Spelling suggestions: "subject:"vesicles transport""
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Elucidating the mechanism of prickle associated epilepsy in fliesEhaideb, Salleh Nasser 01 May 2015 (has links)
About 5% to 10% of epileptic patients suffer from Juvenile Myoclonic Epilepsy (JME), which is characterized by spasms of the arms, ataxia (uncoordinated movements), and general tonic-clonic seizures. In a recent study, a group of patients with myoclonic epilepsy was found to harbor mutations in the PRICKLE1 and PRICKLE2 genes. This suggested that PRICKLE genes might be linked to epilepsy, and given that PRICKLE is highly evolutionarily conserved (including in fruit flies), we decided to use Drosophila in order to determine, first, whether flies with prickle mutations were seizure-prone, and if so, to then use the powerful genetic tools of Drosophila to elucidate the underlying mechanism of the prickle-associated epilepsy. In this work, we show that mutation of the pksple isoform (one of the two adult prickle isoforms in flies) lowers the seizure threshold in the mutant flies (resulting in seizure activity), while mutation of the other adult isoform, pkpk, had no effect. This was demonstrated through both behavioral assays (where the pksple mutant flies showed a reduction in recovery of climbing behavior after being subjected to mechanical stimulation while the pkpk mutant flies did not) as well as electrophysiological analysis (where pksple mutants were shown to be hyperexcitable after electrical stimulation, while the pkpk allele showed no change in spiking activity). We demonstrated that the underlying mechanism of the hyperexcitability seen in the pksple flies was due to enhanced anterograde transport on microtubule (MT) tracks in neurons, the main route for transport in neurons, which could be suppressed by reducing the dose of either of two Kinesin motor proteins, the motors involved in anterograde transport in neurons. On the other hand, the pkpk mutants showed the reverse effect, exhibiting a significant reduction in vesicle transport dynamics. We showed that microtubule polarity could be partially reversed by tipping the balance of the pk isoforms similar to what is seen in the pkpk mutants (such that a large percentage of MTs now had their plus ends oriented towards the cell body, which is extremely rare in axons), suggesting that the vesicle transport defects seen in the pkpk mutants might be due to mixed polarity of MTs.
Next, we showed that the seizure-prone pksple mutants, but not the pkpk mutants, exhibited a myoclonic form of epilepsy, as well as abnormal walking patterns and uncoordinated movements, paralleling the ataxia phenotype seen in the epileptic patients with PRICKLE mutations. These data suggest that the primary aspects of the epilepsy-ataxia syndrome seen in patients with PRICKLE mutations are recapitulated in flies, which underscores the utility of using the fruit fly genetic system to model this disorder. Finally, our preliminary results suggest that the pk alleles have different effects on neuronal morphology due to changes in sizes of terminal boutons at the neuromuscular junction (NMJ) in larvae. These data suggest that pk is having a direct effect on synaptic formation and likely function.
In conclusion, by using our Drosophila model system, we were able to link prickle mutations to epilepsy as well as identify the cellular mechanism of the prickle-associated epilepsy, a novel epilepsy mechanism previously associated with neurodegeneration. To our knowledge, this is the first example of a gene that, when mutated, will cause seizures in flies, zebrafish, mice, and humans, indicating that the role of prickle in controlling seizure activity is remarkably conserved in animals. Significantly, since about one third of patients with epilepsy do not respond to current AEDs, our fly model and the techniques we have developed will enable us to conduct drug screens for testing potential chemical compounds as new AEDs.
