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D W Cleveland

Publications and source records attributed to D W Cleveland.

At least 109 records · Page 6Linked to original sources
PubMed1991

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.

Anaphase↗
PubMed1991

Neuronal and glial cytoskeletons.

Long-awaited evidence for in vivo functions of the major neuronal microtubule associated proteins indicates that they are directly involved in neurite extension. Companion evidence reveals an intrinsic role for glial intermediate filaments in glial cell extension along neurites and for neurofilaments in establishing axonal caliber. New fluorescence and photoactivation experiments require a re-thinking of models of slow axonal transport and of the part the cytoskeleton plays in axonal guidance.

Animals↗
PubMed1991

Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds.

We describe the generation of 11 monoclonal antibodies that bind to the centromere/kinetochore region of human mitotic chromosomes. These antibodies were raised against mitotic chromosome scaffolds and screened for centromere/kinetochore binding by indirect immunofluorescence against purified chromosomes. Immunoblot analyses with these antibodies revealed that all of the antigens are greater than 200 kD and are components of nuclei, chromosomes, and/or chromosome scaffolds. Comparison of the immunolocalization of the antigens with that observed for the centromere-associated protein CENP-B revealed that each of these centromere/kinetochore proteins lies more peripherally to the DNA than does CENP-B. In cells normally progressing through the cell cycle, these antigens displayed four distinct patterns of centromere/kinetochore association, corresponding to a minimum of four novel centromere/kinetochore-associated proteins.

Animals↗
PubMed1991

Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein.

Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.

Amino Acid Sequence↗
PubMed1991

Involvement of neurofilaments in the radial growth of axons.

The control of radial growth of axons is of functional importance because caliber is a principal determinant of conduction velocity in myelinated nerve fibers. Neurofilaments, the major cytoskeletal protein in myelinated nerves, appear to be intrinsic determinants of caliber. Evidence supporting this derives first from the linear relationship between neurofilament content and axonal diameter. Further, following distal axonal injury in a peripheral nerve, caliber is reduced in the proximal axonal stumps. This reduction in caliber is itself due to selective suppression of neurofilament gene expression, thereby leading to lower levels of newly synthesized neurofilament subunits transported into the axon and a consequent decrease in axonal neurofilament content. To demonstrate directly the physiological consequence of altering normal neurofilament accumulation, we have elevated neurofilament expression by introducing additional genes into transgenic mice. The clear result is that increases in NF-L content alone are not sufficient to increase axonal caliber. To test the consequence of disruption of normal filament accumulation, we have identified dominant assembly-disrupting mutants in NF-L and NF-M and have used these to produce transgenic animals in which neurofilament assembly should be disrupted.

Animals↗
PubMed1991

Going public.

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National Institutes of Health (U.S.)↗
PubMed1990

Use of molecular cloning methods to map the distribution of epitopes on topoisomerase I (Scl-70) recognized by sera of scleroderma patients.

We report the initial molecular characterization of the autoimmune response against DNA topoisomerase I (topo I; Scl-70). Sera from 36 patients with scleroderma and 4 healthy control subjects were studied using 6 subcloned portions of topo I. Twenty-three sera recognized at least 2 independent epitopes on the molecule. Therefore, anti-topo I, like other non-organ-specific autoantibodies characterized to date, is polyclonal and multifocal. The cloned protein should prove suitable for sensitive early detection of anti-topo I in the clinical setting.

Autoantibodies↗
PubMed1990

The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis.

Five beta-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene c beta 4 and has been assigned to an isotypic family designated as Class III (beta III). In the nervous system of higher vertebrates, beta III is synthesized exclusively by neurons. A beta III-specific monoclonal antibody was used to determine when during chick embryogenesis c beta 4 is expressed, the cellular localization of beta III, and the number of charge variants (isoforms) into which beta III can be resolved by isoelectric focusing. On Western blots, beta III is first detectable at stages 12-13. Thereafter, the relative abundance of beta III in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of beta III isoforms increases from one to three during neural development. This evidence indicates that beta III is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while c beta 4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that c beta 4 expression occurs either during or immediately following terminal mitosis, and suggests that beta III may have a unique role during early neuronal differentiation and neurite outgrowth.

Animals↗
PubMed1990

Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neurofilament density in axons without affecting caliber.

We have generated transgenic mice containing additional copies of the murine NF-L gene in order to examine the consequences of neurofilament-L overexpression on axonal morphology. Founder mice were constructed to carry a transgene in which the presumptive 5' promoter sequences of NF-L were replaced with the strong murine sarcoma virus long terminal repeat promoter. The transgenes were expressed prominently in several tissues, including skeletal muscle and kidney where NF-L accumulated to approximately 2% of cell protein. This was not accompanied by an overt phenotype, except that expression in lens led to cataract formation. In the brains of these animals, transgene RNA levels exceeded the endogenous NF-L RNAs by up to 20-fold, although no additional protein accumulated, indicating posttranscriptional regulation of NF-L expression. However, in peripheral neurons transgene RNA was approximately fourfold higher than endogenous NF-L mRNAs, and a corresponding increase in NF-L subunits was found in axons arising from these neurons. Myelinated nerve fibers of transgenic animals contained increased numbers of NFs, assembled predominantly of NF-L. This was reflected in an increase in the density of axonal NFs; axonal caliber was not affected.

