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Analysis of mammalian Rab27a and Rab27b and implications for diseaseRamalho, Jose da Silva January 2001 (has links)
No description available.
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Membrane Dynamics During CytokinesisGudejko, Heather F.M. January 2013 (has links)
Thesis advisor: David R. Burgess / Cytokinesis is the final step in cell division, culminating in the formation of two daughter cells from a single mother cell. Previous studies from our lab have shown that lipid rafts are dynamic during cytokinesis in sea urchin embryos, migrating into the ingressing cleavage furrow then moving back outwards towards the poles prior to abscission. Here, I quantitated the mobility of GM1, a ganglioside enriched in lipid rafts, using cholera toxin subunit B (CTB). Despite previous observations of raft movement during cell division, I have found lipid rafts to be immobile throughout the cell cycle. Lipid raft stability is dependent on the activity of myosin light chain kinase (MLCK), most likely due to the dramatic reorganization of actin filaments upon MLCK inhibition. While further investigating the immobility of lipid rafts during cytokinesis using confocal microscopy, I have found that new membrane is added to the cell poles during anaphase, causing the plasma membrane to expand coincident with the constriction of the contractile ring. This membrane addition is dependent on actin and astral microtubules and occurs significantly earlier during mitosis than membrane addition at the furrow. The membrane that is added at the polar regions is compositionally distinct from the original cell membrane in that it is devoid of GM1, a component of lipid rafts. I also found that Rab11 vesicles are trafficked to the polar plasma membrane during the time of this new membrane event, suggesting that the growth of the plasma membrane at the cell poles during cell division is not due to stretching as previously thought, but due to the addition of new membrane through exocytosis. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
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Investigating the Role of Fwd and Potential Role of the Rab11-interacting Protein dRip11 in Drosophila Spermatocyte CytokinesisCyprys, Anya 25 July 2012 (has links)
Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.
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Investigating the Role of Fwd and Potential Role of the Rab11-interacting Protein dRip11 in Drosophila Spermatocyte CytokinesisCyprys, Anya 25 July 2012 (has links)
Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.
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Molecular mechanisms of biphasic insulin secretionGandasi, Nikhil R. January 2015 (has links)
Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.
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Characterisation of LITAF, a protein associated with Charcot-Marie-Tooth disease type 1CHo, Hon Kwan January 2018 (has links)
Charcot-Marie-Tooth disease (CMT) is the commonest inherited neuromuscular disorder, which affects the peripheral nervous system leading to nerve degeneration. CMT is categorised into two forms, ‘axonal’ and ‘demyelinating’, which reflects the main site of pathology as the axon or Schwann cells respectively. Over 90 genes have been identified associated with the disease. Among the genes associated with demyelinating CMT, the focus of my thesis is LITAF, mutations in which lead to an autosomal dominant demyelinating CMT known as CMT type 1C. LITAF is a 17 kDa protein likely to be involved in endocytic degradation and trafficking of specific cargo proteins. It contains an N-terminal proline-rich region mediating protein-protein interactions and a C-terminal LITAF domain consisting of a zinc-ribbon structure with a hydrophobic region incorporated. Most of the CMT1C mutations are clustered in this highly conserved LITAF domain. LITAF was predicted to play roles in recruiting ESCRT components and exosome formation, but the precise function remains unclear. Furthermore, why mutations in LITAF lead to CMT is not known. My work therefore focused on characterising the function of the LITAF protein both in health and disease. In my thesis, I first tried to determine the subcellular localisation of LITAF proteins and also investigated the function of the highly conserved C-terminal LITAF domain in targeting protein to the membrane. With regards to the function of LITAF, potential binding partners were screened using the traditional GST pull-down assay and the in-situ proximity labelling assay, BioID. A number of novel potential binding partners were identified in both assays. Among the list of potential binding partners, BAG3 was captured in both pull-down assays and was chosen for further studies. The interaction with LITAF was characterised and the potential role of this interaction in autophagy was investigated. Integrin, which was also captured in the BioID assay, was another protein chosen for further studies. Internalisation and recycling assay were developed to investigate the function of LITAF in integrin trafficking. The potential of CMT mutations in impairing the internalisation of integrin in A431 cells and the difficulty in performing the assays were discussed. Lastly, with patient fibroblasts available in our lab, disease phenotypes were analysed using two types of imaging technique: transmission electron microscopy (TEM) (in collaboration with J. Edgar) and immunofluorescence microscopy. Swollen vacuoles were observed in the TEM images of the patient fibroblasts only, and various uptake assays were performed to identify these enlarged compartments. In summary, this work offers an insight into the function of LITAF both in health and disease as well as identifying potential intracellular binding partners that might shed light on pathogenesis. Furthermore, the modified trafficking assays described in this thesis can be applied to Schwann cell and patient fibroblasts, providing further tools to probe the underlying membrane trafficking pathways that are dysfunctional in CMT.
