Dissertations / Theses on the topic 'Secretory granula'

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75.
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.

In neuroendocrine cells, immature secretory granules (ISGs) bud from the trans Golgi network (TGN), and then undergo a maturation process that includes ISG-ISG homotypic fusion, removal of excess membranes via clathrin-coated vesicles (CCVs) and acidification of the granules. These mature secretory granules (MSGs) can then fuse with the plasma membrane upon receiving an external signal, and finally release their content in the extracellular space. In this thesis, I will present evidence that Synaptotagmin IV (Syt IV) is one of the components involved in the regulation of granule maturation in neuroendocrine cells. Syt IV is a neuron and neuroendocrine cell-specific isoform, which belongs to the synaptotagmin family of membrane trafficking proteins. After confirming that Syt IV is localised on ISGs and absent from MSGs in PCI2 cells, I investigated whether Syt IV is involved in ISG homotypic fusion by adding the purified Syt IV cytoplasmic domain into an in vitro assay, where it functions as a dominant negative. Addition of this domain, but not a similar domain from Syt I, resulted in a dose-dependent inhibition of ISG-ISG fusion. Furthermore, I found that Syt IV binds to the ISG-SNARE Syntaxin 6 (Stx6), suggesting that the two proteins might be part of the same machinery that regulates ISG maturation. In addition, I used an in vivo approach based on the processing of secretogranin II (Sgll) into its degradation product pi8, which occurs during ISG maturation, to assay Syt IV function. I show that the Syt IV cytoplasmic domain, as well as siRNA-mediated knockdown of Syt IV inhibits Sgll processing by prohormone convertase 2 (PC2). Interestingly, PC2 is found mostly in the pro-form, suggesting that activation of PC2 is also inhibited. We conclude that Syt IV is an essential component for the process of secretory granule maturation. Lastly, I found that Syt IV binds preferentially to membrane-bound clathrin adaptor protein-1 (AP-1). This suggests that Syt IV might be sorted from maturing SGs in a complex, possibly via an ISG-SNARE protein that interacts directly with AP-1.

Atrial natriuretic factor (ANF) is a polypeptide hormone produced in heart atria, stored in atrial secretory granules and released into the circulation in response to various stimuli. Proper sorting of ANF at the level of the trans-Golgi network (TGN) is required for the storage of ANF in these specific granules, and this sorting of hormones has been found to be associated with G-proteins. Specifically, the Go protein alpha-subunit (Gαo) was established to participate in the stretch-secretion coupling of ANF, but may also be involved in the transporting of ANF from the TGN into atrial granules for storage and maturation. Based on knowledge of Gαo involvement in hormone production in other endocrine tissues, protein-protein interactions of Gαo and proANF and their immunochemical co-localization in granules, the direct involvement of these two proteins in atrial granule biogenesis is probable. In this study, mice were created using the Cre/lox recombination system with a conditional Gαo knockout in cardiocytes to study and characterize ANF production, secretion and granule formation. Deletion of this gene was successful following standard breeding protocols. Characterization and validation of cellular and molecular content of the knockout mice through mRNA levels, protein expression, peptide content, electron microscopy, and electrocardiography determined that a significant phenotypic difference was observed in the abundance of atrial granules. However, Gαo knockout mice did not significantly alter the production and secretion of ANF and only partially prevented granule biogenesis, likely due to incomplete Gαo knockout. These studies demonstrate an involvement of Gαo in specific atrial granule formation.

