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K M Johansen

Publications and source records attributed to K M Johansen.

29 records · Page 2Linked to original sources
PubMed1996

Dynamic remodeling of nuclear architecture during the cell cycle.

The nuclear matrix is an integral part of nuclear structure which undergoes a profound reorganization during the cell cycle reflecting major changes in functional requirements. This includes the processes of DNA replication and gene expression at interphase and partitioning of the nuclear contents during mitosis. Using a monoclonal antibody (mAb2A) which specifically stains a novel nuclear meshwork which reorganizes during the cell cycle in Drosophila, we have initiated a study to: 1) more closely analyze this structural reorganization; 2) clone and characterize the antigens recognized by this antibody; and 3) isolate other interacting proteins in order to gain insight into the regulation of this process. The mAb2A-labeled structure changes from what appears as a diffuse meshwork at interphase to a distinct spindle-like scaffold at prophase. Since at metaphase the microtubules of the mitotic apparatus co-localize with the mAb2A spindle structure, a model is considered whereby the nuclear mAb2A-labeled scaffolding reorganizes during the cell cycle to provide a guide for the establishment of the mitotic apparatus. The mAb2A has identified two separate antigens, each of which shows similar distribution patterns. One of these antigens has been partially cloned and contains an unusual tandem ser-thr kinase domain. The association of this kinase homologue with a nuclear scaffold which reorganizes during the cell cycle suggests that it may be involved in regulating changes in nuclear architecture during the cell cycle and/or in mediating the downstream consequences of such changes.

Animals↗
PubMed1996

Differential expression of the EF-hand calcium-binding protein calsensin in the central nervous system of hirudinid leeches.

By immunocytochemistry the distribution and developmental expression of the small EF-hand calcium-binding protein calsensin in the peripheral (PNS) and central nervous system (CNS) of the three hirudinid leech species Haemopis, Hirudo, and Macrobdella was compared. Labeling with calsensin-specific antibodies demonstrated that there was a pronounced difference in the distribution of calsensin immunoreactivity in the CNS of these leeches. In Haemopis more than 70 neurons were labeled, whereas the number in Hirudo was 51 and in Macrobdella only 8. Furthermore, the expression of calsensin in identified cells common to all three leech species also differed. Immunoblot analysis indicated that this variability was not likely to be due to multiple proteins or isoforms being recognized by the calsensin antibody. Labeling of embryos in various stages of development shows that the ontogeny of calsensin expression in the CNS is a gradual process with some neurons expressing calsensin immediately after completion of neurogenesis, about one-third of the way through embryogenesis, and others expressing calsensin only postembryonically. In contrast to the variability in the pattern and temporal expression by CNS neurons, the early embryonic calsensin expression in a small subgroup of sensillar PNS neurons was a shared feature by all three leech species. These findings suggest that calsensin may have different functional properties in CNS and PNS neurons.

Animals↗
PubMed1995

Filarin, a novel invertebrate intermediate filament protein present in axons and perikarya of developing and mature leech neurons.

The lan 3-8 monoclonal antibody recognizes a 63 kD antigen that is associated with the cytoskeleton in leech neurons. We have used this antibody to clone a novel invertebrate neuronal intermediate filament protein, filarin, by screening an expression vector library. A full-length clone of 2.2 kb identified by the antibody was isolated and sequenced. The protein contains a coiled-coil rod domain typical of the superfamily of intermediate filament proteins flanked by unique N- and C-terminal domains. The highest homology of filarin is to the alternatively spliced squid brain intermediate filament protein (Szaro et al., 1991, J. Biol. Chem. 266:15035-15041), the only other invertebrate neuronal intermediate filament in the data bank. However, apart from extensive homology in the two end regions of the rod domain, the similarity of the two proteins is limited to the general coiled-coil structure of intermediate filaments. Thus, filarin may represent a novel type of invertebrate neuronal intermediate filament protein. Filarin contains the extra 6 heptads characteristic of lamins and of all cytoplasmic invertebrate intermediate filaments analyzed so far. By Northern analysis, it appears that filarin is not alternatively spliced, since only a single transcript of 2.2 kb is recognized by the clone. Using the lan 3-8 antibody to follow its developmental expression, we found that filarin is present in all known neurons in the central and peripheral nervous system.

