线虫中细胞分泌特性和蛋白质功能的研究
|
摘要
Caenorhabditis elegans(C. elegans,线虫),一种体长仅1 mm的土壤线虫,最早被英国科学家西德尼·布雷纳作为一种模式生物,用于研究发育和遗传学。
线虫是一种非常理想的遗传学研究模型。首先,它的基因组已经被完全解析,具有5对常染色体和一对性染色体。其次,它生活史短,个体小,可以自体受精,可以方便快捷地进行正义、反义遗传筛选;第三,易于用各种遗传学技术对其进行操作和研究。
基于线虫在多方面的优势,我们希望建立起一套线虫培养、操作的实验平台,尝试在线虫中开展细胞分泌特性和相关蛋白质功能的研究。
我们介绍了线虫培养过程中的各项操作方法。在实验室内,线虫可以在琼脂平板上进行培养,以大肠杆菌OP50为食。线虫雌雄同体共有959个体细胞,胚胎发育的细胞谱系已经完整地被描绘出来。而且,关于线虫302个神经元的位置、形态等也了解得比较清楚。
因此,我们通过线虫胚胎细胞原代培养技术,成功地在体外培养了多种神经细胞和肌肉细胞,并对其形态特征进行了观察和分析。
然后,我们选择了tph-1::GFP融合蛋白特异标记的虫系GR1366进行胚胎细胞的体外培养,得到了有荧光标记的线虫咽部NSM神经元。NSM神经元主要分泌的神经递质是5-HT。利用全细胞膜片钳和膜电容检测技术,记录了NSM神经元上的通道电流,该电流表现出明显的外向整流的特性。利用光解释放钙离子刺激,首次在体外培养的线虫细胞上记录到细胞分泌引起的膜电容增加,增加量平均在10 fF左右,具有良好的信噪比。采用碳纤电极安培测量技术,记录到NSM在高钾刺激下5-HT的电流信号。
在本文中,我们还进行了线虫基因突变库的构建,采用TMP和紫外照射,对大量的线虫进行随机突变。突变库建成之后,可以选择感兴趣的基因,特别是在细胞分泌过程中起作用的基因,设计相应的PCR反应的引物,通过一系列的筛选,即有可能从突变库中发现相应的突变等位基因。利用突变的线虫个体,就可以从单细胞水平对分泌特性和动力学过程进行分析,也可以从行为学深入研究基因和蛋白质的关系。
The 1 mm large soil nematode Caenorhabditis elegans was first used by Sydney Brenner to study the genetics of development and behavior.
C. elegans is an ideal genetic model system. Firstly, its complete genome, consisting of five pairs of autosomal and one pair of sex chromosomes, has been sequenced and annotated. Secondly, due to the short generation time, self-fertilization and small size of C. elegans, forward and reverse genetic screens are fast, easy, cheap and can be easily scaled up. Thirdly, C. elegans is susceptible to manipulation using a range of genetic techniques. Because the advantages of C. elegans are numerous, we attempt to establish a research stage for the culture and manipulation of C. elegans, and study on the characteristic of secretion and protein function related secretory vesicles and membrane.
We described all the techiniques using in the project, including maintaining a culture of a worm strain. In the laboratory, C. elegans is cultured on Petri dishes containing buffered agar and OP50 that serves as food.
The C. elegans hermaphrodite consists of 959 somatic cells. Of all cells, the complete embryonic lineage has been determined. Furthermore, the positions, morphologies, synapses and gap junctions of all 302 neurons have been described. The primary culture system that allows culture of C. elegans embryonic cells had been described. We observed several types of cells and compared their morphological characterization.
And using tph-1::GFP transgenic strain GR1366, we cultured the NSM neurons located on the pharynx in C. elegans. The major neurotramitter of NSM neuron is 5-HT. We undertook a series of patch clamp studies to in vitro electrophysiological analysis. The cultured NSM neuron expressed slowly inactivating, outwardly rectifying currents. By using high time resolution measurements of membrane capacitance, flash photolysis of caged Ca2+ and Carbon-fiber amperometric measurement, we first characterize ~10 fF Cm increasing and the 5-HT release spike current in vitro neurons in C. elegans.
In this dissertation, we also present the construction and screening of deletion mutant libraries to generate C. elegans knockouts. This gene knockout strategy used a random mutagen, TMP and UV to mutagenize a very large number of worms. If PCR primers flanking an area of the gene of our interest are used to amplify from the genomic DNA samples, deletions between the primes can be detected. Once a DNA sample containing a deletion is identified, one can work back to identify the subculture of worms. Individual live animals carrying the deletion mutation can be used to study the molecular and cellular basis of secretory, even in the relationship of gene and behavior.
