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L Y Jan

Publications and source records attributed to L Y Jan.

At least 91 records · Page 5Linked to original sources
PubMed1997

Cloned potassium channels from eukaryotes and prokaryotes.

Potassium channels contribute to the excitability of neurons and signaling in the nervous system. They arise from multiple gene families including one for voltage-gated potassium channels and one for inwardly rectifying potassium channels. Features of potassium permeation, channel gating and regulation, and subunit interaction have been analyzed. Potassium channels of similar design have been found in animals ranging from jellyfish to humans, as well as in plants, yeast, and bacteria. Structural similarities are evident for the pore-forming alpha subunits and for the beta subunits, which could potentially regulate channel activity according to the level of energy and/or reducing power of the cell.

Animals↗
PubMed1997

Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis.

During Drosophila neurogenesis, asymmetric cell divisions are achieved by differential segregation of Numb (d-Numb) into one of the daughter cells to cause a bias in the Notch mediated cell-cell interaction. We have isolated a second mammalian gene with significant sequence similarity to d-numb, mouse numblike. When expressed in dividing neural precursors in Drosophila, Numblike is symmetrically distributed in the cytoplasm, unlike endogenous d-Numb or expressed mouse Numb (m-Numb), both of which are asymmetrically localized to one half of the cell membrane. In d-numb loss-of-function mutant embryos, expression of Numblike allows both daughter cells of a neural precursor to adopt the fate of the cell that normally inherits d-Numb. In mice, numblike mRNA is preferentially expressed in adult and embryonic nervous system. In the developing neocortex, Numblike is expressed in postmitotic neurons in the cortical plate, but not in progenitors within the ventricular zone where m-Numb and Notch1 are expressed. We have also found that, in dividing cortical progenitors, Notch1 is distributed around the entire membrane, unlike m-Numb which is asymmetrically localized to the apical membrane. We propose that an interplay between cell-intrinsic mechanisms (executed by m-numb and numblike) and cell-extrinsic mechanisms (mediated by Notch1) may be involved in both progenitor cell proliferation and neuronal differentiation during mammalian cortical neurogenesis.

Amino Acid Sequence↗
PubMed1997

The Drosophila neurogenic gene big brain, which encodes a membrane-associated protein, acts cell autonomously and can act synergistically with Notch and Delta.

In the developing nervous system of Drosophila, cells in each proneural cluster choose between neural and epidermal cell fates. The neurogenic genes mediate the cell-cell communication process whereby one cell adopts the neural cell fate and prevents other cells in the cluster from becoming neural. In the absence of neurogenic gene function, most, if not all of the cells become neural. big brain is a neurogenic gene that encodes a protein with sequence similarity to known channel proteins. It is unique among the neurogenic genes in that previous genetic studies have not revealed any interaction between big brain and the other neurogenic genes. Furthermore, the neural hypertrophy in big brain mutant embryos is less severe than that in embryos mutant for other neurogenic genes. In this paper, we show by antibody staining that bib is expressed in tissues that give rise to neural precursors and in other tissues that are affected by loss of neurogenic gene function. By immunoelectron microscopy, we found that bib is associated with the plasma membrane and concentrated in apical adherens junctions as well as in small cytoplasmic vesicles. Using mosaic analysis in the adult, we demonstrate that big brain activity is required autonomously in epidermal precursors to prevent neural development. Finally, we demonstrate that ectopically expressed big brain acts synergistically with ectopically expressed Delta and Notch, providing the first evidence that big brain may function by augmenting the activity of the Delta-Notch pathway. These results are consistent with bib acting as a channel protein in proneural cluster cells that adopt the epidermal cell fate, and serving a necessary function in the response of these cells to the lateral inhibition signal.

Amino Acid Sequence↗
PubMed1997

Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle.

Polarization of the microtubule cytoskeleton is an early event in establishment of anterior-posterior polarity for the Drosophila oocyte. During stages 8-9 of oogenesis, when oskar mRNA is transported to the posterior pole of the oocyte, a fusion protein consisting of the plus-end-directed microtubule motor kinesin and beta-galactosidase (Kin:beta gal) similarly localizes to the posterior pole, thereby suggesting that plus ends of microtubules are pointed to the posterior. In this paper, we have substituted the motor domain of Kin:beta gal with the putative motor domain (head) from the kinesin-related protein Nod. In cells with defined microtubule polarity, the Nod:beta gal fusion protein is an in vivo minus-end reporter for microtubules. Nod:beta gal localizes to apical cytoplasm in epithelial cells and to the poles of mitotic spindles in dividing cells. In stage 8-10 oocytes, the Nod fusion localizes to the anterior margin, thus supporting the hypothesis that minus ends of microtubules at these stages are primarily at the anterior margin of the oocyte. The fusion protein also suggests a polarity to the microtubule cytoskeleton of dendrites and muscle fibers, as it accumulates at the ends of dendrites in the embryonic PNS and is excluded from terminal cytoplasm in embryonic muscle. Finally, the reciprocal in vivo localization of Nod:beta gal and Kin:beta gal suggests that the head of Nod may be a minus-end-directed motor.

