Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión
Dynamin is a GTPase required for the vesicle pinching off from the plasma membrane during the late steps of endocytosis. Growing evidences suggest that dynamin also participates duringregulated exocytosis; however its specific contribution is still elusive. Here, using bovine chromaffincells, we sho...
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| Format: | Doctoral thesis |
| Status: | Published version |
| Publication date: | 2013 |
| Country: | Chile |
| Institution: | ANID |
| Repository: | Tesis_ANID |
| Language: | English |
| OAI Identifier: | oai:repositorio.anid.cl:10533/179881 |
| Online Access: | http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ https://hdl.handle.net/10533/179881 |
| Access Level: | Open access |
| _version_ | 1878404583348240384 |
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| author | González-Jamett, Arlek Marion |
| author_browse | González-Jamett, Arlek Marion |
| author_facet | González-Jamett, Arlek Marion |
| author_role | author |
| bitstream_url_str_mv | https://repositorio.anid.cl/bitstreams/39651906-e895-46c6-9223-d838a0c739be/download https://repositorio.anid.cl/bitstreams/4b46eee1-918e-45c3-9a6f-2d6504eb759a/download https://repositorio.anid.cl/bitstreams/7e2b95fe-3eb8-477c-a647-cfabcf57a6d0/download |
| collection | Tesis_ANID |
| description | Dynamin is a GTPase required for the vesicle pinching off from the plasma membrane during the late steps of endocytosis. Growing evidences suggest that dynamin also participates duringregulated exocytosis; however its specific contribution is still elusive. Here, using bovine chromaffincells, we show for the first time that the ubiquitously expressed isoform dynamin-2 controls differentstages of the exoytosis in a stimulus-dependent manner. Thus, we observed that the inhibition of dynamin-2 GTP-ase activity by the expression of a dominant negative mutant (Dyn2K44A), or theknock-down of endogenous dynamin-2 by small interfering RNA (iRNADyn2) prolonged the lifetime of the initial fusion pare, increased the catecholamine quantal release and lengthened the duration of the exocytotic events evoked by low concentration of the nicotinic agonist 1, 1-dimethyl-4-phenylpiperazinium (DMPP) (10 uM), but not during exocytotic events triggered by higher DMPP concentration (50 uM).The expression of Dyn2K44A and iRNADyn2 also dramatically disrupted cortical actin organization by displaying a punctuated pattern. Moreover, the acute inhibition of the dynamin GTP-ase activity with dynasore or dynole 34-2 completely suppressed the de novo Ca2+-dependent polymerization, resembling the effects of cytochalasin D (CytoD), an actin-disrupting drug. The treatment ofchromaffin cells with CytoD also increased the lifetime of the fusion pare, the quantal size and duration of the exocytotic events induced by 10 uM DMPP, without affecting the exocytosis induced by 50 uM DMPP. The effects of dynamin-2 disruption and cytochalasin D on the exocytosis were non-additive, suggesting that dynamin-2 and F-actin work through a common pathway controllingdifferent stages of the exocytosis. |
| eu_rights_str_mv | openAccess |
| format | doctoralThesis |
| id | CL_9fbe00a40d4844d7aa250b92b687320e |
| institution | ANID |
| instname_str | Agencia Nacional de Investigación y Desarrollo |
| language | eng |
| network_acronym_str | CL |
| network_name_str | Chile |
| oai_identifier_str | oai:repositorio.anid.cl:10533/179881 |
| publishDate | 2013 |
| publishDateSort | 2013 |
| reponame_str | Tesis_ANID |
| spelling | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión González-Jamett, Arlek Marion Dynamin is a GTPase required for the vesicle pinching off from the plasma membrane during the late steps of endocytosis. Growing evidences suggest that dynamin also participates duringregulated exocytosis; however its specific contribution is still elusive. Here, using bovine chromaffincells, we show for the first time that the ubiquitously expressed isoform dynamin-2 controls differentstages of the exoytosis in a stimulus-dependent manner. Thus, we observed that the inhibition of dynamin-2 GTP-ase activity by the expression of a dominant negative mutant (Dyn2K44A), or theknock-down of endogenous dynamin-2 by small interfering RNA (iRNADyn2) prolonged the lifetime of the initial fusion pare, increased the catecholamine quantal release and lengthened the duration of the exocytotic events evoked by low concentration of the nicotinic agonist 1, 1-dimethyl-4-phenylpiperazinium (DMPP) (10 uM), but not during exocytotic events triggered by higher DMPP concentration (50 uM).The expression of Dyn2K44A and iRNADyn2 also dramatically disrupted cortical actin organization by displaying a punctuated pattern. Moreover, the acute inhibition of the dynamin GTP-ase activity with dynasore or dynole 34-2 completely suppressed the de novo Ca2+-dependent polymerization, resembling the effects of cytochalasin D (CytoD), an actin-disrupting drug. The treatment ofchromaffin cells with CytoD also increased the lifetime of the fusion pare, the quantal size and duration of the exocytotic events induced by 10 uM DMPP, without affecting the exocytosis induced by 50 uM DMPP. The effects of dynamin-2 disruption and cytochalasin D on the exocytosis were non-additive, suggesting that dynamin-2 and F-actin work through a common pathway controllingdifferent stages of the exocytosis. PFCHA-Becas Doctor en Ciencias Mención Neurociencia 135p. TERMINADA http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ https://hdl.handle.net/10533/179881 |
| spellingShingle | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión González-Jamett, Arlek Marion |
| status_str | publishedVersion |
| title | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión |
| title_full | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión |
| title_fullStr | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión |
| title_full_unstemmed | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión |
| title_short | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión |
| title_sort | Dynamin-2 regulates the late steps of exocytosis in adrenal chromaffin cells through a mechanism that involves actin polymerizatión |
| url | http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ https://hdl.handle.net/10533/179881 |