线虫是一种非常理想的遗传学研究模型。首先,它的基因组已经被完全解析,具有5对常染色体和一对性染色体。其次,它生活史短,个体小,可以自体受精,可以方便快捷地进行正义、反义遗传筛选;第三,易于用各种遗传学技术对其进行操作和研究。
基于线虫在多方面的优势,我们希望建立起一套线虫培养、操作的实验平台,尝试在线虫中开展细胞分泌特性和相关蛋白质功能的研究。
我们介绍了线虫培养过程中的各项操作方法。在实验室内,线虫可以在琼脂平板上进行培养,以大肠杆菌OP50为食。线虫雌雄同体共有959个体细胞,胚胎发育的细胞谱系已经完整地被描绘出来。而且,关于线虫302个神经元的位置、形态等也了解得比较清楚。
因此,我们通过线虫胚胎细胞原代培养技术,成功地在体外培养了多种神经细胞和肌肉细胞,并对其形态特征进行了观察和分析。
然后,我们选择了tph-1::GFP融合蛋白特异标记的虫系GR1366进行胚胎细胞的体外培养,得到了有荧光标记的线虫咽部NSM神经元。NSM神经元主要分泌的神经递质是5-HT。利用全细胞膜片钳和膜电容检测技术,记录了NSM神经元上的通道电流,该电流表现出明显的外向整流的特性。利用光解释放钙离子刺激,首次在体外培养的线虫细胞上记录到细胞分泌引起的膜电容增加,增加量平均在10 fF左右,具有良好的信噪比。采用碳纤电极安培测量技术,记录到NSM在高钾刺激下5-HT的电流信号。
在本文中,我们还进行了线虫基因突变库的构建,采用TMP和紫外照射,对大量的线虫进行随机突变。突变库建成之后,可以选择感兴趣的基因,特别是在细胞分泌过程中起作用的基因,设计相应的PCR反应的引物,通过一系列的筛选,即有可能从突变库中发现相应的突变等位基因。利用突变的线虫个体,就可以从单细胞水平对分泌特性和动力学过程进行分析,也可以从行为学深入研究基因和蛋白质的关系。
The 1 mm large soil nematode Caenorhabditis elegans was first used by Sydney Brenner to study the genetics of development and behavior.
C. elegans is an ideal genetic model system. Firstly, its complete genome, consisting of five pairs of autosomal and one pair of sex chromosomes, has been sequenced and annotated. Secondly, due to the short generation time, self-fertilization and small size of C. elegans, forward and reverse genetic screens are fast, easy, cheap and can be easily scaled up. Thirdly, C. elegans is susceptible to manipulation using a range of genetic techniques. Because the advantages of C. elegans are numerous, we attempt to establish a research stage for the culture and manipulation of C. elegans, and study on the characteristic of secretion and protein function related secretory vesicles and membrane.
We described all the techiniques using in the project, including maintaining a culture of a worm strain. In the laboratory, C. elegans is cultured on Petri dishes containing buffered agar and OP50 that serves as food.
The C. elegans hermaphrodite consists of 959 somatic cells. Of all cells, the complete embryonic lineage has been determined. Furthermore, the positions, morphologies, synapses and gap junctions of all 302 neurons have been described. The primary culture system that allows culture of C. elegans embryonic cells had been described. We observed several types of cells and compared their morphological characterization.
And using tph-1::GFP transgenic strain GR1366, we cultured the NSM neurons located on the pharynx in C. elegans. The major neurotramitter of NSM neuron is 5-HT. We undertook a series of patch clamp studies to in vitro electrophysiological analysis. The cultured NSM neuron expressed slowly inactivating, outwardly rectifying currents. By using high time resolution measurements of membrane capacitance, flash photolysis of caged Ca2+ and Carbon-fiber amperometric measurement, we first characterize ~10 fF Cm increasing and the 5-HT release spike current in vitro neurons in C. elegans.
In this dissertation, we also present the construction and screening of deletion mutant libraries to generate C. elegans knockouts. This gene knockout strategy used a random mutagen, TMP and UV to mutagenize a very large number of worms. If PCR primers flanking an area of the gene of our interest are used to amplify from the genomic DNA samples, deletions between the primes can be detected. Once a DNA sample containing a deletion is identified, one can work back to identify the subculture of worms. Individual live animals carrying the deletion mutation can be used to study the molecular and cellular basis of secretory, even in the relationship of gene and behavior.
引文
[1] Brenner, S. The genetics of Caenorhabditis elegans. Genetics, 1974, 77:71–94.
[2] Sulston J.E., Schierenberg, E. White, et.al. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol., 1983, 100:64–119.
[3] Golstein P. Controlling cell death. Science, 1997, 275:1081-1082.
[4] Boyd L, Guo S, Levitan D, et.al. PAR-2 is asymmetrically distributed and promotes association of P granules and PAR-1 with the cortex in C. elegans embryos. Dev, 1996, 122:3075-3084.
[5] Fire A., Xu S., Montgomery M.K., et.al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806–811.
[6] Bernstein E, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001, 409(6818): 363-6.
[7] Hammond S. M., Kobayashi R, Hannon G. et.al. Link Between Genetic and Biochemical Analyses of RNAi. Science. 2001, 293(5532): 1146-50.
[8] Bosher, J.M., and Labouesse, M. (). RNA interference: genetic wand and genetic watchdog. Nat. Cell Biol. 2000, 2: 31-36.
[9] B. Sonnichsen, L.B.Koski, A.Walsh1, et.al. Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature, 434: 462-469.
[10] Zhaoyang Feng, Christopher J Cronin, John H Wittig Jr, et.al. An imaging system for standardized quantitative analysis of C. elegans behavior. BMC Bioinformatics, 2004, 5:115-121.
[11] Rankin CH. From gene to identified neuron to behavior. Nature Reviews Genetics, 2002 3:622-630.
[12] De Bono M. Molecular approaches to aggregation behavior and social attachment. Journal of Neurobiology, 2003, 54:78-92.
[13] Ailion M, Thomas JH. Isolation and characterization of high-temperature-induced dauer formation mutants in Caenorhabditis elegans. Genetics, 2003, 165:127-144.
[14] Capowski EE, Martin PR, Garvin CM, et.al. Identification of grandchildless lociwhose products are required for normal germ-line development in the nematode Caenorhabditis elegans. Genetics Genetics, 1991, 1061-1072.