Animals↗
PubMed1997

Only a subset of the binary cell fate decisions mediated by Numb/Notch signaling in Drosophila sensory organ lineage requires Suppressor of Hairless.

In Drosophila, an adult external sensory organ (bristle) consists of four distinct cells which arise from a sensory organ precursor cell via two rounds of asymmetric divisions. The sensory organ precursor cell first divides to generate two secondary precursor cells, IIa and IIb. The IIa cell then divides to produce the hair cell and the socket cell. Shortly after, the IIb cell divides to generate the neuron and the sheath cell. The membrane-associated protein Numb has been shown to be required for the first two asymmetric divisions. We now report that a new hypomorphic numb mutant not only displays a double-socket phenotype, due to a hair cell to socket cell transformation, but also a double-sheath phenotype, due to a neuron to sheath cell transformation. This provides direct evidence that numb functions in the neuron/sheath cell lineage as well. Those results, together with our observation from immunofluorescence analysis that Numb forms a crescent in the dividing IIa and IIb cells suggest that asymmetric localization of Numb is important for the cell fate determination in all three asymmetric cell divisions in the sensory organ lineage. Interestingly, we found that in the hair/socket cell lineage but not the neuron/sheath cell lineage, a Suppressor of Hairless mutation acts as a dominant suppressor of numb mutations whereas Hairless mutations act as enhancers of numb. Moreover, epistasis analysis indicates that Suppressor of Hairless acts downstream of numb, and results from in vitro binding analysis suggest that the genetic interaction between numb and Hairless may occur through direct protein-protein interaction. These studies reveal that Suppressor of Hairless is required for only a subset of the asymmetric divisions that depend on the function of numb and Notch.

Animals↗
PubMed1996

Regions responsible for the assembly of inwardly rectifying potassium channels.

Inwardly rectifying potassium channels have an important role in determining the resting potential of the cell. They are tetrameric proteins with two transmembrane segments (M1 and M2), a pore-forming loop (H5), a cytoplasmic N-terminal, and longer C-terminal domain. We have used biochemical and electrophysiological methods to identify regions required for homotypic interactions and those responsible for the incompatibility between IRK1 and two other members of the same subfamily (IRK2 and IRK3) and two members from other subfamilies (ROMK1 and 6.1 uK(ATP)). The data indicate that, in contrast to the voltage-gated class of potassium channel, the proximal C-terminus and the transmembrane segment M2 determine homo-and heteromultimerization and that heteromultimerization between members of the same or different subfamilies is case specific.

Animals↗
PubMed1996

Heteromultimerization of G-protein-gated inwardly rectifying K+ channel proteins GIRK1 and GIRK2 and their altered expression in weaver brain.

The weaver (wv) gene (GIRK2) is a member of the G-protein-gated inwardly rectifying potassium (GIRK) channel family, known effectors in the signal transduction pathway of neurotransmitters such as acetylcholine, dopamine, opioid peptides, and substance P in modulation of neurotransmitter release and neuronal excitability. GIRK2 immunoreactivity is found in but not limited to brain regions known to be affected in wv mice, such as the cerebellar granule cells and dopaminergic neurons in the substantia nigra pars compacta. It is also observed in the ventral tegmental area, hippocampus, cerebral cortex, and thalamus. GIRK2 and GIRK1, a related family member, have overlapping yet distinct distributions in rat and mouse brains. In regions where both channel proteins are expressed, such as the cerebral cortex, hippocampus, and cerebellum, they can be co-immunoprecipitated, indicating that they interact to form heteromeric channels in vivo. In the brain of the wv mouse, GIRK2 expression is decreased dramatically. In regions where GIRK1 and GIRK2 distributions overlap, both GIRK1 and GIRK2 expressions are severely disrupted, probably because of their co-assembly. The expression patterns of these GIRK channel subunits provide a basis for consideration of the machinery for neuronal signaling as well as the differential effects of the wv mutation in various neurons.

Animals↗
PubMed1996

Neuronal type information encoded in the basic-helix-loop-helix domain of proneural genes.