Orientador: Aureo Tatsumi Yamada
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-07T02:54:51Z (GMT). No. of bitstreams: 1 Copi_Cecilia_M.pdf: 12156283 bytes, checksum: 93ce542963e020dcfb1af6c2bf9d5283 (MD5) Previous issue date: 2006
Resumo: Durante a gestação normal em primatas e em roedores observa-se o acúmulo de linfócitos natural killer no ambiente uterino (uNK). A funções comprovadas das células uNK estão relaciondas com o reconhecimento das células trofoblásticas alogênicas e produção de citocinas imunomoduladoras do ambiente uterino que garantem o sucesso da gestação. Além da produção de citocinas envolvidas na homeostase do ambiente uterino, as uNK produzem e estocam nos seus grânulos moléculas citotóxicas como a perforina e granzimas. Estas moléculas estão envolvidas na resposta imune inata das células NK, com potencial de promover a lise de células alvo. Porém, se ocorre a ativação desta atividade citolítica, ou mesmo se as uNK promovem a secreção deste arsenal de moléculas citolíticas no ambiente uterino em gestação normal, ou em casos de interrupção da gestação, não são conhecidas. Os conhecimentos nesta área não avançam pelas limitações éticas de estudar experimentalmente o ambiente uterino em gestantes humanos e a falta de modelos experimentais estabelecidos em animais laboratoriais. No presente trabalho, propos-se utilizar o modelo experimental de indução de lesões embrionárias para provocar o desequilíbrio do ambiente uterino e avaliar as alterações nas células uNK. Para tanto, foram utilizados camundongos prenhes no 9° dia de gestação que foram submetidas à procedimentos cirúrgicos de lesão do embrião. Amostras uterinas dos sítios de desenvolvimento embrionários de animais de gestação normal, dos animais com embriões lesionados nos intervalos de 10, 30 e 60 minutos póslesão, e dos animais manipulados cirurgicamente sem a lesão embrionária foram coletados e processados para avaliações através de métodos citoquímicos e imunocitoquímicos em microscopia de luz e eletrônica de transmissão. Os sítios uterinos de embriões lesionados apresentaram reação hiperêmica na região mesometrial já aos 10 minutos pós-lesão, que se acentuava nos períodos mais longos de pós-lesão. As células uNK presentes nesta região apresentaram alteração na reatividade pela citoquimica de lectina DBA e immunocitoquímica com a anti-perforina, sendo evidente a gradual redução na intensidade das reações no decurso do tempo pós-lesão. Na análise ultra-estrutural verificou-se a desestruturação dos grânulos lisossomo-secretores, sendo notória a perda do conteúdo do compartimento secretor evidenciada pela reação com o azul-cuprolínico. O compartimento lisossomal do grânulo embora sofra também uma desestruturação pronunciada, mantêm a reatividade para anti-catepsina D, sugerindo a preservação de parte da sua funcionalidade. A perda do conteúdo do compartimento secretor, notadamente a perforina e os proteoglicanos no período de 10 minutos pós-lesão, sugere a degranulação das uNK e portanto a ativação da atividade citotóxica destas células, numa resposta aguda frente ao desequilíbrio do ambiente uterino afetado pela lesão embrionária. Porém, não foi constatado qualquer indício morfológico da ocorrência da degranulação através do mecanismo exocitose-símile ou ainda, o efeito da ação citolítica sobre as células circunjacentes às uNK ativadas
Abstract: In the uterine environment during normal pregnancy of primates and rodents is seen the accumulation of natural killer lymphocites (uNK). The confirmed functions of uNK cells are related to recognition of allogeneic trophoblast cells and production of immunomodulating cytokines of uterine environment that assure the successful pregnancy. Besides the production of cytokines related to homeostasis of uterine environment, the uNK produce and store citotoxic molecules of perforin and granzymes in their granules. These molecules are involved in the innate immune response of NK cells with potential to promote lysis of target cells. However, whether the lytic activity is triggered or even the uNK promotes the secretion of these sets Df cytolitic molecules into the uterus of normal pregnancy or in the miscarriages are not known. The advances of knowledge in this field are slow down due to ethical limitation of experimental studies with human pregnant uterus and lack of well established experimental models in laboratory animal. In the present work it was proposed to use the experimental model of induction of embryo lesions to promote the unbalance of uterine environment and evaluated the changes in uNK cells. Pregnant mice on 9° gestational day were submitted to surgical procedure of embryo lesion and uterine samples from these embryo lesioned sites were collected after 10, 30 and 60 minutes, as did the normal pregnant and sham operated animais. The samples were processed for cytochemical and immunocytochemical evaluations in light and transmission electron microscopy. The embryo lesioned uterine sites showed hyperemic reaction in the mesometrial region as soon as 10 min after lesion which increased as increased the time lapse after lesion. The uNK cells found in these regions showed reactivity changes with DBA lectin cytochemistry and with anti-perforin immunocytochemistry, being noticed the gradual reduction on reaction intensities on course of time after lesion. In the ultrastructural analysis were seen the disruptions of secretory-Iysosomes granules with notorious lost of secretory compartment contents as were evidenced by cuprolinic blue cytochemical reaction. In spite of structural organization of lysosome compartment of the granules was also affected it was maintained the anti-cathepsin D positive reaction which suggest the preservation of part of its functionality. The secretory compartment contents lost, notably the perforin and proteoglycans at 10 min after embryo lesion suggest degranulations of uNK and therefore an acute response of these cells by activation of citotoxic activity, under effects of unbalance of uterine environment affected by embryo lesion. However, it was not detected any morphological evidence of degranulation by exocytosis-like mechanism or even, the cytolitic effect on neighbor cells around the activated uNK cells
Mestrado
Histologia
Mestre em Biologia Celular e Estrutural

