Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking

Endomembrane trafficking (ET) is required for maintaining cell homeostasis in eukaryotes. Endocytic recycling, endomembrane vesicular transport, autophagy, mitophagy are considered important aspects of ET process. LRRK2/LRK-1, PINK1/PINK-1, two genes associated with hereditary Parkinson?s Disease, a...

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Detalles Bibliográficos
Autor principal: Gangurde, Dipak
Otros Autores: Simonetta, Sergio
Formato: Tesis de maestría acceptedVersion
Lenguaje:Inglés
Publicado: Facultad de Farmacia y Bioquímica 2015
Materias:
PINK-1
LRK-1
Parkinson hereditario
Parkinson?s Disease
Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2893
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2893.dir/2893.PDF
Aporte de:
Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_2893
record_format dspace
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Inglés
orig_language_str_mv eng
topic PINK-1
LRK-1
Parkinson hereditario
PINK-1
LRK-1
Parkinson?s Disease
Ciencias de la vida
spellingShingle PINK-1
LRK-1
Parkinson hereditario
PINK-1
LRK-1
Parkinson?s Disease
Ciencias de la vida
Gangurde, Dipak
Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking
topic_facet PINK-1
LRK-1
Parkinson hereditario
PINK-1
LRK-1
Parkinson?s Disease
Ciencias de la vida
description Endomembrane trafficking (ET) is required for maintaining cell homeostasis in eukaryotes. Endocytic recycling, endomembrane vesicular transport, autophagy, mitophagy are considered important aspects of ET process. LRRK2/LRK-1, PINK1/PINK-1, two genes associated with hereditary Parkinson?s Disease, and SGK1/SGK-1, in distinct experimental settings, have been suggested to play either direct or indirect roles in regulating these processes. Previous work of my lab had indicated that these genes and their encoded proteins may be mechanistically linked. In addition, C. elegans PINK-1 and LRK-1 have been shown to function antagonistically in stress response and neurite outgrowth and also interaction between SGK-1 and hLRRK2/CeLRK-1 has been suggested in controlling cytoskeletal dynamics which may affect endocytosis and intracellular cell sorting. Our hypothesis is that PINK1, LRRK2, and SGK1 may share a common function in ET. Currently available readouts for the function of these genes do not relate to ET. Studying genetic interaction of these genes in C. elegans requires screenable phenotype associated with ET, and this was the focus of my master project.\nIn this work with mutant analysis and pharmacological studies I have found several new phenotypic readouts using pink-1 mutant animals. Vacuole phenotype observed in pink-1 animals was found to be rescued by loss of lrk-1 in consistency with the anatagonistic function of PINK-1 and LRK-1, although present results suggest that this vacuole phenotype may be independent of ET perturbations. In another readout, modulation of RAB-10 marker protein (rab-10::gfp) in the intestinal cells of pink-1 animals suggested the potential role of PINK-1 in controlling transport of early endosomes during endomembrane trafficking. Furthermore I have also used the vacuole phenotype in SGK-1 transgene for an RNAi test to study putative interaction between LRK-1, PINK-1 and SGK-1, resulting in the identification of several modulators of this phenotype. Moreover lrk-1 downregulation was sufficient to reduce the levels of transgenic SGK-1 expression, suppressing its phenotypic consequences. This data further support the existence of a mechanistic connection between LRK-1 and SGK-1.
author2 Simonetta, Sergio
author_facet Simonetta, Sergio
Gangurde, Dipak
format Tesis de maestría
Tesis de maestría
acceptedVersion
author Gangurde, Dipak
author_sort Gangurde, Dipak
title Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking
title_short Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking
title_full Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking
title_fullStr Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking
title_full_unstemmed Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking
title_sort establishing a readout for validating genetic interactions of c. elegans pink-1, lrk-1 and sgk-1 and their putative roles in regulating endomembrane trafficking
publisher Facultad de Farmacia y Bioquímica
publishDate 2015
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2893
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2893.dir/2893.PDF
work_keys_str_mv AT gangurdedipak establishingareadoutforvalidatinggeneticinteractionsofceleganspink1lrk1andsgk1andtheirputativerolesinregulatingendomembranetrafficking