The proneural genes encode basic-helix-loop-helix (bHLH) proteins and promote the formation of distinct types of sensory organs. In Drosophila, two sets of proneural genes, atonal (ato) and members of the achaete-scute complex (ASC), are required for the formation of chordotonal (ch) organs and external sensory (es) organs, respectively. We assayed the production of sensory organs in transgenic flies expressing chimeric genes of ato and scute (sc), a member of ASC, and found that the information that specifies ch organs resides in the bHLH domain of ato; chimeras containing the b domain of ato and the HLH domain of sc also induced ch organ formation, but to a lesser extent than those containing the bHLH domain of ato. The b domains of ato and sc differ in seven residues. Mutations of these seven residues in the b domain of ato suggest that most or perhaps all of these residues are required for induction of ch organs. None of these seven residues is predicted to contact DNA directly by computer simulation using the structure of the myogenic factor MyoD as a model, implying that interaction of ato with other cofactors is likely to be involved in neuronal type specification.

Amino Acid Sequence↗
PubMed1996

frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance.

We have identified a Drosophila member of the deleted in colorectal cancer (DCC) gene family. The frazzled gene encodes transmembrane proteins that contain four immunoglobulin C2 type domains, six fibronectin type III repeats, and a cytoplasmic domain of 278 amino acids. Like vertebrate members of the DCC family, Frazzled is expressed on axons in the embryonic central nervous system and on motor axons in the periphery. Frazzled is also expressed on epidermis and gut epithelium. Null mutants in frazzled are defective in axon guidance in the central nervous system and in motor axon guidance and targeting in the periphery. The phenotypes strongly resemble those of a deletion of the two Drosophila Netrin genes. We have rescued the frazzled CNS and motor axon defects by expressing Frazzled specifically in neurons; expression in target tissues does not rescue the phenotype. These data, together with vertebrate studies showing binding of DCC to netrin, suggest that Frazzled may function in vivo as a receptor or component of a receptor mediating Netrin-dependent axon guidance.

Animals↗
PubMed1996

The Drosophila Numb protein inhibits signaling of the Notch receptor during cell-cell interaction in sensory organ lineage.

Specification of unequal daughter cell fates in the Drosophila external sense organ lineage requires asymmetric localization of the intrinsic determinant Numb as well as cell-cell interactions mediated by the Delta ligand and Notch receptor. Previous genetic studies indicated that numb acts upstream of Notch, and biochemical studies revealed that Numb can bind Notch. For a functional assay of the action of Numb on Notch signaling, we expressed these proteins in cultured Drosophila cells and used nuclear translocation of Suppressor of Hairless [Su(H)] as a reporter for Notch activity. We found that Numb interfered with the ability of Notch to cause nuclear translocation of Su(H); both the C-terminal half of the phosphotyrosine binding domain and the C terminus of Numb are required to inhibit Notch. Overexpression of Numb during wing development, which is sensitive to Notch dosage, revealed that Numb is also able to inhibit the Notch receptor in vivo. In the external sense organ lineage, the phosphotyrosine binding domain of Numb was found to be essential for the function but not for asymmetric localization of Numb. Our results suggest that Numb determines daughter cell fates in the external sense organ lineage by inhibiting Notch signaling.

Amino Acid Sequence↗
PubMed1996

Role of inscuteable in orienting asymmetric cell divisions in Drosophila.

Drosophila neuroblasts and epithelial cells in the procephalic neurogenic region divide perpendicular to the surface, and segregate the proteins Numb and Prospero into the basal daughter cell. We demonstrate here that orientation of the mitotic spindle and correct localization of Numb and Prospero in these cells require the inscuteable gene. Moreover, ectopic expression of inscuteable in other epithelial cells leads to spindle reorientation. The Inscuteable protein localizes to the apical cell cortex before mitosis, suggesting that Inscuteable functions in establishing polarity for asymmetric cell division.

Actins↗
PubMed1996

Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH.

Potassium (K+) homeostasis is controlled by the secretion of K+ ions across the apical membrane of renal collecting duct cells through a low-conductance inwardly rectifying K+ channel. The sensitivity of this channel to intracellular pH is particularly high and assumed to play a key role in K+ homeostasis. Recently, the apical K+ channel has been cloned (ROMK1,2,3 = Kir1.1a, Kir1.1b and Kir1.1c) and the pH dependence of ROMK1 was shown to resemble closely that of the native apical K+ channel. It is reported here that the steep pH dependence of ROMK channels is determined by a single amino acid residue located in the N-terminus close to the first hydrophobic segment M1. Changing lysine (K) at position 80 to methionine (M) removed the sensitivity of ROMK1 channels to intracellular pH. In pH-insensitive IRK1 channels, the reverse mutation (M84K) introduced dependence on intracellular pH similar to that of ROMK1 wild-type. A detailed mutation analysis suggests that a shift in the apparent pKalpha of K80 underlies the pH regulation of ROMK1 channels in the physiological pH range.