Orientador: Áureo Tatsumi Yamada
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-20T04:02:31Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_D.pdf: 2840118 bytes, checksum: 470a2cf0b7d63e81fd8f48aba5af44fc (MD5) Previous issue date: 2012
Resumo: Células Natural Killer uterina (uNK) produzem moléculas angiogênicas e citocinas críticas ao sucesso da gestação , assim como proteínas citolíticas relacionadas à resposta imune inata. Contudo, se as capacidades angiogênicas e citolíticas são provenientes de diferentes subpopulações de células uNK não é conhecido; da mesma forma, estes fenótipos ainda não são estabelecidos. Assim, a proposta inicial deste trabalho foi avaliar...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: Angiogenic and cytokine molecules produced by uterine natural killer (uNK) cells are critical for successful pregnancy. Cytolytic proteins are also express by uNK cells. However, it is unknown whether the angiogenic and cytolytic capacities are from different uNK subsets, or the same cells. Thus, we initially proposed to evaluate...Note: The complete abstract is available with the full electronic document
Doutorado
Histologia
Doutor em Biologia Celular e Estrutural

Secretory granule biogenesis describes the events leading up to the budding of a nascent granule from the trans Golgi network. Literature surrounding secretory granule biogenesis is conflicting and has generated much debate. This thesis aims to address the important issues of this debate by utilizing the insulin-producing liver cell line HUH7-ins. This cell line has been shown to synthesize, store and secrete mature insulin in response to glucose via the possession of secretory granules. Using microarray technology the gene expression profile of HUH7-ins cells was compared with parental HUH7 cells, hoping to identify possible candidate genes contributing to secretory granule biogenesis. 164 genes were shown to be differentially expressed although no known granulogenic protein exhibited a change in expression. The data did suggest a nervous system differentiation event and implicates myosin Vc in the regulated secretion of insulin. HUH7-ins cells express a number of granulogenic protein mRNAs and while chromogranin B (CgB) protein level remained constant upon insulin expression, a significant increase in the level of chromogranin A (CgA) was observed, though the significance of this increase in expression is unknown. The over-expression of CgA in a clone of HUH7-ins that did not possess the regulated secretory pathway was unable to rescue the regulated secretory pathway, suggesting that CgA expression alone is unable to form secretory granules in our model. To determine if the secretory granule biogenesis seen in HUH7-ins cells was specific to insulin, three prohormones of different neuroendocrine origin were over-expressed in HUH7 cells; amylin ( cell), pancreatic polypeptide (pancreatic islet) and proopiomelanocortin (pituitary). None of these prohormones were able to form structures in the cytoplasm that resembled secretory granules by immunofluorescent microscopy, nor did they induce the expression of CgA. No prohormone was detected in cell lysates or conditioned media, raising the possibility that these exogenous prohormone aggregates are trafficked to the lysosomal/endosomal system for degradation. This study provides significant information regarding the genome-wide expression changes induced upon secretory granule biogenesis in a liver cell line, describes the lack of effect of CgA in this event and suggests that secretory granule biogenesis in this liver cell line is specific to insulin.

