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K Zinn

Publications and source records attributed to K Zinn.

At least 37 records · Page 2Linked to original sources
PubMed1997

Functional expression of the heteromeric "olfactory" cyclic nucleotide-gated channel in the hippocampus: a potential effector of synaptic plasticity in brain neurons.

Cyclic nucleotide-gated (cng) channels are important components of signaling systems mediating sensory transduction. In vertebrate photoreceptors, light activates a signaling cascade that causes a decrease in intracellular cGMP concentrations, closing retinal cng channels. Signal transduction in olfactory receptor neurons is believed to proceed via G-protein-mediated elevation of intracellular cAMP in response to odorant binding by 7-helix receptors. cAMP opens the olfactory cng channel, which is highly permeable to Ca2+. Here we demonstrate by in situ hybridization and immunohistochemistry with subunit-specific antibodies that both subunits of the heteromeric rat olfactory cng channel are also widely expressed in the brain. Expression of the retinal rod cng channel, however, can be detected only in the eye. In the adult hippocampus, the olfactory cng channel is expressed on cell bodies and processes of CA1 and CA3 neurons. In cultured embryonic hippocampal neurons, the channel is localized to a subset of growth cones and processes. We recorded conductances with the electrophysiological characteristics of the heteromeric olfactory cng channel in excised inside-out patches from these cultured neurons. We also show that Ca2+ influx into hippocampal neurons in response to cyclic nucleotide elevation can be detected using fura-2 imaging. Cyclic nucleotide elevation has been implicated in several mechanisms of synaptic plasticity in the hippocampus, and these mechanisms also require elevation of intracellular Ca2+. Our results suggest that the "olfactory" cng channel could regulate synaptic efficacy in brain neurons by modulating Ca2+ levels in response to changes in cyclic nucleotide concentrations.

Animals↗
PubMed1997

Tyrosine phosphorylation and axon guidance: of mice and flies.

Recent genetic evidence suggests that tyrosine kinases and tyrosine phosphatases can control the guidance of specific growth cones. Within a family of related phosphatases or kinases, individual members can have partially redundant functions. Receptor phosphatases can work together at one guidance choice point, but in opposition at another. The specific combination of kinases and phosphatases active in a growth cone may be an important determinant of pathway choice. One mechanism by which these proteins could control guidance decisions is through regulation of adhesion between growth cones and axons.

Animals↗
PubMed1997

Transmembrane glycoprotein gp150 is a substrate for receptor tyrosine phosphatase DPTP10D in Drosophila cells.

We have begun to explore the downstream signaling pathways of receptor protein tyrosine phosphatases (RPTPs) that control axon guidance decisions in the Drosophila central nervous system. We have focused our studies on the adhesion molecule-like gp150 protein, which binds directly to and is an in vitro substrate for the RPTP DPTP10D. Here we show that gp150 and DPTP10D form stable complexes in Drosophila Schneider 2 (S2) cells and in wild-type larval tissue. We also demonstrate that the DPTP10D cytoplasmic domain is sufficient to confer binding to gp150. gp150 has a short cytoplasmic domain containing four tyrosines, all found within sequences similar to immunoreceptor family tyrosine-based activation motifs (ITAMs). We demonstrate that gp150 is tyrosine phosphorylated in wild-type larvae. In S2 cells, gp150 becomes tyrosine phosphorylated following incubation with PTP inhibitors or upon coexpression of the Dsrc tyrosine kinase. Phosphorylated Dsrc and an unknown 40-kDa phosphoprotein form stable complexes with gp150, thereby implicating them in a putative gp150 signaling pathway. When coexpressed with gp150, either full-length DPTP10D or its cytoplasmic domain mediates gp150 dephosphorylation whereas a catalytically inactive DPTP10D cytoplasmic domain does not. The neural RPTP DPTP99A can also induce gp150 dephosphorylation but does not coimmunoprecipitate with gp150. Taken together, the results suggest that gp150 transduces signals via phosphorylation of its ITAM-like elements. Phosphotyrosines on gp150 might function as binding sites for downstream signaling molecules, thereby initiating a signaling cascade that could be modulated in vivo by RPTPs such as DPTP10D.

Animals↗
PubMed1997

Competition and cooperation among receptor tyrosine phosphatases control motoneuron growth cone guidance in Drosophila.