Amino Acid Sequence↗
PubMed1996

Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines.

Neurons contain distinct compartments including dendrites, dendritic spines, axons and synaptic terminals. The molecular mechanisms that generate and distinguish these compartments, although largely unknown, may involve the small GTPases Rac and Cdc42, which appear to regulate actin polymerization. Having shown that perturbations of Rac1 activity block the growth of axons but not dendrites of Drosophila neurons, we investigated whether this also applies to mammals by examining transgenic mice expressing constitutively active human Rac1 in Purkinje cells. We found that these mice were ataxic and had a reduction of Purkinje-cell axon terminals in the deep cerebellar nuclei, whereas the dendritic trees grew to normal height and branched extensively. Unexpectedly, the dendritic spines of Purkinje cells in developing and mature cerebella were much reduced in size but increased in number. These 'mini' spines often form supernumerary synapses. These differential effects of perturbing Rac1 activity indicate that there may be distinct mechanisms for the elaboration of axons, dendrites and dendritic spines.

Animals↗
PubMed1996

Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation.

Wing margin formation in Drosophila requires the Notch receptor and, in the dorsal compartment, one of its ligands, Serrate. We provide evidence that Delta, the other known ligand for Notch, is also essential for this process. Delta is required in ventral cells at the dorsal/ventral compartment boundary, where its expression is specifically elevated in second-instar wing discs during wing margin formation. Moreover, ectopic Delta expression induces wingless, vestigial, and cut and causes adult wing tissue outgrowth in the dorsal compartment. The effect is mediated by Notch, because loss of Notch activity suppresses Delta-induced ectopic wing outgrowth. Whereas ectopic expression of Notch or the truncated activated Notch induces cut in both dorsal and ventral compartments, ectopic Delta expression induces cut only in the dorsal compartment and ectopic Serrate induces cut only in the ventral compartment. These observations indicate that Notch-expressing cells in a given compartment have different responses to Delta and Serrate. We propose that Delta and Serrate function as compartment-specific signals in the wing disc, to activate Notch and induce downstream genes required for wing formation.

Animals↗
PubMed1996

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.

Animals↗
PubMed1996

Functional effects of the mouse weaver mutation on G protein-gated inwardly rectifying K+ channels.

The weaver mutation corresponds to a substitution of glycine to serine in the H5 region of a G protein-gated inwardly rectifying K+ channel gene (GIRK2). By studying mutant GIRK2 weaver homomultimeric channels and heteromultimeric channels comprised of GIRK2 weaver and GIRK1 in Xenopus oocytes, we found that GIRK2 weaver homomultimeric channels lose their selectivity for K+ ions, giving rise to inappropriate receptor-activated and basally active Na+ currents, whereas heteromultimers of GIRK2 weaver and GIRK1 appeared to have reduced current. Immunohistochemical localization indicates that GIRK2 and GIRK1 proteins are expressed in the cerebellar neurons of mice at postnatal day 4, at a time when these neurons normally undergo differentiation. Thus, the aberrant behavior of mutant GIRK2 weaver channels could affect the development of weaver mice in at least two distinct ways.

Aging↗
PubMed1996

Control of daughter cell fates during asymmetric division: interaction of Numb and Notch.

During development of the Drosophila peripheral nervous system, a sensory organ precursor (SOP) cell undergoes rounds of asymmetric divisions to generate four distinct cells of a sensory organ. Numb, a membrane-associated protein, is asymmetrically segregated into one daughter cell during SOP division and acts as an inherited determinant of cell fate. Here, we show that Notch, a transmembrane receptor mediated cell-cell communication, functions as a binary switch in cell fate specification during asymmetric divisions of the SOP and its daughter cells in embryogenesis. Moreover, numb negatively regulates Notch, probably through direct protein-protein interaction that requires the phosphotyrosine-binding (PTB) domain of Numb and either the RAM23 region or the very C-terminal end of Notch. Notch then positively regulates a transcription factor encoded by tramtrack (ttk). This leads to Ttk expression in the daughter cell that does not inherit Numb. Thus, the inherited determinant Numb bestows a bias in the machinery for cell-cell communication to allow the specification of distinct daughter cell fates.

Animals↗