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Characterizing dynein in T cellsTan, Sarah Youngsun 23 November 2010 (has links)
T cells play pivotal roles in the immune system and focused secretion of either cytokines or cytotoxic molecules toward its target is crucial for T cell functions. This directional secretion involves two critical steps: the movement of the microtubule organizing center (MTOC) up to the cell-cell contact site and the directed movement of secretory vesicles towards the MTOC. The minus end-directed microtubule motor protein dynein was previously shown in our studies and those of others to accumulate and anchor at the contact site where it then draws the MTOC up to the contact site. A variety of studies led to the suggestion that there were two functionally different pools of dynein in Jurkat cells, one a ring-like structure that pulled the MTOC to the contact site and the other one uniquely corresponding to the distribution of dynactin. This led to the hypothesis that the second pool of dynein drove vesicle transport. To address this possibility, we used siRNA to deplete the cell of dynactin. These studies showed that almost complete knockdown of dynactin (p150[superscript Glued]) had little effect on MTOC translocation but it also had little effect on a panel of Golgi vesicle markers, whose movement the literature suggested was dynein dependent. As an alternative, a Jurkat cell line expressing fluorescent CTLA4, a known marker for the secretory lysosomes was generated. CTLA4 accumulated at the contact site when Jurkat cells made contact with synthetic target cells. When we repeated the p150[superscript Glued] knockdown in these cells, we found that vesicle transport was blocked, whereas MTOC polarization remained normal. These studies suggest that dynein serves critical roles in both aspects of T cell effector function, the movement of the MTOC up to the cell-cell contact site and the movement of a special class of secretory vesicles up to the MTOC. By the combined processes of MTOC translocation and the minus end-directed movement of vesicles, T cells make it so that a concentrated pool of secretory vesicles are aimed to secrete locally only towards target cells. This ensures that the antigen-specificity of T cell activation is followed by a localized response aimed at the intended target cell. / text
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The Defense Response of Glycine Max to its Major Parasitic Nematode Pathogen Heterodera GlycinesPant, Shankar R 12 August 2016 (has links)
Heterodera glycines, soybean cyst nematode (SCN) causes more than one billion dollars soyben production loss in the U.S. annually. SCN is an obligate parasite of specialized feeding cells within the host root known as syncytium. The SCN resistance genes and signaling pathways in soybean have not been fully characterized. Gene expression analysis in syncytium from compatible and incompatible interactions identified candidate genes that might involve conferring resistance to SCN. This dissertation aimed to investigate the biological functions of the candidate resistance genes to confirm the roles of these genes in resistance to SCN. The study demonstrated a role of syntaxin 31-like genes (Gm-SYP38) in resistance to SCN. Overexpression of Gm-SYP38 induced the transcriptional activity of the cytoplasmic receptor-like kinase BOTRYTIS INDUCED KINASE 1 (Gm-BIK1-6). Overexpression of Gm-BIK1-6 rescued the resistant phenotype. In contrast, Gm-BIK1-6 RNAi increased parasitism. In another experiment, the expression of a Glycine max homolog of LESION SIMULATING DISEASE1 (LSD1) resulted in the transcriptional activation of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and NONEXPRESSOR OF PR1 (NPR1), that function in salicylic acid (SA) signaling, implicating the involvement of the antiapoptotic, environmental response gene LESION SIMULATING DISEASE1 (LSD1) in defense that is demonstrated here. The study also investigated the role of SNARE components (genes functioning in membrane fusion) in resistance to SCN. Experiments showed that SNARE functions in concert with a beta-glucosidase having homology to PEN2 and an ATP binding cassette transporter having homology to PEN3. This study provides novel information for the genetic improvement of soybean for enhanced disease resistance.