Secretory processes underpin the emergence of cellular diversity in complex multicellular organisms. However, our understanding of the basic mechanisms controlling the different secretory and endosomal compartments involved remains surprisingly incomplete. During my DPhil I have studied a specialised epithelial cell type in the male Drosophila accessory glands, the secondary cell, which contains unusually large intracellular compartments that are accessible to detailed morphological study. I characterise the organisation, ultrastructure and molecular composition of this cell's secretory and endosomal compartments, and I employ specific Rab GTPases, conserved coordinators of membrane trafficking and identity, to define multiple compartmental subtypes. By developing super-resolution and time-lapse microscopy approaches in these cells, I show that numerous intraluminal vesicles (ILVs) are formed within Rab11-labelled secretory compartments and released into the accessory gland lumen as exosomes, the first clear demonstration in eukaryotic cells of exosome biogenesis within a non-late endosomal compartment. Biogenesis of these ILVs is dependent on evolutionarily conserved Endosomal Sorting Complexes Required for Transport (ESCRT) 0-III genes and involves loading of compartment-specific cargoes. Work by others, some in collaboration with me, has shown that these novel mechanisms are conserved in human cells. I show that dense-core granules, the structures employed to package proteins and other molecules destined for regulated secretion, form within large non-cored Rab6- positive compartments, in a process that seems to involve inputs from both the Golgi and recycling endosomal pathways. Further analysis has revealed roles for specific Rabs, for ILVs, and for the conserved fibrillar protein Mfas/TGFBI in different aspects of DCG formation. I also show that DCGs are not only secreted, but can also be degraded by fusion to acidic endosomal compartments. Remarkably, there is evidence that mammalian cells may employ all of these mechanisms and defects in these processes may be linked to diseases like cancer, diabetes and neurodegenerative disorders. Hence my work has established a new system to study complex secretory mechanisms, which can now be developed to model specific disease processes in the future. In summary, I have discovered several novel cell biological mechanisms controlling exosome biology, dense-core granule biogenesis, regulated secretion, and endolysosomal trafficking. Some of these already appear relevant to human health and disease, suggesting that the secondary cell system has considerable further potential for unravelling the fundamental processes underlying eukaryotic secretion in the future.

Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0°C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared to 37°C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with Bis (sulfosuccinimidyl) suberate (BS3) covalently crosslinked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC using the 1gG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. Intact RMC were pretreated with serine esterase inhibitors prior to 125I-C3a and BS3 exposure. The cells to which 125I-C3a had been crosslinked to were solublized and analyzed by SDS PAGE and autoradiography. There were three bands visualized, a 35,000 dalton band which was defined as chymase, and two undefined 45,000 and 55,000 dalton bands. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan. In addition, this proteolysis of 125I-C3a by chymase must be blocked in order to detect plasma membrane C3a binding components on RMC.