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The involvement of ARF6 in rapid membrane recycling during Drosophila spermatocyte cytokinesis / Die Bedeutung von ARF6 für das rapide Membranrecycling während der Cytokinese der Spermatocyten von DrosophilaFoster, Naomi 14 February 2007 (has links) (PDF)
Cytokinesis involves constriction of the cell at the equator. Without decreasing in volume, a spherical cell requires a net increase in the surface area during this constriction. The constriction is driven by formation of an actomyosin contractile ring, and the surface increase by addition of membrane during the formation of the cleavage furrow. Both events depend on the central spindle microtubules at the midzone of the spindle and, in particular, on the centralspindlin protein complex. The communication between the central spindle microtubules and the actomyosin ring involves binding of a GAP and a GEF for RhoA to the centralspindlin kinesin Pavarotti/MKLP1. However, it is still unclear which molecular machinery connects the mitotic spindle to membrane trafficking during cleavage furrow ingression. ARF6 is a member of the ARF family of small GTPases, and previous studies suggest that it is an important regulator of membrane trafficking through the endocytic pathway, and cortical Actin remodelling. I generated an arf6 null mutant in Drosophila. arf6 null mutants survive to adulthood without obvious morphological defects, indicating that ARF6 is not required for Drosophila somatic development. However, ARF6 is required for cytokinesis in Drosophila spermatocytes. The centralspindlin kinesin Pavarotti, identified as an ARF6 interactor in a Yeast-2-Hybrid assay, binds ARF6 in GST pulldowns, and interacts genetically with the arf6 mutant. ARF6 localizes to the plasma membrane and a population of early and recycling endosomes. During cytokinesis, ARF6 is enriched on recycling endosomes at the central spindle. arf6 mutants form a cleavage furrow during cytokinesis, which later regresses. Cytokinesis in arf6 mutant spermatocytes lacks the rapid plasma membrane expansion observed during normal divisions. The results of this study suggest that ARF6 might promote rapid recycling of endosomal membrane stores at the central spindle to the plasma membrane during cytokinesis. ARF6 might be recruited to the central spindle via its interaction with Pavarotti, and act as part of the molecular link between the central spindle cytoskeleton and the rapid plasma membrane addition necessary for cytokinesis. Für die Ansicht der quick-time-Movies mit der Endung "avi" ist die Installation des "Apple QuickTime-Players" erforderlich.
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Quantitative Analysis of Synaptic Vesicle Membrane TraffickingSeitz, Katharina Johanna 10 August 2017 (has links)
No description available.
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Hypoxia stimulates retrograde membrane trafficking to the trans-Golgi network via recruitment of T-plastinNaas, Stephanie 25 October 2018 (has links)
No description available.
