PubMed Health⌕ Search

Biomedical subjects

K Broadie

Publications and source records attributed to K Broadie.

36 records · Page 2Linked to original sources
PubMed1998

Forward and reverse genetic approaches to synaptogenesis.

Genetic approaches at the neuromuscular synapse are leading investigation into the mechanisms of synaptogenesis. The marriage of classical (forward) and reverse genetic techniques allows the isolation and analysis of novel proteins involved in synaptic maturation and the functional in vivo characterization of previously identified synaptic proteins. Of particular interest are recent advances using mouse reverse genetics and Drosophila forward genetics.

Animals↗
PubMed1997

Leonardo, a Drosophila 14-3-3 protein involved in learning, regulates presynaptic function.

The leonardo gene encodes a conserved member of the 14-3-3 protein family, which plays a role in Drosophila learning. Immunological localization of the protein shows that it is expressed at synaptic connections and enriched in presynaptic boutons of the neuromuscular junction (NMJ). Null leonardo mutants die as mature embryos. Electrophysiological assays of the mutant NMJ demonstrate that basal synaptic transmission is reduced by 30% and that transmission amplitude, fidelity, and fatigue resistance properties are reduced at elevated stimulation frequencies and in low external [Ca2+]. Moreover, transmission augmentation and post-tetanic potentiation (PTP) are disrupted in the mutant. These results suggest that Leonardo plays a role in the regulation of synaptic vesicle dynamics, a function which may underlie synaptic modulation properties enabling learning.

14-3-3 Proteins↗
PubMed1996

A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.

Septate and tight junctions are thought to seal neighboring cells together and to function as barriers between epithelial cells. We have characterized a novel member of the neurexin family, Neurexin IV (NRX), which is localized to septate junctions (SJs) of epithelial and glial cells. NRX is a transmembrane protein with a cytoplasmic domain homologous to glycophorin C, a protein required for anchoring protein 4.1 in the red blood cell. Absence of NRX results in mislocalization of Coracle, a Drosophila protein 4.1 homolog, at SJs and causes dorsal closure defects similar to those observed in coracle mutants. nrx mutant embryos are paralyzed, and electrophysiological studies indicate that the lack of NRX in glial-glial SJs causes a breakdown of the blood-brain barrier. Electron microscopy demonstrates that nrx mutants lack the ladder-like intercellular septa characteristic of pleated SJs (pSJs). These studies identify NRX as the first transmembrane protein of SJ and demonstrate a requirement for NRX in the formation of septate-junction septa and intercellular barriers.

Amino Acid Sequence↗
PubMed1996

Presynaptic development at the Drosophila neuromuscular junction: assembly and localization of presynaptic active zones.

We describe the extent to which presynaptic structures at the embryonic neuromuscular junction of Drosophila can form in mutants where development of postsynaptic somatic muscles is affected. Although twist mutant embryos lack mesoderm, motor axons still grow out of the CNS and form morphologically normal presynaptic active zones, independent of their target cells. In myoblast city mutant embryos, myoblasts do not fuse but form fully differentiated mononucleate muscles, which make functional neuromuscular synapses with correctly localized presynaptic active zones. Myoblasts also fail to fuse but still attract appropriate innervation in mef2 mutant embryos. However, these myoblasts fail to differentiate into muscles and presynaptic active zones fail to localize at neuromuscular contacts. Thus, the process of synapse formation can be genetically separated from the process of target recognition, revealing that localization of presynaptic active zones requires mef2-dependent muscle differentiation.

Animals↗
PubMed1996

The drosophila neuromuscular junction: a model system for studying synaptic development and function.

The Drosophila neuromuscular junction has attracted widespread attention as an excellent model system for studying the cellular and molecular mechanisms of synaptic development and neurotransmission. In Drosophila the advantages of invertebrate small systems, where individual cells can be examined with single-cell resolution, are combined with the powerful techniques of patch-clamp analysis and molecular genetics. In this review we examine myogenesis and motoneuron development, the problems of axon outgrowth and target selection, the differentiation of the synapse, and the mechanisms of both synaptic function and plasticity in this model genetic system.

Animals↗
PubMed1995

Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila.

Peripheral glia help ensure that motor and sensory axons are bathed in the appropriate ionic and biochemical environment. In Drosophila, peripheral glia help shield these axons against the high K+ concentration of the hemolymph, which would largely abolish their excitability. Here, we describe the molecular genetic analysis of gliotactin, a novel transmembrane protein that is transiently expressed on peripheral glia and that is required for the formation of the peripheral blood-nerve barrier. In gliotactin mutant embryos, the peripheral glia develop normally in many respects, except that ultrastructurally and physiologically they do not form a complete blood-nerve barrier. As a result, peripheral motor axons are exposed to the high K+ hemolymph, action potentials fail to propagate, and the embryos are nearly paralyzed.

