protein to compare the distributions of phosphorylated and total MAP1B during neurite outgrowth. Phosphorylated MAP1B progressively accumulated in both the soluble and cytoskeletal fractions of differentiating cells. Similar proportions of total and phosphorylated MAP1B were associated with the cytoskeletons of differentiating PC12 cells. Within individual cells, phosphorylated MAP1B, in comparison with total MAP1B, was not limited to a particular intracellular domain. Phosphorylated MAP1B was present in both neurites and cell bodies. It was associated with fibrillar microtubules in neurites and growth cones, but it appeared nonfibrillar within cell bodies. In some cells that differentiated rapidly, there was little phosphorylated MAP1B in the early neurites despite the presence of extensive microtubules. In addition, although phosphorylated MAP1B increased in populations of mature PC12 cell cultures, increases in phosphorylated MAP1B did not always correlate with neurite outgrowth in individual cells. These results suggest that the phosphorylated isoform of MAP1B recognized by mAb 7-1.1 may not be required for neurite outgrowth.


Subject(s)
Microtubule-Associated Proteins/metabolism , Neurites/metabolism , Animals , Antibodies, Monoclonal , Cell Differentiation , Epitopes , Microtubule-Associated Proteins/immunology , Neurites/immunology , PC12 Cells , Phosphorylation , Rats
18.
Mol Biol Cell ; 5(1): 57-70, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8186465

ABSTRACT

Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins.


Subject(s)
Cilia/chemistry , Cytoplasm/chemistry , Dyneins/genetics , Genes , Multigene Family , Phylogeny , Sea Urchins/genetics , Amino Acid Sequence , Animals , Consensus Sequence , Dictyostelium/genetics , Drosophila melanogaster/genetics , Embryo, Nonmammalian/chemistry , Embryo, Nonmammalian/ultrastructure , Gene Expression Regulation , Molecular Sequence Data , Sea Urchins/embryology , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
19.
Trends Cell Biol ; 3(11): 398-402, 1993 Nov.
Article in English | MEDLINE | ID: mdl-14731658

ABSTRACT

The translocation of dynein along microtubules is the basis for a wide variety of essential cellular movements. Dynein was first discovered in the ciliary axoneme, where it causes the directed sliding between outer doublet microtubules that underlies ciliary bending. The initiation and propagation of ciliary bends are produced by a precisely located array of different dyneins containing eight or more different dynein heavy chain isoforms. The detailed clarification of the structural and functional diversity of axonemal dynein heavy chains will not only provide the key to understanding how cilia function, but also give insights applicable to the study of non-axonemal microtubule motors.

20.
Cell Motil Cytoskeleton ; 24(1): 29-38, 1993.
Article in English | MEDLINE | ID: mdl-8319265

ABSTRACT

The ciliate Paramecium tetraurelia presents a powerful system to define the structural basis for dynein functional diversity within a single cell. This analysis will depend on the biochemical resolution of the dynein proteins. As an important first step, the three heavy chains of the ciliary outer arm dynein of paramecium were characterized. Sucrose density gradient centrifugation in a high salt buffer separated the dynein into a 22S species, which contained the alpha and beta heavy chains, and a 12S species, which contained the gamma chain as well as the inner arm dynein heavy chains. Both the 22S and 12S species retained enzymatic latency as indicated by stimulation of MgATPase activity by 0.1% Triton X-100. An unusual ATP-independent V1-like photolysis of only the beta chain provided the basis for estimating that the beta chain contributes almost half of the 22S MgATPase activity that is susceptible to V1 photolysis. The combination of the density gradient separation of the partially dissociated dynein and the ATP-independent V1-like photolysis of only the beta chain led to the unambiguous assignment of the V1 photolytic products to the appropriate parent heavy chains. An estimate of the molecular sizes of the three heavy chains was obtained. The photolytic peptide maps, which define the ATP-binding domains, were determined for the three heavy chains.


Subject(s)
Dyneins/chemistry , Paramecium tetraurelia/enzymology , Amino Acid Sequence , Animals , Blotting, Western , Cilia/enzymology , Dyneins/isolation & purification , Molecular Sequence Data , Peptide Mapping/methods , Photolysis
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