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The role of novel pro-viral cellular proteins in the replication of Vaccinia virusHarrison, Kate January 2018 (has links)
Vaccinia virus (VACV), the prototypic poxvirus, undergoes a complex life cycle, with multiple stages that are not yet fully understood. This work studied two cellular proteins which had previously been identified by siRNA screens as playing proviral roles in the replication cycle of VACV: the dual specificity mitogen-activated protein kinase kinase 3 (MKK3) and vacuolar protein sorting 52 (Vps52). MKK3 is an upstream regulator in the p38 pathway which, along with MKK6, phosphorylates and therefore activates p38. In HeLa cell cultures, siRNA depletion experiments confirmed that MKK3 supported VACV replication. MKK3 knockdown reduced production of both early and late-class VACV proteins, suggesting that it facilitates viral gene expression. However, this difference did not translate to an in vivo model, as comparison between wild type and MKK3 knockout mice infected with VACV revealed no significant differences in virus replication or overall disease. The Golgi-associated retrograde protein complex (GARP) is composed of four large heteromeric proteins: Vps51, Vps52, Vps53 and Vps54, and plays a key role in retrograde transport from endosomes to the TGN. The effects of loss of GARP function were investigated using three techniques: mouse embryonic fibroblasts (MEFs) containing the hypomorphic Vps54 “wobbler” mutation, Vps52-targetting siRNA in HeLa cells and pharmacological inhibition of retrograde transport using the drug Retro-2. GARP loss resulted in a marked reduction in VACV spread due to a reduction specifically in “double wrapped” extracellular enveloped virion (EEV) production. Investigation of the mechanism by which GARP facilitates EEV production revealed a disruption of the VACV morphogenesis pathway prior to the double wrapping event, resulting in mislocalisation and aggregation of the viral membrane protein B5 within the cytoplasm. The effects of GARP loss translated to an in vivo model, as mice infected with VACV and treated with Retro-2 exhibited reduced viral replication and overall disease. These results identify GARP as a pro-viral host complex required for EEV production, and suggest that cellular retrograde transport pathways are required for double-wrapping of VACV virions. Overall, the study illustrates both the potential pitfalls of carrying out genetic screens in a transformed cell line and the power of such studies to nevertheless identify novel features of virus biology as well as druggable targets for antiviral intervention.
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Identifying the Effects of a Human Dynein Mutation on GFP-Rab7 Axonal Transport in Embryonic Mouse NeuronsWilson, Natalie E 01 January 2017 (has links)
The first dynein mutation found in humans that caused disease was a cytoplasmic dynein 1 heavy chain (DYNC1H1 in humans) p.His306Arg mutation, first described by Weedon et al. in 2011. This mutation caused Charcot-Marie-Tooth (CMT) subtype 2O. CMT has a prevalence of approximately 1 in 2500 people, making it the most common hereditary neuromuscular disorder. Cytoplasmic dynein 1 is used by eukaryotic cells for minus-end directed microtubule-based transport of cargo. One such cargo is Rab7, a late endosomal marker. The purpose of this study is to identify the effects of this mutation on the transport of GFP-tagged Rab7 cargo in neurons from wild type (HH), heterozygous mutant (HR), and homozygous mutant (RR) mice harboring a DYNC1HI His306Arg mutation. Mouse embryos were euthanized, dissected to collect the hippocampal and cortical brain tissues, and these tissues were digested to isolate neurons. Nucleofection was used to introduce the exogenous GFP-Rab7 gene construct. These neurons were plated and imaged at 10 days in vitro using wide-field epifluorescence microscopy to generate image stacks of fluorescent GFP-Rab7 vesicles. Kymograph analysis was performed on the image stacks using MetaMorph software to measure several characteristics of movement. Statistical analysis of the data from each of the three genotypes shows there is no significant difference in Rab-7 transport between the three genotypes.
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Secretory Homeostasis and Fungal Pathogenesis: Characterization of the Contribution of Calnexin, SrgA, and the IreA Kinase to the Growth and Virulence of Aspergillus fumigatusPowers-Fletcher, Margaret MV 16 September 2013 (has links)
No description available.
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Rolle der d-COP-Untereinheit im frühen sekretorischen Pfad / Role of the d-COP-subunit in the early secretory pathwayRichter, Kora Pauline 16 June 2015 (has links)
No description available.
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The identification and investigation of neurochondrin as a novel interactor of the survival of motor neuron protein, through analysis of the interactomes of Sm family proteins and cell fractionationThompson, Luke January 2018 (has links)
Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.
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Visualization of procollagen IV reveals ER-to-Golgi transport by ERGIC-independent carriers / IV型プロコラーゲン輸送を可視化して解析し、小胞体からゴルジ装置への輸送はERGIC非依存性であることを解明したMatsui, Yuto 24 November 2020 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22832号 / 医博第4671号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 安達 泰治, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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