[15] Albertson DG. Mapping chromosome rearrangement breakpoints to the physical map of Caenorhabditis elegans by fluorescent in situ hybridization. Genetics, 1993, 134: 211-219.
[16] Alfonso A, Grundahl K, Duerr JS, et.al. The Caenorhabditis elegans unc-17 gene: A putative vesicular acetylcholine transporter. Science, 1993, 261:617-619.
[17] Alkema MJ, Hunter-Ensor M, Ringstad N, et.al. Tyramine Functions Independently of Octopamine in the Caenorhabditis elegans Nervous System. Neuron, 2005, 46: 247-260.
[18] Bargmann CI. Genetic and cellular analysis of behavior in C. elegans. Annual Review of Neuroscience, 1993, 16: 47-71.
[19] Ahnn JH, Fire A. A screen for genetic loci required for body wall muscle development during embryogenesis in Caenorhabditis elegans. Genetics, 1994, 137: 483-498.
[20] Beitel GJ, Tuck SP, Greenwald IS, et.al. The Caenorhabditis elegans gene lin-1 encodes an ETS-domain protein and defines a branch of the vulval induction pathway. Genes and Development, 1995, 9: 3149-3162.
[21] Bulik DA, Robbins PW. The Caenorhabditis elegans sqv genes and functions of proteoglycans in development. Biochimica et Biophysica Acta, 2002, 1573: 247-257.
[22] Basson M, Horvitz HR. The Caenorhabditis elegans gene sem-4 controls neuronal and mesodermal cell development and encodes a zinc finger protein. Genes and Development, 1996, 10: 1953-1965.
[23] Bean CJ, Schaner CE, Kelly WG. Meiotic pairing and imprinted X chromatin assembly in Caenorhabditis elegans. Nature Genetics, 2004, 36: 100-105.
[24] Chasnov JR, Chow KL. Why are there males in the hermaphroditic species. Genetics, 2002, 160: 983-994.
[25] Ailion M, Thomas JH. Dauer formation induced by high temperatures in Caenorhabditis elegans. Genetics, 2000, 156: 1047-1067.
[26] Alcedo J, Kenyon C. Regulation of C. elegans longevity by specific gustatory and olfactory neurons. Neuron, 2003, 41: 45-55.
[27] Antebi A. Long life: a matter of taste (and smell). Neuron, 2004, 41:1-6.
[28] Adams JM, Cory S. Apoptosomes: engines for caspase activation. Current Opinion in Cell Biology, 2002, 14: 715-720.
[29] Bany IA, Dong MQ, Koelle MR. Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior. Journal of Neuroscience, 2003, 23: 8060-8069.
[30] Gerald Karp. Cell and Molecular Biology:Concepts and Experiments. 2002, Third Edition. John Wiley & Sons, Inc.
[31] Juan S. Bonifacino and Benjamin S. Glick. The mechanisms of vesicle budding and fusion. Cell, 2004, 116: 153-166.
[32] Nudelman G, Louzoun Y. Cell surface dynamics: the balance between diffusion, aggregation and endocytosis. Syst Biol (Stevenage), 2006, 153(1): 34-42.
[33] Granseth B, Odermatt B, Royle SJ, Lagnado L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron, 2006, 51(6): 773-86.
[34] Sudhof TC. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature, 1995, 375(6533): 645-653.
[35] Bennett MK, Scheller RH. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci U S A, 1993, 90(7): 2559-2563.
[36] Ferro-Novick S, Jahn R. Vesicle fusion from yeast to man. Nature 1994, 370(6486): 191-193.
[37] Sutton RB, Fasshauer D, Jahn R, et al. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature, 1998, 395(6700): 347-353.
[38] Gerona RR, Larsen EC, Kowalchyk JA, et al. The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. J Biol Chem, 2000, 275(9): 6328-6336.
[39] Xu T, Rammner B, Margittai M, et al. Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis. Cell, 1999, 99(7): 713-722.
[40] Rettig J, Neher E. Emerging roles of presynaptic proteins in Ca2+-triggered exocytosis. Science, 2002, 298(5594): 781-785.
[41] Tooze SA, Martens GJ, Huttner WB. Secretory granule biogenesis: rafting to the SNARE. Trends Cell Biol, 2001, 11(3): 116-122.
[42] Tooze SA. Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion. FEBS Lett, 1991, 285(2): 220-224.
[43] Chen YA, Scales SJ, Patel SM, et al. SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell, 1999, 97(2): 165-174.
[44] Hanson PI, Heuser JE, Jahn R. Neurotransmitter release-four years of SNARE complexes. Curr Opin Neurobiol, 1997, 7(3): 310-315.
[45] Ungermann C, Sato K, Wickner W. Defining the functions of trans-SNARE pairs. Nature, 1998, 396(6711): 543-548.
[46] Xu T, Binz T, Niemann H, et.al. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci, 1998, 1(3): 192-200.
[47] Smith C, Moser T, Xu T, et al. Cytosolic Ca2+ acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells. Neuron, 1998, 20(6): 1243-1253.
[48] Voets T, Neher E, Moser T. Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices. Neuron, 1999, 23(3): 607-615.
[49] Lauer J.M., Dalal S, Marz K.E, et.al. SNARE complex zero layer residues are not critical for NSF-mediated disassembly. J Biol Chem, 2006, 281: 14823-14832.
[50] Staunton, J., Ganetzky, B. and Nonet, M. L. Rabphilin potentiates SNARE function independently of rab3. J Neurosci, 2001, 21: 9255-9564.