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spelling I28-R145-HWA_28932020-02-04 Endomembrane trafficking (ET) is required for maintaining cell homeostasis in eukaryotes. Endocytic recycling, endomembrane vesicular transport, autophagy, mitophagy are considered important aspects of ET process. LRRK2/LRK-1, PINK1/PINK-1, two genes associated with hereditary Parkinson?s Disease, and SGK1/SGK-1, in distinct experimental settings, have been suggested to play either direct or indirect roles in regulating these processes. Previous work of my lab had indicated that these genes and their encoded proteins may be mechanistically linked. In addition, C. elegans PINK-1 and LRK-1 have been shown to function antagonistically in stress response and neurite outgrowth and also interaction between SGK-1 and hLRRK2/CeLRK-1 has been suggested in controlling cytoskeletal dynamics which may affect endocytosis and intracellular cell sorting. Our hypothesis is that PINK1, LRRK2, and SGK1 may share a common function in ET. Currently available readouts for the function of these genes do not relate to ET. Studying genetic interaction of these genes in C. elegans requires screenable phenotype associated with ET, and this was the focus of my master project.\nIn this work with mutant analysis and pharmacological studies I have found several new phenotypic readouts using pink-1 mutant animals. Vacuole phenotype observed in pink-1 animals was found to be rescued by loss of lrk-1 in consistency with the anatagonistic function of PINK-1 and LRK-1, although present results suggest that this vacuole phenotype may be independent of ET perturbations. In another readout, modulation of RAB-10 marker protein (rab-10::gfp) in the intestinal cells of pink-1 animals suggested the potential role of PINK-1 in controlling transport of early endosomes during endomembrane trafficking. Furthermore I have also used the vacuole phenotype in SGK-1 transgene for an RNAi test to study putative interaction between LRK-1, PINK-1 and SGK-1, resulting in the identification of several modulators of this phenotype. Moreover lrk-1 downregulation was sufficient to reduce the levels of transgenic SGK-1 expression, suppressing its phenotypic consequences. This data further support the existence of a mechanistic connection between LRK-1 and SGK-1. Fil: Gangurde, Dipak. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Simonetta, Sergio Facultad de Farmacia y Bioquímica Baumeister, Ralf Gangurde, Dipak 2015-06-01 El tráfico endomembranoso (TE) es necesario para el mantener la homeostasis celular en eucariontes. El reciclaje endocítico, el transporte vesicular endomembranoso, la autofagia y mitofagia se consideran aspectos importantes en el proceso de TE. Y se ha propuesto que LRRK2/LRK-1, PINK1/PINK-1, genes asociados a la enfermedad de Parkinson hereditaria; y SGK1/SGK-1 desempeñan un rol directo o indirecto en la regulación de este proceso en distintas condiciones experimentales. Trabajos previos realizados en mi grupo han demostrado que estos genes y las proteinas que codifican podrían estar implicadas en mismo mecanismo de interacción. Por otra parte se demostró que PINK-1 y LRK-1 en C. elegans funcionan antagónicamente en respuesta al estrés y en el crecimiento de neuritas, también se ha propuesto que la interacción entre SGK-1 and hLRRK2/CeLRK-1 participa en el control de la dinámica del citoesqueleto que a su vez afecta la endocitosis y clasificación celular interna. Nuestra hipótesis es que PINK1, LRRK2 y SGK1 pueden compartir una función común en el TE. Las lecturas disponibles hasta ahora para la función de estos genes no se relacionan con TE. El estudio de la interacción genética de estos genes en C. elegans requiere fenotipos detectables asociados con TE, por lo tanto este fue el principal foco de mi proyecto de maestría.\nEn este trabajo mediante el análisis de mutantes y estudios farmacológicos encontré nuevas lecturas de pink-1 mutado animal. Se halló que el fenotipo vacuola observado en pink-1 animal puede ser rescatado por la pérdida de función de lrk-1 que es consistente con la función antagónica de PINK-1 y LRK-1, aunque resultados aquí presentados mostraron que el fenotipo vacuola podría ser independiente de las perturbaciones de TE. En otra lectura, la modulación de la proteína marcadora RAB-10 en pink-1 animal sugiere que PINK-1 tiene un rol potencial en el control del transporte de los endosomas tempranos durante el tráfico endomembranoso. Además también he utilizado el fenotipo vacuola ya identificado en el transgén SGK-1 para una prueba de RNAi y poder estudiar la interacción putativa entre LRK-1, PINK-1 y SGK-1 donde se identificaron varios moduladores del fenotipo. La supresión de lrk-1 fue suficiente para reducir los niveles de expresion del SGK-1 trangenico, suprimiendo su fenotipo. Estos datos soportan la existencia de un mecanismo de conexion entre LRK-1 y SGK-1. application/pdf Munarriz, Eliana Morelli, Laura Borner, Christoph PINK-1 LRK-1 Parkinson hereditario PINK-1 LRK-1 Parkinson?s Disease eng Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Establishing a readout for validating genetic interactions of C. elegans PINK-1, LRK-1 and SGK-1 and their putative roles in regulating endomembrane trafficking info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2893 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2893.dir/2893.PDF

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