Amino Acid Sequence↗
PubMed1995

Peripheral neurons depend on CNS-derived guidance cues for proper navigation during leech development.

In leech, major nerve pathways are pioneered by CNS neurons and evidence from dye-injection and antibody experiments suggest that they serve as guides for later differentiating neurons. In this study we have directly tested this hypothesis by examining the consequences of CNS ablation on the navigation in the periphery of a well-defined population of afferent sensory neurons. We show that in the absence of CNS-derived axons the axonal growth cones of this population of peripheral neurons extend with little directionality and instead of forming orderly projections, default into forming circular fasciculated pathways with each other. This suggests that CNS-derived guidance cues are absolutely required for the correct navigation of these peripheral sensory neurons.

Animals↗
PubMed1995

Calsensin: a novel calcium-binding protein expressed in a subset of peripheral leech neurons fasciculating in a single axon tract.

The mAb lan3-6 recognizes a cytosolic antigen which is selectively expressed in the growth cones and axons of a small subset of peripheral sensory neurons fasciculating in a single tract common to all hirudinid leeches. We have used this antibody to clone a novel EF-hand calcium-binding protein, calsensin, by screening an expression vector library. A full-length clone of 1.1 kb identified by the antibody was isolated and sequenced. In situ hybridizations with calsensin probes and antibody staining using new polyclonal antisera generated against calsensin sequence demonstrate that calsensin indeed corresponds to the lan3-6 antigen. Calsensin consists of 83 residues with a calculated molecular mass of 9.1 kD that contains two helix-loop-helix domains. The calcium-binding domains are likely to be functional in vivo since a fusion protein derived from the calsensin clone binds 45Ca2+ in vitro. Immunoaffinity purification experiments with the lan3-6 antibody shows that a large 200,000 M(r) protein selectively copurifies with calsensin in two different leech species. These results suggest that calsensin may be functioning as a trigger protein which interacts with the larger protein. These data are consistent with the hypothesis that calsensin may mediate calcium-dependent signal transduction events in the growth cones and axons of this small group of sensory neurons which fasciculate in a single axon tract.

Amino Acid Sequence↗
PubMed1993

Selective pathway choice of a single central axonal fascicle by a subset of peripheral neurons during leech development.

In the CNS of leech, the central projections of peripheral sensory neurons segregate into three distinct axonal tracts during early development. We have previously shown that a subset of these neurons, recognized by the monoclonal antibody lan 4-2, projects axons into only one of these fascicles (Johansen et al., 1992, Neuron 8, 599). Here we report on a developmental and biochemical characterization of another fascicle-specific antigen labeled by the monoclonal antibody lan 3-6. By immunocytochemistry and double labelings we demonstrate that the lan 3-6 epitope is expressed only by a small subgroup of the peripheral neurons in Macrobdella embryos. The axons of these neurons selectively fasciculate in the CNS, but to only one of the three lan 3-2-positive tracts, which is different from the previously described lan 4-2-positive tract. These observations support the existence of a hierarchy of guidance cues mediating specific tract formation in this system. A biochemical analysis of the antigen suggests that it is likely to be a glycosylated protein with a molecular weight of approximately 200 kDa. Thus, the restricted expression of the lan 3-6 antigen and its biochemical properties are consistent with the hypothesis that this antigen may be playing a role in axonal guidance.

Animals↗
PubMed1992

Tract formation and axon fasciculation of molecularly distinct peripheral neuron subpopulations during leech embryogenesis.

In leech, the central projections of peripheral sensory neurons segregate into specific axonal tracts, which are distinguished by differential expression of surface antigens recognized by the monoclonal antibodies Lan3-2 and Lan4-2. Lan3-2 recognizes an epitope expressed on axons that segregate into three distinct axon fascicles. In contrast, the Lan4-2-positive axons selectively project into only one of the Lan3-2-positive axon tracts. These observations provide evidence for a hierarchy of guidance cues mediating specific pathway formation in this system. Since the Lan3-2 antibody has been shown to perturb this process and since, as shown here, the Lan3-2 and Lan4-2 antigens are closely molecularly interrelated, these antibodies may help define molecules and epitopes mediating neuronal recognition and axonal guidance.