The neural receptor tyrosine phosphatases DPTP69D, DPTP99A and DLAR are involved in motor axon guidance in the Drosophila embryo. Here we analyze the requirements for these three phosphatases in growth cone guidance decisions along the ISN and SNb motor pathways. Any one of the three suffices for the progression of ISN pioneer growth cones beyond their first intermediate target in the dorsal muscle field. DLAR or DPTP69D can facilitate outgrowth beyond a second intermediate target, and DLAR is uniquely required for formation of a normal terminal arbor. A different pattern of partial redundancy among the three phosphatases is observed for the SNb pathway. Any one of the three suffices to allow SNb axons to leave the common ISN pathway at the exit junction. When DLAR is not expressed, however, SNb axons sometimes bypass their ventrolateral muscle targets after leaving the common pathway, instead growing out as a separate bundle adjacent to the ISN. This abnormal guidance decision can be completely suppressed by also removing DPTP99A, suggesting that DLAR turns off or counteracts a DPTP99A signal that favors the bypass axon trajectory. Our results show that the relationships among the tyrosine phosphatases are complex and dependent on cellular context. At growth cone choice points along one nerve, two phosphatases cooperate, while along another nerve these same phosphatases can act in opposition to one another.

Animals↗
PubMed1996

Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo.

The receptor tyrosine phosphatases DPTP69D and DPTP99A are expressed on motor axons in Drosophila embryos. In mutant embryos lacking DPTP69D protein, motor neuron growth cones stop growing before reaching their muscle targets, or follow incorrect pathways that bypass these muscles. Mutant embryos lacking DPTP99A are indistinguishable from wild type. Motor axon defects in dptp69D dptp99A double mutant embryos, however, are much more severe than in embryos lacking only DPTP69D. Our results demonstrate that DPTP69D and DPTP99A are required for motor axon guidance and that they have partially redundant functions during development of the neuro-muscular system.

Animals↗
PubMed1995

Activation of cAMP-dependent protein kinase triggers a glial-to-neuronal cell-fate switch in an insect neuroblast lineage.

BACKGROUND: The grasshopper median neuroblast (MNB) is a multipotent progenitor cell that produces neurons and midline glia in distinct temporal phases. The MNB generates pioneer neurons during its first few divisions, and then switches to production of midline glial precursors. After the glia have been produced, the MNB reverts to generating neurons. We have investigated the molecular mechanism underlying the transition from glia production back to neuron production in the MNB lineage. RESULTS: We report evidence that this second transition in the MNB lineage is triggered by the activation of cAMP-dependent protein kinase (PKA). PKA is a heterodimer of a catalytic (PKA-C) and a cAMP-binding regulatory (R) subunit. The R subunit dissociates from PKA-C on binding cAMP, and free PKA-C than translocates into the nucleus. Nuclear localization of PKA-C can thus be used as an indicator of PKA activation within a cell. We have found that PKA-C is translocated into the nucleus at the time of the second switch in the MNB lineage. When PKA is prematurely activated in the MNB by microinjection of purified PKA-C, or by pharmacological agents that elevate intracellular cAMP levels, the glial-to-neuronal cell-fate switch takes place prematurely. Inhibition of PKA activity by microinjection of a peptide inhibitor, or by a non-hydrolyzable cAMP analog, blocks the glial-to-neuronal switch. CONCLUSIONS: Our results imply that elevation of cAMP in the MNB, and the resultant activation of PKA, is likely to be a trigger for the glial-to-neuronal cell-fate transition within the MNB lineage.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Tenascin-C mRNA is expressed in cranial neural crest cells, in some placodal derivatives, and in discrete domains of the embryonic zebrafish brain.

A partial zebrafish tenascin-C cDNA clone was isolated from an embryonic zebrafish cDNA library on the basis of homology to mouse tenascin-C. The expression pattern in the head of embryonic zebrafish was analyzed by in situ hybridization. Tenascin-C mRNA was detected in neural crest cells during the period of their migration and differentiation. Expression also occurred in differentiating placodal tissues and in mesodermal cells. In the developing brain, tenascin-C mRNA was expressed in specific domains. In the hindbrain the pattern of the domains was dynamic. At 18 to 22 h postfertilization, expression was widespread in rhombomeres 3, 5, and 6, confined to periventricular cells in rhombomere 2, and not detectable in rhombomere 4. At 32 h postfertilization, tenascin-C was expressed at the rhombomere boundaries. In contrast to the hindbrain, the pattern in the forebrain and midbrain did not show any major changes between 22 and 32 h postfertilization. Domains expressing tenascin-C alternated with regions devoid of it. The most anterior domain of expression was observed at the telencephalic-diencephalic border, surrounding the optic recess. A second domain, at the border between the diencephalon and the midbrain, and a third domain, in the caudal midbrain tegmentum, appeared restricted to the basal plate. Additionally, expression of tenascin-C mRNA was detected in the hypothalamus and in the developing epiphysis. These expression patterns suggest that tenascin-C may play a role in neural crest cell migration and during the differentiation of neural crest, placodal, and mesodermal derivatives. In the developing brain, tenascin-C may be involved in the consolidation of different regional identities.

Amino Acid Sequence↗
PubMed1995

Use of 82Br- radiotracer to study transmembrane halide flux: the effect of a tranquilizing drug, chlordiazepoxide on channel opening of a GABAA receptor.