Amino Acid Sequence↗
PubMed1995

Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission.

Cloning and characterization of the Drosophila syntaxin-1A gene, syx-1A, reveal that it is present in several tissues but is predominantly expressed in the nervous system, where it is localized to axons and synapses. We have generated an allelic series of loss-of-function mutations that result in embryonic lethality with associated morphological and secretory defects dependent on the severity of the mutant allele. Electrophysiological recordings from partial loss-of-function mutants indicate absence of endogenous synaptic transmission at the neuromuscular junction and an 80% reduction of evoked transmission. Complete absence of syx-1A causes subtle morphological defects in the peripheral and central nervous systems, affects nonneural secretory events, and entirely abolishes neurotransmitter release. These data demonstrate that syntaxin plays a key role in nonneuronal secretion and is absolutely required for evoked neurotransmission.

Alleles↗
PubMed1995

Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila.

In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.

Animals↗
PubMed1995

Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.

Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and the ensuing loss of neurotransmitter exocytosis has implicated synaptobrevin in this process. To further the study of synaptic function in a genetically tractable organism and to generate a tool to disable neuronal communication for behavioural studies, we have expressed a gene encoding tetanus toxin light chain in Drosophila. Toxin expression in embryonic neurons removes detectable synaptobrevin and eliminates evoked, but not spontaneous, synaptic vesicle release. No other developmental or morphological defects are detected. Correspondingly, only synaptobrevin (n-syb), but not the ubiquitously expressed syb protein, is cleaved by tetanus toxin in vitro. Targeted expression of toxin can produce specific behavioral defects; in one case, the olfactory escape response is reduced.

Amino Acid Sequence↗
PubMed1994

Absence of synaptotagmin disrupts excitation-secretion coupling during synaptic transmission.

Synaptotagmin is an integral synaptic vesicle protein proposed to be involved in Ca(2+)-dependent exocytosis during synaptic transmission. Null mutations in synaptotagmin have been made in Drosophila, and the protein's in vivo function has been assayed at the neuromuscular synapse. In the absence of synaptotagmin, synaptic transmission is dramatically impaired but is not abolished. In null mutants, evoked vesicle release is decreased by a factor of 10. Moreover, the fidelity of excitation-secretion coupling is impaired so that a given stimulus generates a more variable amount of secretion. However, this residual evoked release shows Ca(2+)-dependence similar to normal release, suggesting either that synaptotagmin is not the Ca2+ sensor or that a second, independent Ca2+ sensor exists. While evoked transmission is suppressed, the rate of spontaneous vesicle fusion is increased by a factor of 5. We conclude that synaptotagmin is not an absolutely essential component of the Ca(2+)-dependent secretion pathway in synaptic transmission but is necessary for normal levels of transmission. Our data support a model in which synaptotagmin functions as a negative regulator of spontaneous vesicle fusion and acts to increase the efficiency of excitation-secretion coupling during synaptic transmission.

Animals↗
PubMed1994

Mutations in the Drosophila Rop gene suggest a function in general secretion and synaptic transmission.

The Drosophila protein Rop shows similarity with the Sec1p protein of S. cerevisiae. Sec1p has an essential role in secretion, whereas most related proteins from higher organisms are hypothesized to function in neurotransmitter release. We show that, like the latter proteins, Rop is expressed in the nervous system, but it is expressed in other tissues as well, many of which are actively engaged in secretion. We have isolated mutations in the Rop gene and find that the extracellular accumulation of a number of normally secreted cellular products fails to occur in null mutant animals, which subsequently die at a late embryonic stage. Electrophysiological recordings on temperature-sensitive Rop mutants show that reductions in Rop activity result in a loss of the normal synaptic response to a light stimulus. These data suggest that a member of the Sec1p class of proteins has an in vivo function in both general secretion and synaptic transmission.

Amino Acid Sequence↗
PubMed1994

Sequential fates in a single cell are established by the neurogenic cascade in the Malpighian tubules of Drosophila.

In each Malpighian tubule of Drosophila, one cell is singled out, the tip cell, whose function during embryogenesis is to promote cell division in its neighbours. We follow the segregation of this cell, explore the genetic interactions that underlie its specification and demonstrate that tip cell allocation closely resembles neurogenesis. The tip cell arises by division of a tip mother cell, which is selected from a cluster of equivalent cells in each tubule primordium. Each cluster is marked out by the expression of proneural genes and the selection of a single cell from each group involves lateral inhibition, mediated by the neurogenic genes. We confirm the mitogenic role of the tip cell during embryogenesis by mutational analysis and show that it subsequently adopts a second fate, differentiating neural characteristics. We demonstrate that both stages in the differentiation of this cell are established by the same sequence of genetic interactions, which have not previously been shown to occur outside the neurogenic ectoderm.

Animals↗
PubMed1994

The cell adhesion molecule, connectin, and the development of the Drosophila neuromuscular system.

