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41 Publications

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    Fetter Lab
    11/01/09 | A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia.
    Abodeely M, DuBois KN, Hehl A, Stefanic S, Sajid M, DeSouza W, Attias M, Engel JC, Hsieh I, Fetter RD, McKerrow JH
    Eukaryotic Cell. 2009 Nov;8(11):1665-76. doi: 10.1128/EC.00123-09

    The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

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    Fetter Lab
    11/01/09 | Wnt-Ror signaling to SIA and SIB neurons directs anterior axon guidance and nerve ring placement in C. elegans.
    Kennerdell JR, Fetter RD, Bargmann CI
    Development. 2009 Nov;136(22):3801-10. doi: 10.1242/dev.038109

    Wnt signaling through Frizzled proteins guides posterior cells and axons in C. elegans into different spatial domains. Here we demonstrate an essential role for Wnt signaling through Ror tyrosine kinase homologs in the most prominent anterior neuropil, the nerve ring. A genetic screen uncovered cwn-2, the C. elegans homolog of Wnt5, as a regulator of nerve ring placement. In cwn-2 mutants, all neuronal structures in and around the nerve ring are shifted to an abnormal anterior position. cwn-2 is required at the time of nerve ring formation; it is expressed by cells posterior of the nerve ring, but its precise site of expression is not critical for its function. In nerve ring development, cwn-2 acts primarily through the Wnt receptor CAM-1 (Ror), together with the Frizzled protein MIG-1, with parallel roles for the Frizzled protein CFZ-2. The identification of CAM-1 as a CWN-2 receptor contrasts with CAM-1 action as a non-receptor in other C. elegans Wnt pathways. Cell-specific rescue of cam-1 and cell ablation experiments reveal a crucial role for the SIA and SIB neurons in positioning the nerve ring, linking Wnt signaling to specific cells that organize the anterior nervous system.

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    Fetter Lab
    09/01/09 | Negative regulation of active zone assembly by a newly identified SR protein kinase.
    Johnson EL, Fetter RD, Davis GW
    PLoS Biology. 2009 Sep;7(9):e1000193. doi: 10.1371/journal.pbio.1000193

    Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc) that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

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    Fetter Lab
    04/22/09 | Caenorhabditis elegans innexins regulate active zone differentiation.
    Yeh E, Kawano T, Ng S, Fetter R, Hung W, Wang Y, Zhen M
    The Journal of Neuroscience. 2009 Apr 22;29(16):5207-17. doi: 10.1523/JNEUROSCI.0637-09.2009

    In a genetic screen for active zone defective mutants in Caenorhabditis elegans, we isolated a loss-of-function allele of unc-7, a gene encoding an innexin/pannexin family gap junction protein. Innexin UNC-7 regulates the size and distribution of active zones at C. elegans neuromuscular junctions. Loss-of-function mutations in another innexin, UNC-9, cause similar active zone defects as unc-7 mutants. In addition to presumptive gap junction localizations, both UNC-7 and UNC-9 are also localized perisynaptically throughout development and required in presynaptic neurons to regulate active zone differentiation. Our mosaic analyses, electron microscopy, as well as expression studies suggest a novel and likely nonjunctional role of specific innexins in active zone differentiation in addition to gap junction formations.

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    Hess LabFetter Lab
    03/03/09 | Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.
    Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW, Fetter RD, Hess HF
    Proceedings of the National Academy of Sciences of the United States of America. 2009 Mar 3;106:3125-30. doi: 10.1073/pnas.0813131106

    Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

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    Fetter Lab
    10/24/08 | A parasite cysteine protease is key to host protein degradation and iron acquisition.
    O'Brien TC, Mackey ZB, Fetter RD, Choe Y, O'Donoghue AJ, Zhou M, Craik CS, Caffrey CR, McKerrow JH
    Journal of Biological Chemistry. 2008 Oct 24;283(43):28934-43. doi: 10.1074/jbc.M805824200

    Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

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    Fetter Lab
    04/24/08 | A presynaptic giant ankyrin stabilizes the NMJ through regulation of presynaptic microtubules and transsynaptic cell adhesion.
    Pielage J, Cheng L, Fetter RD, Carlton PM, Sedat JW, Davis GW
    Neuron. 2008 Apr 24;58(2):195-209. doi: 10.1016/j.neuron.2008.02.017

    In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.

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    Fetter Lab
    03/25/08 | Clathrin dependence of synaptic-vesicle formation at the Drosophila neuromuscular junction.
    Heerssen H, Fetter RD, Davis GW
    Current Biology. 2008 Mar 25;18(6):401-9. doi: 10.1016/j.cub.2008.02.055

    BACKGROUND: Among the most prominent molecular constituents of a recycling synaptic vesicle is the clathrin triskelion, composed of clathrin light chain (Clc) and clathrin heavy chain (Chc). Remarkably, it remains unknown whether clathrin is strictly necessary for the stimulus-dependent re-formation of a synaptic vesicle and, conversely, whether clathrin-independent vesicle endocytosis exists at the neuronal synapse.

    RESULTS: We employ FlAsH-FALI-mediated protein photoinactivation to rapidly (3 min) and specifically disrupt Clc function at the Drosophila neuromuscular junction. We first demonstrate that Clc photoinactivation does not impair synaptic-vesicle fusion. We then provide electrophysiological and ultrastructural evidence that synaptic vesicles, once fused with the plasma membrane, cannot be re-formed after Clc photoinactivation. Finally, we demonstrate that stimulus-dependent membrane internalization occurs after Clc photoinactivation. However, newly internalized membrane fails to resolve into synaptic vesicles. Rather, newly internalized membrane forms large and extensive internal-membrane compartments that are never observed at a wild-type synapse.

    CONCLUSIONS: We make three major conclusions. (1) FlAsH-FALI-mediated protein photoinactivation rapidly and specifically disrupts Clc function with no effect on synaptic-vesicle fusion. (2) Synaptic-vesicle re-formation does not occur after Clc photoinactivation. By extension, clathrin-independent "kiss-and-run" endocytosis does not sustain synaptic transmission during a stimulus train at this synapse. (3) Stimulus-dependent, clathrin-independent membrane internalization exists at this synapse, but it is unable to generate fusion-competent, small-diameter synaptic vesicles.

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    Fetter Lab
    02/07/08 | GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems.
    Feinberg EH, Vanhoven MK, Bendesky A, Wang G, Fetter RD, Shen K, Bargmann CI
    Neuron. 2008 Feb 7;57(3):353-63. doi: 10.1016/j.neuron.2007.11.030

    The identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed GRASP, a system to label membrane contacts and synapses between two cells in living animals. Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.

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    Fetter LabSimpson Lab
    09/20/07 | NF-κB, IκB, and IRAK control glutamate receptor density at the Drosophila NMJ.
    Heckscher ES, Fetter RD, Marek KW, Albin SD, Davis GW
    Neuron. 2007 Sep 20;55(6):859-73. doi: 10.1016/j.neuron.2007.08.005

    NF-κB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-κB signaling within the nervous system remains to be clearly defined. Here, we show that the NF-κB and IκB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. We then show that mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, we provide evidence that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon our data we speculate that Dorsal, Cactus, and Pelle could function together, locally at the postsynaptic density, to specify GluR levels.

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