[51] Weimer, R. M., Richmond J. E., Davis, W. S., et.al. Defects in synaptic vesicle docking in unc-18 mutants. Nat Neurosci, 2003, 6: 1023.
[52] Hata Y, Slaughter CA, Sudhof TC. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature, 1993, 366(6453): 347-351.
[53] Verhage M, Maia AS, Plomp JJ, et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science, 2000, 287(5454): 864-869.
[54] Wu MN, Schulze KL, Lloyd TE, et.al. The ROP-syntaxin interaction inhibits neurotransmitter release. Eur J Cell Biol, 2001, 80(2): 196-199.
[55] Voets T, Toonen RF, Brian EC, et.al. Munc18-1 promotes large dense-core vesicle docking. Neuron, 2001, 31(4): 581-591.
[56] Bryant NJ, James DE. Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation. Embo J., 2001, 20(13): 3380-3388.
[57] Weimer RM, Richmond JE, Davis WS, et.al. Defects in synaptic vesicle docking in unc-18 mutants. Nat Neurosci, 2003, 6(10): 1023-1030.
[58] Fisher RJ, Pevsner J, Burgoyne RD. Control of fusion pore dynamics during exocytosis by Munc18. Science, 2001, 291(5505): 875-878.
[59] Shuang R, Zhang L, Fletcher A, et.al. Regulation of Munc-18/syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings. J Biol Chem, 1998, 273(9): 4957-4966.
[60] Bryant NJ, James DE. The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport. J Cell Biol, 2003, 161(4): 691-696.
[61] Craig TJ, Evans GJ, Morgan A. Physiological regulation of Munc18/nSec1 phosphorylation on serine-313. J Neurochem, 2003, 86(6): 1450-1457.
[62] Janet E. Richmond, Robby M. Weimer, Erik M. Jorgensen. An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature, 2001, 412: 338-341.
[63] Jorgensen, E. M., Hartweig, E, Schuske, K, et.al. Horvitz. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature, 1995, 378: 196-199.
[64] Nonet, M. L., K. Grundahl, B. J. Meyer et.al. Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell, 1993, 73: 1291-1305.
[65] Li C, Ullrich B, Zhang JZ, et al. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature, 1995, 375(6532): 594-599.
[66] Fukuda M, Aruga J, Niinobe M, et al. Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II. J Biol Chem, 1994, 269(46): 29206-29211.
[67] Robinson IM, Ranjan R, Schwarz TL. Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain. Nature, 2002, 418(6895): 336-340.
[68] Mackler JM, Drummond JA, Loewen CA, et.al. The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature, 2002, 418(6895):340-344.
[69] Voets T, Moser T, Lund PE, et.al. Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I. Proc Natl Acad Sci U S A, 2001, 98(20): 11680-11685.
[70] Bai J, Wang CT, Richards DA, et.al. Fusion pore dynamics are regulated by synaptotagmin t-SNARE interactions. Neuron, 2004, 41(6): 929-942.
[71] Ann K, Kowalchyk JA, Loyet KM, et al. Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis. J Biol Chem, 1997, 272(32): 19637-19640.
[72] Walent JH, Porter BW, Martin TF. A novel 145 kd brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells. Cell, 1992, 70(5): 765-775.
[73] M. Gerard Waters and Frederick M. Hughson. Membrane tethering and fusion in the secretory and endocytic pathways. Traffic, 2000, 1: 588-597.
[74] S. L. Deken, R. Vincent, G. Hadwiger, et.al. Redundant Localization Mechanisms of RIM and ELKS in Caenorhabditis elegans. J. Neurosci., 2005, 25(25): 5975-5983.
[75] Reinhard Jahn and Thomas C. Sudhof. Membrane fusion and exocytosis. Annu. Rev. Biochem, 1999, 68: 863~911.
[76] Pierce KL, Lefkowitz RJ. Classical and new roles of beta-arrestins in the regulation of G-protein-coupled receptors. Nat. Rev. Neurosci., 2001, 2(10): 727-733.
[77] Chalfie M., Tu Y., Euskirchen G., et.al. Green fluorescent protein as a marker for gene expression. Science, 1994, 263: 802-805.
[78] Maduro, M., and Pilgrim, D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics, 1995, 141: 977-988.
[79] Maduro M.F., Gordon M., Jacobs R., et.al. The UNC-119 family of neural proteins is functionally conserved between humans, Drosophila and C. elegans. J. Neurogenet, 2000, 13: 191-212.
[80] Sakmann B, Neher E. Geometric parameters of pipette and membrane patches. In: Sakmann B, Neher E, editors. Single-Channel Recording, 2nd edition. New York, NY: Plenum Press, 1995, 637-650.
[81] Rae JL, Levis RA. Glass technology for patch clamp electrodes. Methods Enzymol, 1992, 207: 66–92.
[82] Miriam B. Goodman, Shawn R. Lockery. Pressure polishing: a method for re-shaping patch pipettes during fire polishing. Journal of Neuroscience Methods, 2000, 100: 13-15
[83] Goodman MB, Hall DH, Avery L, et.al. Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron, 1998, 20:763-772.
[84] Neher E. and Sakmann B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature, 1976, 260(5554): 799-802.
[85] Gentet L. J., Stuart G. J. and Clememts J. D. Direct measurement of specific membrane capacitance in neurons. Biophys J., 2000, 79(1): 314-320.