The connectin gene of Drosophila has been identified as a candidate direct target of homeotic gene control and has also been implicated in the formation of specific neuromuscular connections. The gene product, connectin, is a member of the leucine-rich repeat protein family and we show that it is attached to the cell surface via a glycosylphosphatidylinositol linkage and that it can mediate homotypic cell-cell adhesion in vitro. The expression of connectin protein during Drosophila embryogenesis provides support for a role in adhesion in vivo. In the central nervous system, it is initially expressed on longitudinal glia and on a few identified neurons. These cells extend processes and connect up to form a continuous scaffold of connectin-expressing cells, presaging the development of axonal pathways. Later, connectin is expressed on specific axons as they track along the connectin scaffold. Glial expression then declines and connectin appears on axons that fasciculate with pre-existing connectin-positive bundles. Thus scaffold formation, axon pathfinding and fasciculation involve specific contacts between connectin-positive cells. The timing and pattern of connectin expression suggest that it may play an important role in mediating specific interactions through homotypic cell adhesion.

Animals↗
PubMed1993

Innervation directs receptor synthesis and localization in Drosophila embryo synaptogenesis.

In the Drosophila embryo, motor neurons form stereotyped synapses (neuromuscular junctions) on identified muscles. We have used a mutant (prospero) that removes or delays innervation to assay the role of the presynaptic motor neuron in the development of the receptive field of the postsynaptic muscle. prospero (pros) is not expressed in the muscles or their precursors. Here we find that the muscle defines the correct synaptic zone in the absence of the motor neuron by restricting putative guidance molecules to this specialized membrane region. Furthermore, the muscle expresses functional transmitter receptors at the correct developmental time without innervation. On the other hand, the muscle does not localize receptors to the synapse without instruction from the motor neuron, nor does a second, much larger, synthesis of receptors occur in muscles deprived of innervation. In muscles receiving delayed innervation, or muscles innervated at aberrant synaptic sites, both receptor clustering and receptor synthesis are delayed or redirected, consistent with the new pattern of innervation. We conclude that the muscle autonomously defines the synaptic site, whereas the motor neuron directs the development of the muscle's receptive field by stimulating the synthesis and localization of transmitter receptors.

Animals↗
PubMed1993

Activity-dependent development of the neuromuscular synapse during Drosophila embryogenesis.

In Drosophila, mutations in specific ion channel genes can increase or decrease the level of neural/synaptic activity. We have used these genetic tools, in combination with classical pharmacological agents, to modulate neural activity during embryogenesis and examined effects on the differentiation of an identified neuromuscular junction. We find that electrical activity is required for the neural induction of transmitter receptor expression during synaptogenesis. Likewise, neural electrical activity is required to localize transmitter receptors to the synaptic site. In muscles with activity-blocked synapses, a low level of receptors is expressed homogeneously in the muscle membrane as in muscles developing without innervation. Thus, presynaptic electrical activity is required to mediate the neural induction of the transmitter receptor field in the postsynaptic membrane.

Animals↗
PubMed1993

Muscle development is independent of innervation during Drosophila embryogenesis.

We have examined the role of innervation in directing embryonic myogenesis, using a mutant (prospero), which delays the pioneering of peripheral motor nerves of the Drosophila embryo. In the absence of motor nerves, myoblasts fuse normally to form syncytial myotubes, myotubes form normal attachments to the epidermis, and a larval musculature comparable to the wild-type pattern is generated and maintained. Likewise, the twist-expressing myoblasts that prefigure the adult musculature segregate normally in the absence of motor nerves, migrate to their final embryonic positions and continue to express twist until the end of embryonic development. In the absence of motor nerves, myotubes uncouple at the correct developmental stage to form single cells. Subsequently, uninnervated myotubes develop the mature electrical and contractile properties of larval muscles with a time course indistinguishable from normally innervated myotubes. We conclude that innervation plays no role in the patterning, morphogenesis, maintenance or physiological development of the somatic muscles in the Drosophila embryo.

Animals↗
PubMed1993

From growth cone to synapse: the life history of the RP3 motor neuron.

In Drosophila, the ability to analyze the development of individually identified neurons with a variety of imaging and biophysical techniques can be complemented by sophisticated genetics and molecular biology. This powerful combination is allowing the development and function of single neurons and their synaptic connections to be unraveled at an unparalleled level of resolution. In this article, we focus on a single, identified motoneuron--RP3--arguably the best understood neuron in the fruitfly. Many events in the life history of RP3 are well characterized, including cell migration, axon outgrowth and pathfinding within the central nervous system, pathfinding in the periphery to its appropriate muscle target domain, the specific recognition of its muscle targets, the events of synapse formation and maturation, and its mature function in the locomotion of the fly larva. Genetic analysis has revealed mutations in a number of different genes which affect specific aspects of RP3 development from axon outgrowth to synapse formation.

Animals↗