We used the short-lived radionuclide, 82Br- to follow gamma-aminobutyrate (GABA) receptor-mediated halide exchange into membrane vesicles from rat cerebral cortex in millisecond and second time regions using quench-flow technique. The radioisotope was prepared by neutron capture [81Br-(n,gamma)82Br-] on irradiation of a natural isotope of bromine, 81Br- in a neutron flux. 82Br- decays by beta-emission with secondary gamma-emission. Possible advantages of 82Br- over 36Cl- in anion tracer measurements include, (a) a short lifetime (t1/2 = 35.3 hr), which alleviates contamination and disposal problems, (b) high counting efficiency (1.54) due to the secondary radiation, (c) measurement with a gamma-counter as well as a beta-counter, (d) a simple preparation not requiring subsequent purification steps giving a specific activity depending on the irradiation time. With 6 hr irradiation time the specific activity was sufficient to make measurements with < 1 mM Br-, which is less than the bromide concentration known to affect the properties of GABAA receptor. The radiotracers, 82Br- and 36Cl- could be compared with the same solution composition. In conditions where a direct effect of binding of halide to receptor does not contribute to a difference in measured ion-flux, 82Br- was translocated only marginally faster than 36Cl-. The effect of chlordiazepoxide (CDPX) (2-250 microM) on the progress of GABA (10 microM)-mediated 82Br- uptake was measured in a time range of 200 msec to 20 sec using quench-flow technique. The two phases of anion exchange previously reported in this experimental model with GABA alone were observed. The rate of 82Br- exchange was increased 2.3-fold at 30-60 microM CDPX and was not further increased with increasing [CDPX]. The rate of halide exchange is a measure of open channel concentration. The isotope exchange rate constant, J, in a membrane vesicle preparation, is a measure of the membrane permeability per internal volume/surface area, J = PmA/V. Receptor desensitization rate was also increased by CDPX, but unlike the isotope exchange rate, it continued to increase up to at least 250 microM CDPX.

Animals↗
PubMed1994

An adhesion molecule-like protein that interacts with and is a substrate for a Drosophila receptor-linked protein tyrosine phosphatase.

Receptor-linked protein tyrosine phosphatases (R-PTPs) are a large and diverse group of transmembrane signaling molecules. In Drosophila, four R-PTPs are localized to central nervous system axons in the embryo and may participate in assembly of the central nervous system axon array. In this paper, we identify and characterize a transmembrane glycoprotein, gp150, that selectively interacts with the catalytic domain of the axonal R-PTP DPTP10D. gp150 does not bind to a cysteine-to-serine active site mutant, and binding is inhibited by vanadate, suggesting that it interacts with the active site. It has an extracellular domain composed of 18 leucine-rich repeats, which are found in many adhesion molecules. Its short cytoplasmic domain contains 4 tyrosine residues in sequence contexts that suggest that they could interact with SH2 domain-containing effector molecules. The overall organization of the tyrosine motifs resembles that of antigen recognition activation motif signaling elements from receptors in the vertebrate immune system. The cytoplasmic domain of gp150 is a good substrate for v-Abl tyrosine kinase, and phosphorylated gp150 can be dephosphorylated efficiently in vitro by DPTP10D. We suggest that DPTP10D may function in vivo to regulate phosphorylation of gp150 and thereby control its interactions with downstream effectors.

Amino Acid Sequence↗
PubMed1994

Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP.

Olfactory receptor neurons respond to odorant stimulation with a rapid increase in intracellular cAMP that opens cyclic nucleotide-gated (cng) cation channels. cng channels in rat olfactory neurons are activated by cAMP in the low micromolar range and are outwardly rectifying. The cloned rat olfactory cng channel (rOCNC1), however, is much less sensitive to cAMP and exhibits very weak rectification. Here we describe the cloning and characterization of a second rat cng channel subunit, denoted rOCNC2. rOCNC2 does not form functional channels when expressed alone. When rOCNC1 and rOCNC2 are coexpressed, however, an outwardly rectifying cation conductance with cAMP sensitivity near that of the native channel is observed. In situ hybridization with probes specific for the two subunits shows that they are coexpressed in olfactory receptor neurons. These data indicate that the native olfactory cng channel is likely to be a heterooligomer of the rOCNC1 and rOCNC2 subunits.

Amino Acid Sequence↗
PubMed1994

Pair-rule expression patterns of even-skipped are found in both short- and long-germ beetles.

Now that the genes controlling embryonic patterning have been identified in several model organisms, long-standing questions concerning the evolution of developmental systems are open to investigation. Examination of the expression of even-skipped in a variety of insects reveals that Insect germ-type designations apparently do not reflect the variations in the mechanisms of segmentation evident throughout insect phylogeny.