[86] Lollike K. and Lindau M. Membrane capacitance techniques to monitor granule exocytosis in neutrophils. J. Immunol methods, 1999, 232(1-2): 111-120.
[87] Bloom L. Genetic and molecular ananlysis of genes required for axon outgrowth in C. elegans. Ph.D. thesis, 1993, Massachusetts Institute of Technology.
[88] Edgar,L.G. Blastomere culture and analysis. Methods Cell Biol., 1995, 48: 303-321.
[89] Christensen M., Estevez A., Yin X., et.al. A Primary Culture System for Functional Analysis of C. elegans Neurons and Muscle Cells. Neuron, 2002, 33: 503-514.
[90] Theresa Stiernagle. Maintenance of C. elegans.
[91] Molecular, Genetic and Informatic Methods for C. elegans. The Sanger Centre, Wellcome Trust Genome Campus Hinxton, Cambridge.
[92] Kerr R., Lev-Ram V., Baird G., et.al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron, 2000, 26: 583-594.
[93] Nonet, M. L. Visualization of synaptic specializations in live C. elegans using synaptic vesicle-GFP protein fusions. J. Neurosci. Methods, 1999, 89: 33-40.
[94] Fire A, Xu S, Montgomery MK, et.al. Potent and specific interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806-811.
[95] Gally C, Bessereau JL. GABA is dispensable for the formation of juncational GABA receptor clusters in Caenorhabditis elegans. Journal of Neuroscience, 2003, 23: 2591-2599.
[96] Lockery, S.R., and Goodman, M.B. Tight-seal whole-cell apical membrane patch clamping of Caenorhabditis elegans neurons. Methods Enzymol., 1998, 293: 201-217.
[97] White J.G., Southgate E., Thomson, J.N., et.al. The structure of the nervous system of the nematode C. elegans. Philos. Trans. Royal Soc. Lond. B Biol. Sci., 1986, 314: 1-340.
[98] Rand JB, Nonet ML. Neurotransmitter assignments for specific neurons. In: C. elegans II (Riddle, Blumenthal, Meyer & Priess, editors), 1997, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY: 1049-1052.
[99] Richmond, J.E. and Jorgensen E.M. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci., 1999, 2: 791-797.
[100] Alfonso A, Grundahl K, Duerr JS, et.al. The Caenorhabditis elegans unc-17 gene: A putative vesicular acetylcholine transporter. Science, 1993, 261: 617-619.
[101] James B. Rand, Janet S. Duerr and Dennis L. Frisby. Neurogenetics of vesicular transporters in C. elegans. FASEB J., 2000, 14: 2414-2422.
[102] Duerr J. S., Frisby D. L., Gaskin J., et.al. The cat-1 gene of C. elegans encodes a vesicular monoamine transporter required for specific monoamine-dependent behaviors. J. Neurosci. , 1999, 19: 72-84.
[103] Sze, Victor, Loer, et.al. Feeding and metabolic signalling defects in a Caenorhabditis elegans serotonin-synthesis mutant. Nature, 2000, 403: 560-564.
[104] Loer and Kenyon. Serotonin-deficient mutants and male mating behavior in the nematode Caenorhabditis elegans. J. Neuroscience, 1993, 13: 5407-5417.
[105] Lucia Carvelli, Paul W. McDonald, Randy D. Blakely, et.al. Dopamine transporters depolarize neurons by a channel mechanism.PNAS, 2004, 101(45): 16046-16051.
[106] Xu T, Naraghi M, Kang H, et.al.. Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells. Biophys J., 1997, 73(1): 532-545.
[107] Ana Y. Estevez, Randolph K. Roberts and Kevin Strange. Identification of Store-independent and Store-operated Ca2+ Conductances in Caenorhabditis elegans Intestinal Epithelial Cells. J. Gen. Physiol., 2003, 122: 207-223.
[108] Jorgensen EM, Mango SE. The art and design of genetic screens: Caenorhabditis elegans. Nature Reviews Genetics, 2002, 3: 356-369.
[109] Mark Edgley, Anil D. Souza1, Gary Moulder, et.al. Improved detection of small deletions in complex pools of DNA.Nucleic Acids Research, 2002, 30(12): 52.
[110] A. Wei, A. Yuan, G. Fawcett, et.al. Efficient isolation of targeted Caenorhabditis elegans deletion strains using highly thermostable restriction endonucleases and PCR. Nucleic Acids Res., 2002, 30(20): 101-110.
[111] Wei Geng. Automatic Tracking, Feature Extraction and Classification of C. elegans Phenotypes. IEEE Transactions on Biomedical Engineering, 2004, 51: 1811-1820.
[2] Sulston J.E., Schierenberg, E. White, et.al. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol., 1983, 100:64–119.
[3] Golstein P. Controlling cell death. Science, 1997, 275:1081-1082.
[4] Boyd L, Guo S, Levitan D, et.al. PAR-2 is asymmetrically distributed and promotes association of P granules and PAR-1 with the cortex in C. elegans embryos. Dev, 1996, 122:3075-3084.
[5] Fire A., Xu S., Montgomery M.K., et.al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806–811.
[6] Bernstein E, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001, 409(6818): 363-6.
[7] Hammond S. M., Kobayashi R, Hannon G. et.al. Link Between Genetic and Biochemical Analyses of RNAi. Science. 2001, 293(5532): 1146-50.
[8] Bosher, J.M., and Labouesse, M. (). RNA interference: genetic wand and genetic watchdog. Nat. Cell Biol. 2000, 2: 31-36.