Animals↗
PubMed1990

Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M.

We have generated a set of amino- and carboxy-terminal deletions of the neurofilament NF-M gene and determined the molecular consequences of forced expression of these mutant constructs in mouse fibroblasts. To follow the expression of mutant NF-M subunits in transfected cells, a 12 amino acid epitope (from the human c-myc protein) was expressed at the carboxy terminus of each mutant. We show that NF-M molecules missing up to 90 or 70% of the nonhelical carboxy-terminal tail or amino-terminal head domains, respectively, incorporate readily into an intermediate filament network comprised either of vimentin or NF-L, whereas deletions into either the amino- or carboxy-terminal alpha-helical rod region generate assembly-incompetent polypeptides. Carboxy-terminal deletions into the rod domain invariably yield dominant mutants which rapidly disrupt the array of filaments comprised of NF-L or vimentin. Accumulation of these mutant NF-M subunits disrupts vimentin filament arrays even when present at approximately 1% the level of the wild-type subunits. In contrast, the amino-terminal deletions into the rod produce pseudo-recessive mutants that perturb the wild-type NF-L or vimentin arrays only modestly. The inability of such amino-terminal mutants to disrupt wild-type subunits defines a region near the amino-terminal alpha-helical rod domain (residues 75-126) that is required for the earliest steps in filament assembly.

Amino Acid Sequence↗
PubMed1990

Assembly properties of dominant and recessive mutations in the small mouse neurofilament (NF-L) subunit.

We have generated a set of amino- and carboxy-terminal deletions of the NF-L neurofilament gene and determined the assembly properties of the encoded subunits after coexpression with vimentin or wild-type NF-L. NF-L molecules missing greater than 30% (31 amino acids of the head) or 90% (128 amino acids of the tail) failed to incorporate into intermediate filament networks. Carboxy-terminal deletions into the rod domain yield dominant mutants that disrupt arrays assembled from wild-type subunits, even when present at levels of approximately 2% of the wild-type subunits. Even mutants retaining 55% of the tail (61 amino acids) disrupt normal arrays when accumulated above approximately 10% of wild-type subunits. Since deletion of greater than 90% of the head domain produces "recessive" assembly incompetent subunits that do not affect wild-type filament arrays, whereas smaller deletions yield efficient network disruption, we conclude that some sequence(s) in the head domain (within residues 31-87) are required for the earliest steps in filament assembly. Insertional mutagenesis in the nonhelical spacer region within the rod domain reveals that as many as eight additional amino acids can be tolerated without disrupting assembly competence.

Amino Acid Sequence↗
PubMed1990

A rapidly rearranging retrotransposon within the miniexon gene locus of Crithidia fasciculata.

The tandemly arrayed miniexon genes of the trypanosomatid Crithidia fasciculata are interrupted at specific sites by multiple copies of an inserted element. The element, termed Crithidia retrotransposable element 1 (CRE1), is flanked by 29-base-pair target site duplications and contains a long 3'-terminal poly(dA) stretch. A single 1,140-codon reading frame is similar in sequence to the integrase and reverse transcriptase regions of retroviral pol polyproteins. Cloned lines derived from a stock of C. fasciculata have unique arrangements of CRE1s. In different cloned lines, CRE1s, in association with miniexon genes, are located on multiple chromosomes. By examining the arrangement of CRE1s in subclones, we estimate that the element rearranges at a rate of ca. 1% per generation. These results indicate that the C. fasciculata miniexon locus is the target for a novel retrotransposon.

Amino Acid Sequence↗
PubMed1990

In vivo discrimination among beta-tubulin isotypes: selective degradation of a type IV beta-tubulin isotype following overexpression in cultured animal cells.

Preceding efforts have revealed that tubulin synthesis in animal cells is regulated both by selective expression of individual members of the tubulin multigene families and by a post-transcriptional control pathway that cotranslationally degrades tubulin mRNAs when the concentrations of unassembled subunits are increased. To test the effect of forced expression of a specific beta-tubulin, we constructed Chinese hamster ovary (CHO) cell lines that stably express the chicken class IV (c-IV) beta-tubulin gene. After gene amplification, we obtained lines that synthesize the c-IV polypeptide at a rate two to three times that of all endogenous beta-tubulins. Despite this elevated rate of synthesis, these c-IV polypeptides accumulated to only 4 to 10% of cellular beta-tubulin. Furthermore, when c-IV transcription was further elevated transiently, there was a compensatory loss in the endogenous class IV isotype (m-IV) so that the total level of class IV isotypes remained unchanged. The data indicate that beta-tubulin isotypes I and IV are biochemically distinguished in these cultured cell lines and that the stability of individual isotypes is established in part by isotype-specific interactions with other cellular factors.

Amino Acid Sequence↗