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Identification et caractérisation de GTPases Activating Proteins spécifiques à la petite GTPase RAB21 / Identification and characterization of GTPases Activating Proteins specific to the small GTPase RAB21Normandin, Caroline January 2017 (has links)
L’autophagie est un processus de dégradation et de recyclage des composés cellulaires. Ce mécanisme est nécessaire que ce soit à l’état basal pour éliminer des agrégats protéiques ou des organites endommagés ou en condition de stress, tels que la carence nutritionnelle, l’hypoxie ou encore des traitements anticancéreux. De ce fait, l’autophagie est un processus essentiel à la survie ainsi qu’au maintien de l’homéostasie cellulaire. Connaître les joueurs et comprendre les mécanismes de régulation de l’autophagie sont donc importants. Les GTPases RABs sont des régulateurs importants de ce processus. Celles-ci agissent comme des interrupteurs moléculaires permettant d’exécuter rapidement des fonctions dans la cellule. Les RABs sont activées par des Guanine Nucleotide Exchange Factors (GEF) alors que les GTPase Activating Proteins (GAP) accélèrent la désactivation de la RAB. RAB21 est essentielle dans les étapes tardives de l’autophagie. En effet, RAB21 est activée par la carence nutritionnelle, via sa GEF MTMTR13, et permet le trafic d’une SNARE requise pour le flux autophagique. Lors d’une carence prolongée, l’activité de RAB21 diminue rapidement, suggérant ainsi le rôle d’une GAP dans cette régulation négative. Toutefois, aucune GAP pour RAB21 n’a été identifiée jusqu’à maintenant. Un criblage génétique chez la drosophile a permis d’identifier quelques candidats. Suite à des essais d’interactions protéiques, il s’est avéré que seule la GAP TBC1D25 interagissait avec RAB21. De plus, cette interaction est augmentée en fonction de la carence nutritionnelle. Des immunofluorescences par microscopie confocale ont révélé que l’interaction RAB21-TBC1D25 était située en partie au niveau des endosomes précoces. Par ailleurs, une activation prolongée de RAB5, située sur les endosomes précoces, inhibe l’interaction RAB21-TBC1D25. De plus amples expériences devront être réalisées afin d’expliquer ces résultats. Dans un autre ordre d’idée, RAB21 est surexprimée dans les cellules ayant un flux autophagique élevé ainsi que dans certaines tumeurs de cancer du côlon (données non publiées du laboratoire). L’expression de Tbc1d25 dans ces mêmes tumeurs ne semble pas augmentée, indiquant que TBC1D25 pourrait être un inhibiteur autophagique spécifique aux cellules ayant un flux autophagique élevé. À la lumière des résultats obtenus, TBC1D25 semble être une GAP pour RAB21 qui permet sa régulation négative suivant l’activation de l’autophagie induite par la carence nutritionnelle. / Abstract : Autophagy is defined as the lysosomal degradation and recycling of cellular constituents. At basal levels, autophagy eliminates protein aggregates or damaged organelles. In condition of stress, such as in condition of nutritional deficiency, hypoxia or cancer treatments, autophagy allow cells to adapt and survive. Therefore, autophagy is an essential system required for survival and maintenance of cellular homeostasis. It is thus essential to identify the cellular entities and mechanisms regulating this process. RAB GTPases were identified as master regulators of autophagy. These particular proteins act as molecular switches for the rapid execution of cellular responses. RABs are activated by Guanine Nucleotide Exchange Factors (GEF) whereas GTPase Activating Proteins (GAP) accelerates RAB deactivation. RAB21 is essential in the late stages of autophagy. Indeed, RAB21 is activated by nutritional deficiency, via its GEF MTMTR13, to allow trafficking of a SNARE required for autophagic flux. During starvation, RAB21 is deactivated which suggest that a GAP could negatively regulate RAB21 activity. However, to date no GAP for RAB21 has been identified. An eye modifier genetic screen in Drosophila was performed to identify potential RAB21 GAPs and some candidates were identified. As a result of this screen, the GAP TBC1D25 was identified as interacting with RAB21. Moreover, this interaction was increased by starvation. Proximity ligation assays revealed that the RAB21-TBC1D25 interaction partially localized at early endosomes. Moreover, prolonged activation of RAB5, located at early endosomes, inhibited RAB21-TBC1D25 interaction. Further experiments will be carried out to explain these results. With respect to the roles of autophagy in cancer, RAB21 was shown to be overexpressed in cells with high autophagic flux as well as in some colon cancer tumors. Importantly, the expression of Tbc1d25 in these same tumors does not appear to be increased, indicating that TBC1D25 could be an autophagic inhibitor specific to cells with a high autophagic flow. My work suggests that TBC1D25 could function as a GAP to negatively regulate RAB21 activity in condition of prolonged starvation.
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