[9] B. Sonnichsen, L.B.Koski, A.Walsh1, et.al. Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature, 434: 462-469.
[10] Zhaoyang Feng, Christopher J Cronin, John H Wittig Jr, et.al. An imaging system for standardized quantitative analysis of C. elegans behavior. BMC Bioinformatics, 2004, 5:115-121.
[11] Rankin CH. From gene to identified neuron to behavior. Nature Reviews Genetics, 2002 3:622-630.
[12] De Bono M. Molecular approaches to aggregation behavior and social attachment. Journal of Neurobiology, 2003, 54:78-92.
[13] Ailion M, Thomas JH. Isolation and characterization of high-temperature-induced dauer formation mutants in Caenorhabditis elegans. Genetics, 2003, 165:127-144.
[14] Capowski EE, Martin PR, Garvin CM, et.al. Identification of grandchildless lociwhose products are required for normal germ-line development in the nematode Caenorhabditis elegans. Genetics Genetics, 1991, 1061-1072.
[15] Albertson DG. Mapping chromosome rearrangement breakpoints to the physical map of Caenorhabditis elegans by fluorescent in situ hybridization. Genetics, 1993, 134: 211-219.
[16] Alfonso A, Grundahl K, Duerr JS, et.al. The Caenorhabditis elegans unc-17 gene: A putative vesicular acetylcholine transporter. Science, 1993, 261:617-619.
[17] Alkema MJ, Hunter-Ensor M, Ringstad N, et.al. Tyramine Functions Independently of Octopamine in the Caenorhabditis elegans Nervous System. Neuron, 2005, 46: 247-260.
[18] Bargmann CI. Genetic and cellular analysis of behavior in C. elegans. Annual Review of Neuroscience, 1993, 16: 47-71.
[19] Ahnn JH, Fire A. A screen for genetic loci required for body wall muscle development during embryogenesis in Caenorhabditis elegans. Genetics, 1994, 137: 483-498.
[20] Beitel GJ, Tuck SP, Greenwald IS, et.al. The Caenorhabditis elegans gene lin-1 encodes an ETS-domain protein and defines a branch of the vulval induction pathway. Genes and Development, 1995, 9: 3149-3162.
[21] Bulik DA, Robbins PW. The Caenorhabditis elegans sqv genes and functions of proteoglycans in development. Biochimica et Biophysica Acta, 2002, 1573: 247-257.
[22] Basson M, Horvitz HR. The Caenorhabditis elegans gene sem-4 controls neuronal and mesodermal cell development and encodes a zinc finger protein. Genes and Development, 1996, 10: 1953-1965.
[23] Bean CJ, Schaner CE, Kelly WG. Meiotic pairing and imprinted X chromatin assembly in Caenorhabditis elegans. Nature Genetics, 2004, 36: 100-105.
[24] Chasnov JR, Chow KL. Why are there males in the hermaphroditic species. Genetics, 2002, 160: 983-994.
[25] Ailion M, Thomas JH. Dauer formation induced by high temperatures in Caenorhabditis elegans. Genetics, 2000, 156: 1047-1067.
[26] Alcedo J, Kenyon C. Regulation of C. elegans longevity by specific gustatory and olfactory neurons. Neuron, 2003, 41: 45-55.
[27] Antebi A. Long life: a matter of taste (and smell). Neuron, 2004, 41:1-6.
[28] Adams JM, Cory S. Apoptosomes: engines for caspase activation. Current Opinion in Cell Biology, 2002, 14: 715-720.
[29] Bany IA, Dong MQ, Koelle MR. Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior. Journal of Neuroscience, 2003, 23: 8060-8069.
[30] Gerald Karp. Cell and Molecular Biology:Concepts and Experiments. 2002, Third Edition. John Wiley & Sons, Inc.
[31] Juan S. Bonifacino and Benjamin S. Glick. The mechanisms of vesicle budding and fusion. Cell, 2004, 116: 153-166.
[32] Nudelman G, Louzoun Y. Cell surface dynamics: the balance between diffusion, aggregation and endocytosis. Syst Biol (Stevenage), 2006, 153(1): 34-42.
[33] Granseth B, Odermatt B, Royle SJ, Lagnado L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron, 2006, 51(6): 773-86.
[34] Sudhof TC. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature, 1995, 375(6533): 645-653.
[35] Bennett MK, Scheller RH. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci U S A, 1993, 90(7): 2559-2563.
[36] Ferro-Novick S, Jahn R. Vesicle fusion from yeast to man. Nature 1994, 370(6486): 191-193.
[37] Sutton RB, Fasshauer D, Jahn R, et al. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature, 1998, 395(6700): 347-353.
[38] Gerona RR, Larsen EC, Kowalchyk JA, et al. The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. J Biol Chem, 2000, 275(9): 6328-6336.
[39] Xu T, Rammner B, Margittai M, et al. Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis. Cell, 1999, 99(7): 713-722.
[40] Rettig J, Neher E. Emerging roles of presynaptic proteins in Ca2+-triggered exocytosis. Science, 2002, 298(5594): 781-785.
[41] Tooze SA, Martens GJ, Huttner WB. Secretory granule biogenesis: rafting to the SNARE. Trends Cell Biol, 2001, 11(3): 116-122.
[42] Tooze SA. Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion. FEBS Lett, 1991, 285(2): 220-224.
[43] Chen YA, Scales SJ, Patel SM, et al. SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell, 1999, 97(2): 165-174.