Afferent Pathways↗
PubMed1989

The notch gene product is a glycoprotein expressed on the cell surface of both epidermal and neuronal precursor cells during Drosophila development.

The Notch locus of Drosophila melanogaster is one of a small number of zygotically acting "neurogenic" genes involved in the correct segregation of neural from epidermal lineages during embryogenesis as well as in other postembryonic developmental events. We have generated antibody probes against three regions of the Notch protein to study the expression of Notch and begin a biochemical characterization of the protein. Consistent with predictions based on DNA sequence data, here we gather evidence showing that Notch encodes a large, glycosylated surface protein with an apparent molecular mass of 300 kD: (a) all three antibodies detect Notch on Western blots as a high molecular mass, primarily full-length product; (b) immunoelectron microscopy localizes the Notch protein to the cell membrane; and (c) lentil lectin column binding demonstrates that the protein is glycosylated, indicative of its surface protein nature. In general, the distribution of the Notch protein coincides with that of the Notch transcript determined previously by in situ hybridizations. Notch is expressed in a much wider range of tissue types than those disrupted in the neurogenic mutant, as determined by antibody localization. Early labeling in the blastoderm appears ubiquitous except for the pole cells, but as development proceeds some distinctive features emerge: stronger staining is seen within the germ band layer where neuroblast delamination occurs, and the developing embryonic nervous system shows pronounced axonal staining. In third instar larvae, Notch is expressed in imaginal disks and in the central nervous system. Based on these results, certain models for how Notch controls the neuroblast cell fate choice are eliminated. We discuss how Notch may function in this choice as well as in other lineage fate determinations.

Animals↗
PubMed1989

Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae.

The distribution and morphology of glutamatergic synapses on Drosophila bodywall muscle fibers were examined at the single-synapse level using immunocytochemistry and electrophysiology. We find that glutamate-immunoreactive motor endings innervate the entire larval bodywall musculature, with each muscle fiber receiving at least one glutamatergic ending. The innervation is initiated at stereotyped locations on each muscle fiber from where moderately branched varicose nerve processes project over the internally facing muscle surface. Individual muscle fibers have distinct stereotypic patterns of nerve endings that occupy characteristic regions on the cell surface. The muscle-specific branching pattern of motor endings is reiterated by segmentally homologous fibers. Two morphological types of innervating nerve processes can be distinguished by their bouton size distributions: (1) Type I processes, which have localized branching and a broad size distribution of relatively large varicosities ranging up to 8 microns (mean diameter, 3.1 +/- 1.6 microns; +/- SD, n = 521), and (2) thinner Type II processes, which have a narrower distribution of small varicosities with a mean diameter of only 1.4 +/- 0.6 microns (+/- SD, n = 214). Immunoelectron microscopy with peroxidase-labeled second antibody demonstrates that the varicosities are surrounded by a subsynaptic reticulum, that they contain immunoreactive vesicles of about 30-50 nm, and thus probably represent synaptic release sites. By iontophoretic application of glutamate we mapped the responsive sites on the muscle surface and found an excellent correspondence between transmitter sensitivity and the patterns of endings as described by immunocytochemistry. In contrast to our finding of numerous glutamate iontophoresis-sensitive sites, we did not detect any aspartate-responsive muscles. These data provide strong new evidence for glutamate being an endogenous transmitter at the Drosophila larval neuromuscular junction.

Animals↗
PubMed1985

Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats.

The primary structure of the major embryonic Notch transcript is presented, as determined by sequence analysis of overlapping cDNA clones. The 10,148 bp sequence corresponding to this transcript possesses an 8109 bp open reading frame that potentially codes for a 2703 amino acid protein. We show that this polypeptide contains a repeated structure composed of 36 tandemly arranged 40 amino acid long repeats, which show homology to the epidermal growth factor and other proteins containing EGF-like repeats. Hydropathy plots suggest that the putative Notch protein may span the membrane. We relate these findings to the developmental action of Notch and speculate that the locus may be involved in a cell-cell interaction mechanism that is essential for the differentiation of the ectoderm into neural and epidermal precursors.

Amino Acid Sequence↗