Neospora caninum (filo Apicomplexa) é um parasita obrigatório intracelular como todos os membros deste filo, alguns reconhecidos por causarem doenças com impacto relevante na saúde humana (Plasmodium e Toxoplasma) e veterinária (Babesia, Eimeria e Cryptosporidium). Causador da neosporose, N. caninum vem emergindo como um dos maiores causadores de abortos infecciosos em bovinos, levando a consideráveis perdas econômicas na bovinocultura mundial. Devido à sua recente descoberta, o conhecimento sobre diversos processos bioquímicos de N.caninum ainda é limitado, demandando novas pesquisas para a compreensão de seus mecanismos de sobrevivência e consequente identificação de alvos para intervenção terapêutica. O processo de invasão celular é bastante investigado em pesquisas envolvendo apicomplexas, uma vez que a sobrevivência desses parasitas depende do sucesso de sua entrada na célula hospedeira. Proteínas secretadas de organelas filo-específicas (micronemas, roptrias e grânulos densos) estão intimamente envolvidas com a invasão celular. Elas são responsáveis pela interação inicial com a célula hospedeira, participam da junção de movimento formada no momento da invasão, e contribuem para a estabilização do vacúolo parasitóforo. Neste trabalho as proteínas secretadas por taquizoítas de N. caninum foram investigadas de duas formas: (1) por caracterização molecular de proteínas com domínio Apple; e (2) por estudo do secretoma do parasita. Os domínios proteicos do tipo Apple são caracterizados pela capacidade de interação proteína-proteína e proteína-carboidrato, e estão presentes em algumas proteínas micronêmicas com propriedades adesivas. Neste trabalho três proteínas de N. caninum contendo domínios Apple foram caracterizadas: MIC17A, MIC17B e MIC17C. A análise das sequências proteicas e das estruturas dos domínios Apple, obtidas por modelagem molecular, mostraram alta identidade sequencial e estrutural entre MIC17A e MIC17C. Apesar de ser paráloga às outras duas, MIC17B apresenta diferenças importantes em sua sequência e estrutura. Para MIC17B e MIC17C foram realizados experimentos de detecção das proteínas nativas nos extratos total e secretado do taquizoíta que sugerem diferentes formas de processamento entre essas proteínas no parasita. Para MIC17B foi confirmada a localização em micronemas, num padrão diferente do observado para MIC17C. Os ensaios de invasão combinados aos de localização indicam que estas proteínas estejam relacionadas ao processo de invasão celular, porém, suas funções permanecem desconhecidas. O secretoma é o conjunto de proteínas secretadas pelo parasita e, para explorar a composição deste extrato (ESA) no taquizoíta de N. caninum, duas abordagens complementares foram utilizadas. Na primeira abordagem foram identificadas as proteínas presentes no ESA por espectrometria de massas. Na segunda abordagem realizou-se uma ii quantificação relativa das proteínas, marcadas por dois isótopos, nos extratos totais de taquizoítas submetidos ou não ao estímulo secretório. O resultado esperado seria com as proteínas secretadas diminuídas no parasita estimulado. Em ambas as abordagens foram utilizadas técnicas de espectrometria de massas de alta resolução (nanoLC-MS/MS), o que resultou num alto número de identificações; 615 proteínas no ESA e 2011 proteínas quantificadas. A comparação das duas abordagens permitiu o reconhecimento de proteínas com maior probabilidade de secreção. Uma rede de interação entre as proteínas diferencialmente expressas foi predita, gerando resultados que, associados às informações sobre as proteínas aumentadas, permitiram uma investigação sobre proteínas potencialmente envolvidas com a regulação do metabolismo relacionado à secreção. Os resultados obtidos por ambos os estudos aqui demonstrados somam conhecimento acerca do parasita N. caninum e demonstram ser úteis para guiar a busca e seleção de alvos a serem investigados para o desenvolvimento de terapêutica contra a neosporose.
Neospora caninum (Apicomplexa phylum) is an obligatory intracellular parasite like all members from this phylum, some causing diseases with relevant impact on human (Plasmodium and Toxoplasma) and veterinary (Babesia, Eimeria and Cryptosporidium) health. Causative agent of neosporosis, N. caninum has emerged as one of the leading causes of infectious abortion in cattle, generating huge economical losses in worldwide livestock. Due to its recent discovery, knowledge of N. caninum biochemical processes remains scarce, demanding new research for comprehending its survival mechanisms and, consequently, identifying new targets for therapeutic intervention. The invasion process has often been investigated in apicomplexans since their survival depends on the success of their entry into the host cell. Proteins secreted from phylum-specific organelles (micronemes, rhoptries and dense granules) are deeply involved with invasion. They are responsible for the initial interaction with the host cell; participate of the moving junction formed in the moment of invasion; and contribute for the stabilization of the parasitophorus vacuole. In this study, the proteins secreted by N. caninum tachyzoites were investigated in two ways: (1) the molecular characterization of Apple domaincontaining proteins; and (2) exploring the parasite secretome. The Apple protein domains are characterized by the ability to interact as protein-protein and proteincarbohydrate, and are present in some microneme proteins with adhesive properties. Here three N. caninum proteins containing Apple domains were characterized: MIC17A, MIC17B and MIC17C. Analyses of the Apple domains sequences and structures, obtained by molecular modeling, revealed high sequential and structural identities between MIC17A and MIC17C. Although being a paralog of the other two proteins, MIC17B presents significant differences in its sequence and structure. Experiments were performed for native MIC17B and MIC17C detection in the total and secreted tachyzoite extracts, suggesting different processing forms for these proteins in the parasite. For MIC17B, the microneme localization was confirmed, differently from the pattern observed for MIC17C. Invasion and localization assays indicated that these proteins are related to the cell invasion process; nevertheless, their functions remain unknown. The secretome is the set of proteins secreted by the parasite and, to explore this extract (ESA) composition in N. caninum, two complementary approaches were used. Firstly proteins present in ESA were identified by mass spectrometry. In the second approach, a relative quantification was performed on the proteomes of ethanol stimulated/non stimulated tachyzoites, expecting that the secreted proteins would be down regulated at the stimulated parasite. Both approaches were performed with high resolution mass spectrometry techniques (nanoLC-MS/MS), reaching a high number of identifications: 615 proteins iv in ESA and 2011 quantified proteins. The comparison between both approaches allowed the recognition of the most likely secreted proteins. An interaction network was predicted, involving the differentially expressed proteins. These results, associated with the information of up regulated proteins, allowed the investigation of proteins potentially involved with the secretion metabolism regulation. The findings from our two studies add up knowledge about N. caninum and demonstrate to be useful in guiding the search and selection for new targets for therapeutic development against neosporosis.