[44] Hanson PI, Heuser JE, Jahn R. Neurotransmitter release-four years of SNARE complexes. Curr Opin Neurobiol, 1997, 7(3): 310-315.
[45] Ungermann C, Sato K, Wickner W. Defining the functions of trans-SNARE pairs. Nature, 1998, 396(6711): 543-548.
[46] Xu T, Binz T, Niemann H, et.al. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci, 1998, 1(3): 192-200.
[47] Smith C, Moser T, Xu T, et al. Cytosolic Ca2+ acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells. Neuron, 1998, 20(6): 1243-1253.
[48] Voets T, Neher E, Moser T. Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices. Neuron, 1999, 23(3): 607-615.
[49] Lauer J.M., Dalal S, Marz K.E, et.al. SNARE complex zero layer residues are not critical for NSF-mediated disassembly. J Biol Chem, 2006, 281: 14823-14832.
[50] Staunton, J., Ganetzky, B. and Nonet, M. L. Rabphilin potentiates SNARE function independently of rab3. J Neurosci, 2001, 21: 9255-9564.
[51] Weimer, R. M., Richmond J. E., Davis, W. S., et.al. Defects in synaptic vesicle docking in unc-18 mutants. Nat Neurosci, 2003, 6: 1023.
[52] Hata Y, Slaughter CA, Sudhof TC. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature, 1993, 366(6453): 347-351.
[53] Verhage M, Maia AS, Plomp JJ, et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science, 2000, 287(5454): 864-869.
[54] Wu MN, Schulze KL, Lloyd TE, et.al. The ROP-syntaxin interaction inhibits neurotransmitter release. Eur J Cell Biol, 2001, 80(2): 196-199.
[55] Voets T, Toonen RF, Brian EC, et.al. Munc18-1 promotes large dense-core vesicle docking. Neuron, 2001, 31(4): 581-591.
[56] Bryant NJ, James DE. Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation. Embo J., 2001, 20(13): 3380-3388.
[57] Weimer RM, Richmond JE, Davis WS, et.al. Defects in synaptic vesicle docking in unc-18 mutants. Nat Neurosci, 2003, 6(10): 1023-1030.
[58] Fisher RJ, Pevsner J, Burgoyne RD. Control of fusion pore dynamics during exocytosis by Munc18. Science, 2001, 291(5505): 875-878.
[59] Shuang R, Zhang L, Fletcher A, et.al. Regulation of Munc-18/syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings. J Biol Chem, 1998, 273(9): 4957-4966.
[60] Bryant NJ, James DE. The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport. J Cell Biol, 2003, 161(4): 691-696.
[61] Craig TJ, Evans GJ, Morgan A. Physiological regulation of Munc18/nSec1 phosphorylation on serine-313. J Neurochem, 2003, 86(6): 1450-1457.
[62] Janet E. Richmond, Robby M. Weimer, Erik M. Jorgensen. An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature, 2001, 412: 338-341.
[63] Jorgensen, E. M., Hartweig, E, Schuske, K, et.al. Horvitz. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature, 1995, 378: 196-199.
[64] Nonet, M. L., K. Grundahl, B. J. Meyer et.al. Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell, 1993, 73: 1291-1305.
[65] Li C, Ullrich B, Zhang JZ, et al. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature, 1995, 375(6532): 594-599.
[66] Fukuda M, Aruga J, Niinobe M, et al. Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II. J Biol Chem, 1994, 269(46): 29206-29211.
[67] Robinson IM, Ranjan R, Schwarz TL. Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain. Nature, 2002, 418(6895): 336-340.
[68] Mackler JM, Drummond JA, Loewen CA, et.al. The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature, 2002, 418(6895):340-344.
[69] Voets T, Moser T, Lund PE, et.al. Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I. Proc Natl Acad Sci U S A, 2001, 98(20): 11680-11685.
[70] Bai J, Wang CT, Richards DA, et.al. Fusion pore dynamics are regulated by synaptotagmin t-SNARE interactions. Neuron, 2004, 41(6): 929-942.
[71] Ann K, Kowalchyk JA, Loyet KM, et al. Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis. J Biol Chem, 1997, 272(32): 19637-19640.
[72] Walent JH, Porter BW, Martin TF. A novel 145 kd brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells. Cell, 1992, 70(5): 765-775.
[73] M. Gerard Waters and Frederick M. Hughson. Membrane tethering and fusion in the secretory and endocytic pathways. Traffic, 2000, 1: 588-597.
[74] S. L. Deken, R. Vincent, G. Hadwiger, et.al. Redundant Localization Mechanisms of RIM and ELKS in Caenorhabditis elegans. J. Neurosci., 2005, 25(25): 5975-5983.
[75] Reinhard Jahn and Thomas C. Sudhof. Membrane fusion and exocytosis. Annu. Rev. Biochem, 1999, 68: 863~911.
[76] Pierce KL, Lefkowitz RJ. Classical and new roles of beta-arrestins in the regulation of G-protein-coupled receptors. Nat. Rev. Neurosci., 2001, 2(10): 727-733.
[77] Chalfie M., Tu Y., Euskirchen G., et.al. Green fluorescent protein as a marker for gene expression. Science, 1994, 263: 802-805.
[78] Maduro, M., and Pilgrim, D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics, 1995, 141: 977-988.
[79] Maduro M.F., Gordon M., Jacobs R., et.al. The UNC-119 family of neural proteins is functionally conserved between humans, Drosophila and C. elegans. J. Neurogenet, 2000, 13: 191-212.
