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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2019.tde-01062020-074350
Document
Author
Full name
Johnny Alex Rockenbach Zenzen
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2020
Supervisor
Committee
Espreafico, Enilza Maria (President)
Annichini, Maria Sol Brassesco
Santos, Aline Mara dos
Santos, Marinilce Fagundes dos
Title in Portuguese
Miosina-Va auxilia no transporte de Dinamina-2 e Clatrina para as adesões focais e promove sua desmontagem
Keywords in Portuguese
Adesão focal
Dinamina
FAK
Miosina-Va
Abstract in Portuguese
A montagem e desmontagem das adesões focais (AFs) desempenham um papel fundamental em vários processos celulares, incluindo migração celular, proliferação e sobrevivência. As adesões focais são desmontadas na parte traseira e montadas na parte dianteira, durante a migração celular. Resultados anteriores do nosso laboratório mostram que a perda ou redução da expressão de miosina-Va (MVa) fibroblastos e células de melanoma causam uma redução significativa nas taxas de migração e invasão. Aqui demonstramos que os fibroblastos silenciados para MVa sofrem um atraso dramático na desmontagem das AFs, o que implica que a atividade motora da MVa é essencial para promover a desmontagem. A desmontagem é necessária para que ocorra a reciclagem de componentes das AFs, permitindo que a célula remonte novas AFs na frente migratória e migre corretamente. Os microtúbulos apresentam um papel fundamental na desmontagem das AFs e são necessários para levar proteínas para as AFs, como a dinamina-2, uma GTPase necessária para a endocitose da integrina mediada por clatrina. Os resultados prévios do meu mestrado mostraram que a miosina-V fosforilada se localiza nas AFs, durante a rápida formação dessas estruturas induzidas por estímulo com soro fetal bovino. Uma vez que ocorre a formação das AFs em fibroblastos deficientes para MVa, formulamos a hipótese de que a localização de miosina-Va nas AFs é necessária para fornecer fatores de desmontagem e / ou sinalização, resultando na formação de estruturas mais dinâmicas do que as formadas na ausência de MVa. O presente projeto propôs monitorar a dinâmica molecular das AFs: montagem, desmontagem e reciclagem, em células controle e deficientes em MVa. Mostramos aqui que componentes do soro fetal bovino, como EGF e LPA induzem a fosforilação da MVa em células quiescentes e que a MVa auxilia no transporte de Dinamina-2 e Clatrina para as adesões focais e promove sua desmontagem.
Title in English
Myosin-Va assists in the transport of Dynamin-2 and Clathrin to focal adhesions and promotes their disassembly
Keywords in English
Dynamin
FAK
Focal adhesions
Myosin Va
Abstract in English
Assembly and disassembly of focal adhesions (FAs) play a key role in many cellular processes, including cell migration, proliferation and survival. Focal adhesions are disassembled at the rear and mounted at the front during cell migration. Previous results from our laboratory show that the loss or reduction of myosin-Va (MVa) expression in fibroblast and melanoma cells causes a significant reduction in migration and invasion rates. Here we demonstrate that MVa knockdown fibroblasts experience a dramatic delay in disassembly of FAs, implying that the motor activity of MVa is essential to promote FA disassembly. Disassembly is required for FA component recycling to occur, allowing the cell to reassemble new FAs on the migratory front and migrate properly. Microtubules play a key role in disassembling FAs, they are required to deliver proteins into FAs, such as dinamine-2, a GTPase necessary for clathrin-mediated integrin endocytosis. Previous results from my master's degree dissertation showed that phosphorylated myosin-V is located in FAs during the rapid formation of these structures after fetal bovine serum stimulation. Since FA formation occurs in MVa deficient fibroblasts, we hypothesize that localization of MVa in FAs is necessary to provide disassembly and / or signaling factors, resulting in the formation of more dynamic structures than those formed in the absence of myosin-Va. This project proposed to monitor the molecular dynamics of FAs: assembly, disassembly and recycling, in control and in myosin-Va deficient cells. We show here that components of fetal bovine serum, such as EGF and LPA, induced the phosphorylation of MVa in quiescent cells and that MVa assists in the transport of Dynamin-2 and Clathrin to focal adhesions and promotes their disassembly.
 
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Publishing Date
2020-07-13
 
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