Pancreas is known to be an organ producing a variety of exocrine and endocrine substances, where also insulin belongs. This hormone is produced in the body almost solely by specialized β-cells of the Langerhans islets and is stored here in secretory granules. As the β-cells contain large number of these vesicles, an organism can quickly respond to the glucose stimulation. Completely processed insulin is formed in the secretory granules probably as a hexamer, where six insulin molecules are coordinated along two zinc bivalent cations. Appropriate β-cell response to higher glucose level and following insulin secretion is one of the key processes that regulate metabolism in the body. In order to study insulin production, its effects or secretion, permanent pancreatic cell lines are often used as biological models, out of primary cells from islets of Langerhans. This diploma thesis is focused on two permanent cell lines INS-1E and BRIN-BD11. We searched for the ability of the cells to produce insulin, if the hormone is fully processed, as well as zinc content, which could have a great influence on insulin's processing. Using different methods we compared these two cell lines with cells from the Langerhans islets. We succeeded in isolation of secretory granules from all three cell types and we plan to...

abstract: There is increasing interest and demand in biology studies for identifying and characterizing rare cells or bioparticle subtypes. These subpopulations demonstrate special function, as examples, in multipotent proliferation, immune system response, and cancer diagnosis. Current techniques for separation and identification of these targets lack the accuracy and sensitivity needed to interrogate the complex and diverse bioparticle mixtures. High resolution separations of unlabeled and unaltered cells is an emerging capability. In particular, electric field-driven punctuated microgradient separations have shown high resolution separations of bioparticles. These separations are based on biophysical properties of the un-altered bioparticles. Here, the properties of the bioparticles were identified by ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. As part of this dissertation, high-resolution separations have been applied to neural stem and progenitor cells (NSPCs). The abundance of NSPCs captured with different range of ratio of EK to DEP mobilities are consistent with the final fate trends of the populations. This supports the idea of unbiased and unlabeled high-resolution separation of NSPCs to specific fates is possible. In addition, a new strategy to generate reproducible subpopulations using varied applied potential were employed for studying insulin vesicles from beta cells. The isolated subpopulations demonstrated that the insulin vesicles are heterogenous and showed different distribution of mobility ratios when compared with glucose treated insulin vesicles. This is consistent with existing vesicle density and local concentration data. Furthermore, proteins, which are accepted as challenging small bioparticles to be captured by electrophysical method, were concentrated by this technique. Proteins including IgG, lysozyme, alpha-chymotrypsinogen A were differentiated and characterized with the ratio factor. An extremely narrow bandwidth and high resolution characterization technique, which is experimentally simple and fast, has been developed for proteins. Finally, the native whole cell separation technique has also been applied for Salmonella serotype identification and differentiation for the first time. The technique generated full differentiation of four serotypes of Salmonella. These works may lead to a less expensive and more decentralized new tool and method for transplantation, proteomics, basic research, and microbiologists, working in parallel with other characterization methods.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2020