[80] Sakmann B, Neher E. Geometric parameters of pipette and membrane patches. In: Sakmann B, Neher E, editors. Single-Channel Recording, 2nd edition. New York, NY: Plenum Press, 1995, 637-650.
[81] Rae JL, Levis RA. Glass technology for patch clamp electrodes. Methods Enzymol, 1992, 207: 66–92.
[82] Miriam B. Goodman, Shawn R. Lockery. Pressure polishing: a method for re-shaping patch pipettes during fire polishing. Journal of Neuroscience Methods, 2000, 100: 13-15
[83] Goodman MB, Hall DH, Avery L, et.al. Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron, 1998, 20:763-772.
[84] Neher E. and Sakmann B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature, 1976, 260(5554): 799-802.
[85] Gentet L. J., Stuart G. J. and Clememts J. D. Direct measurement of specific membrane capacitance in neurons. Biophys J., 2000, 79(1): 314-320.
[86] Lollike K. and Lindau M. Membrane capacitance techniques to monitor granule exocytosis in neutrophils. J. Immunol methods, 1999, 232(1-2): 111-120.
[87] Bloom L. Genetic and molecular ananlysis of genes required for axon outgrowth in C. elegans. Ph.D. thesis, 1993, Massachusetts Institute of Technology.
[88] Edgar,L.G. Blastomere culture and analysis. Methods Cell Biol., 1995, 48: 303-321.
[89] Christensen M., Estevez A., Yin X., et.al. A Primary Culture System for Functional Analysis of C. elegans Neurons and Muscle Cells. Neuron, 2002, 33: 503-514.
[90] Theresa Stiernagle. Maintenance of C. elegans.
[91] Molecular, Genetic and Informatic Methods for C. elegans. The Sanger Centre, Wellcome Trust Genome Campus Hinxton, Cambridge.
[92] Kerr R., Lev-Ram V., Baird G., et.al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron, 2000, 26: 583-594.
[93] Nonet, M. L. Visualization of synaptic specializations in live C. elegans using synaptic vesicle-GFP protein fusions. J. Neurosci. Methods, 1999, 89: 33-40.
[94] Fire A, Xu S, Montgomery MK, et.al. Potent and specific interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806-811.
[95] Gally C, Bessereau JL. GABA is dispensable for the formation of juncational GABA receptor clusters in Caenorhabditis elegans. Journal of Neuroscience, 2003, 23: 2591-2599.
[96] Lockery, S.R., and Goodman, M.B. Tight-seal whole-cell apical membrane patch clamping of Caenorhabditis elegans neurons. Methods Enzymol., 1998, 293: 201-217.
[97] White J.G., Southgate E., Thomson, J.N., et.al. The structure of the nervous system of the nematode C. elegans. Philos. Trans. Royal Soc. Lond. B Biol. Sci., 1986, 314: 1-340.
[98] Rand JB, Nonet ML. Neurotransmitter assignments for specific neurons. In: C. elegans II (Riddle, Blumenthal, Meyer & Priess, editors), 1997, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY: 1049-1052.
[99] Richmond, J.E. and Jorgensen E.M. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci., 1999, 2: 791-797.
[100] Alfonso A, Grundahl K, Duerr JS, et.al. The Caenorhabditis elegans unc-17 gene: A putative vesicular acetylcholine transporter. Science, 1993, 261: 617-619.
[101] James B. Rand, Janet S. Duerr and Dennis L. Frisby. Neurogenetics of vesicular transporters in C. elegans. FASEB J., 2000, 14: 2414-2422.
[102] Duerr J. S., Frisby D. L., Gaskin J., et.al. The cat-1 gene of C. elegans encodes a vesicular monoamine transporter required for specific monoamine-dependent behaviors. J. Neurosci. , 1999, 19: 72-84.
[103] Sze, Victor, Loer, et.al. Feeding and metabolic signalling defects in a Caenorhabditis elegans serotonin-synthesis mutant. Nature, 2000, 403: 560-564.
[104] Loer and Kenyon. Serotonin-deficient mutants and male mating behavior in the nematode Caenorhabditis elegans. J. Neuroscience, 1993, 13: 5407-5417.
[105] Lucia Carvelli, Paul W. McDonald, Randy D. Blakely, et.al. Dopamine transporters depolarize neurons by a channel mechanism.PNAS, 2004, 101(45): 16046-16051.
[106] Xu T, Naraghi M, Kang H, et.al.. Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells. Biophys J., 1997, 73(1): 532-545.
[107] Ana Y. Estevez, Randolph K. Roberts and Kevin Strange. Identification of Store-independent and Store-operated Ca2+ Conductances in Caenorhabditis elegans Intestinal Epithelial Cells. J. Gen. Physiol., 2003, 122: 207-223.
[108] Jorgensen EM, Mango SE. The art and design of genetic screens: Caenorhabditis elegans. Nature Reviews Genetics, 2002, 3: 356-369.
[109] Mark Edgley, Anil D. Souza1, Gary Moulder, et.al. Improved detection of small deletions in complex pools of DNA.Nucleic Acids Research, 2002, 30(12): 52.
[110] A. Wei, A. Yuan, G. Fawcett, et.al. Efficient isolation of targeted Caenorhabditis elegans deletion strains using highly thermostable restriction endonucleases and PCR. Nucleic Acids Res., 2002, 30(20): 101-110.
[111] Wei Geng. Automatic Tracking, Feature Extraction and Classification of C. elegans Phenotypes. IEEE Transactions on Biomedical Engineering, 2004, 51: 1811-1820.