La lecture à portée de main
Découvre YouScribe en t'inscrivant gratuitement
Je m'inscrisDécouvre YouScribe en t'inscrivant gratuitement
Je m'inscrisDescription
Sujets
Informations
Publié par | profil-zyak-2012 |
Publié le | 01 décembre 2010 |
Nombre de lectures | 69 |
Langue | English |
Poids de l'ouvrage | 39 Mo |
Extrait
THESE
Présentée pour obtenir le titre de
Docteur de l’Université de Strasbourg
Discipline: Sciences de la vie et de la santé
Spécialité: Aspects moléculaires et cellulaires de la biologie
par
Matthias Spiess
Soutenue le 20 Décembre 2010
Evolution des réseaux d’interaction des domaines
protéiques SH3 dans la levure; fonctions et interactions
d’une famille de protéines composée de Lsb1 et Lsb2
Evolution of the SH3 domain protein interaction networks
in yeast; functions and interactions of the Lsb1 and Lsb2
protein family
Devant le jury composé de:
Dr. B. Winsor Directrice de thèse
Dr. M. Geli Rapporteur externe Dr. M. Knop Rapporteur externe Pr. J. de Montigny Examinateur interne
Dr. N. Entelis Membre invité
Génétique Moléculaire Génomique Microbiologie GMGM, (CNRS, UMR7156)
Acknowledgements
I would like to thank my thesis director Barbara Winsor for giving me the opportunity to do
my thesis in her laboratory, assisting me with the little and big problems of such a work, for
fighting for funding to finish the work and for all the rest. Especially I would like to mention
her support to find and exploit collaborations.
I would like to thank Rudolph Volkmer for accommodating me in his laboratory and
Christiane Landgraf for showing me around and introducing me into the technique SPOT.
Thanks also to Victor Tapia Mancilla who did a great work in the data analysis. Thanks to all
the other lab members as well, I really had a great time in Berlin.
I would like to thank David Drubin for accommodating me in his laboratory and Alphée
Michelot for showing me around and introducing me into the technique of in vitro actin
polymerization. It was a great and fruitful stay in Berkeley.
I would like to thank Danièle Urban-Grimal for accommodating me in Paris and helping me
in the laboratory with ubiquitination experiments and all other lab members of the Rosine
Hagenauer-Tsapis laboratory. I really learned to understand the fascination of and appreciate
the French capital.
I would like to thank the numerous temporal and permanent members of Penelope. Although
it was not all the time the easiest collaboration, I always enjoyed our meetings, had a lot of
fun with you in the rare spare time and looked forward to the next. And Michela did a
fantastic job.
I would like to thank Aline Huber and Audrey Geissler who helped me a lot in the lab, who
were kindly concerned about my security and amused me with their unique alsacian humor.
Thanks to Albert who introduced me into the new environment and the new techniques in the
laboratory with his ability to speak English and thanks to Vincent who helped me to speak French and in return learned that beer can be as good as wine. Many thanks also to the
remaining Winsor lab members Gladys, Guillaume, Lydia and Ray.
I would like to thank Sylvie Friant and her laboratory for discussing and advancing my
project. We passed some journal clubs, some interesting lab meetings and some fantastic food
or Pétanque parties together. Thanks to Johan, Serge, Dimitri and Fanny. Many thanks also to
Joelle Morvan for kindly helping me to correct this work and last but not least I would like to
thank Bruno, who sold me my fist car and helped with his thousand and one tools where he
could.
I would like to thank Cathy and Aline for helping me to get through the jungle of French
administration and for being there for some de-stressing chat.
Bedanken möchte ich mich auch bei Tom und Serge, die nicht nur tagsüber im Labor sondern
auch noch spät Abends für gute Stimmung sorgten. Die kulinarisch hochstehenden
Abendessen waren mir eine wahre Freude und die Diskussionen auf der Terrasse, bei einem
Bier oder einem vom Meistersommelier auserkorenen Wein waren allemal ein kürzere Nacht
wert.
Zum Schluss möchte ich noch all meinen Freunden aus der schönen Schweiz danken, meiner
Familie natürlich und allen voran meinen Eltern Paul und Denise, die all die Jahre über an
mich geglaubt und mich tatkräftig unterstützt haben.
Table of contents
Preface................................................................................................................................................3
Abstract...............4
Résumé................5
Chapter I – Introduction................................................................................................9
I. The Budding Yeast Saccharomyces cerevisiae............9
II. The Actin Cytoskeleton...................10
II.1 The cytoskeleton...........................................................................................................10
II.2 The budding yeast actin cytoskeleton.11
III. Actin.....................................................................................................................13
II.1 Actin polymerization..................................................................14
IV. Actin Nucleation...............................16
IV.1 The Arp2/3 complex.17
V. Nucleation Promoting Factors......................................................................................................19
V.1 The WASP family proteins........................21
VII. Membrane Traffic...........................................................24
VIII. Endocytosis.....25
VIII.1 Historical overview of endocytosis in Saccharomyces cerevisiae.......................................26
VIII.2 Early steps of endocytosis....................................................................................27
IX. Regulation of Endocytosis by Ubiquitin...................................................30
X. Objectives.............................................33
Chapter II Characterization of Lsb1 and Lsb2..................................................................37
I. Introduction to Lsb1 and Lsb2.......................................37
II. Lsb1 and Lsb2 and their Homologs.............................37
III. Characterization of lsb1Δlsb2Δ...39
III.1 Growth of deletion strains......................................................................................39
III.2 Functional analysis of LSB1 and LSB2 deletion strains..............................39
III.3 Biochemical and genetic interactions of LSB1 and LSB2...........................40
III.4 Analysis of budding polarity..................................................................................................................42
III.5 Lat‐A sensitivity...........................................43
III.6 Actin cytoskeleton organization...........44
III.7 Endocytosis...................................................................................................................45
IV. Localization of Lsb1 and Lsb2......47
IV.1 Subcellular Fractionation of Lsb1 and Lsb2....................48
IV.2 Lsb1 and Lsb2 pellet upon centrifugation.......................................................................................49
IV.3 Lsb1 is present in a high molecular weight complex..................................50
V. Colocalization of Lsb1 and Lsb2 with endocytic Proteins...................53
V.1 Lsb1 and Lsb2 localization is not dependent on actin patch proteins..55
V.2 Lsb2 and prion formation.........................................................................................................................56
VI. Ubiquitination of Lsb1 and Lsb2.................................................................58
VI.1 Lsb1 is stable.................................................59
VII. Role of Lsb1 and Lsb2 in Actin Polymerization....60
VII.1 Lsb1 and Lsb2 bind Las17 but do not induce ex vivo actin polymerizat