Animals↗
PubMed1994

Engrailed controls glial/neuronal cell fate decisions at the midline of the central nervous system.

The molecular mechanisms involved in glial/neuronal fate decisions during embryonic development are largely unknown. Here we show that the segment-polarity gene engrailed, which encodes a homeodomain protein, controls these decisions within an insect CNS lineage. The grasshopper median neuroblast (MNB) generates both neurons and midline glia in distinct temporal phases. engrailed expression in MNB progeny can be inhibited by injection of antisense oligodeoxynucleotides into the MNB nucleus. This produces a phenotype in which the midline glia do not develop and extra midline neurons are generated. In the absence of engrailed function, midline glial precursors are apparently converted into neuronal precursors. Thus, engrailed is required for execution of the decision between the glial and neuronal fates.

Animals↗
PubMed1994

Cell fate decisions in the grasshopper central nervous system.

The central nervous system (CNS) of grasshopper embryos is similar in organization to the embryonic Drosophila CNS, but its neurons are much larger. The recent development of a culture system in which extensive CNS development occurs has allowed new types of experiments to be performed, including perturbation of gene expression within single neuroblast lineages.

Animals↗
PubMed1994

A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the "HRP" carbohydrate epitope.

Recent studies have defined several cell surface glycoproteins expressed in the developing nervous system of insect embryos that may be involved in axon outgrowth and guidance processes. These glycoproteins include the fasciclins and a group of receptor-linked protein tyrosine phosphatases (R-PTPs). In embryos, the fasciclins are localized to axonal subsets, while the R-PTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, we have taken a biochemical approach. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted "HRP proteins") apparently bear the HRP carbohydrate epitope. We have used polyclonal anti-HRP antibodies to purify these proteins from Drosophila embryos, and have obtained protein sequences from seven HRP protein bands. These data define three major HRP proteins as neurotactin, fasciclin I, and an R-PTP, DPTP69D. Western blotting data suggest that fasciclin II, neuroglian, DPTP10D, and DPTP99A are also HRP proteins. We show that DPTP69D, like the previously characterized R-PTPs, is localized to CNS axons in the embryo. In third instar larvae, DPTP69D expression is restricted to subsets of neuronal processes in the brain, ventral nerve cord, and eye disk. In the optic lobes, DPTP69D is localized to the neuropils of the lamina and medulla, and to an array of parallel thick bundles that may be the transmedullary fibers of the developing lobula complex.

Amino Acid Sequence↗
PubMed1994

The grasshopper median neuroblast is a multipotent progenitor cell that generates glia and neurons in distinct temporal phases.

The midline of the insect CNS provides a good system for studying the control of neuronal and glial cell fates, because it contains a small number of distinct cell types that arise from a unique set of precursors. In this report we analyze the development of the CNS midline in the grasshopper embryo, focusing on the median neuroblast (MNB) cluster, which contains the majority of midline neurons. We have directly traced cell lineage in the MNB cluster by injecting the MNB with tracer dyes and following the development of its progeny in whole embryo cultures. As least three types of neuronal progeny are labeled with tracer dye in these experiments. In addition, we find that all of the midline glia are also derived from the MNB. Thus, this neuroblast is actually a multipotent progenitor cell. We also examined the temporal and spatial patterns of midline development by staining embryos with antibodies to neuronal and glial markers and to the protein product of the engrailed gene, which is transiently expressed by all MNB progeny. Our data show that neuronal and glial progeny are generated from the MNB in distinct temporal phases. A change in the orientation of the MNB's mitotic spindle correlates with the transition between two of the phases of progeny production.

Animals↗
PubMed1993

Expression and structural studies of fasciclin I, an insect cell adhesion molecule.

Fasciclin I is a lipid-linked cell-surface glycoprotein that can act as a homophilic adhesion molecule in tissue culture cells. It is thought to be involved in growth cone guidance in the embryonic insect nervous system. To facilitate structure-function studies, we have generated Chinese hamster ovary (CHO) cell lines expressing high levels of cell surface grasshopper and Drosophila fasciclin I. Grasshopper fasciclin I released by phospholipase C cleavage was purified on an immunoaffinity column and single crystals were obtained that diffracted to approximately 5-A resolution. We also generated CHO and Drosophila S2 cell lines that produce a secreted form of fasciclin I. Fasciclin I expressed in S2 cells contains significantly less carbohydrate than the protein expressed in CHO cells, and may therefore be more suitable for crystallization. Biochemical characterization of purified fasciclin I indicates that the extracellular portion exists as a monomer in solution. Circular dichroism studies suggest that fasciclin I is primarily alpha-helical. Its structure is therefore different from other known cell adhesion molecules, which are predicted to be elongated beta-sheet structures. This suggests that fasciclin I may define a new structural motif used to mediate adhesive interactions between cell surfaces.

Amino Acid Sequence↗