Lipid raft association and subcellular localization of UNC5 Netrin-1 receptors Associació als lípids raft i localització subcelꞏlular de la familia de receptors de la Netrina-1 UNC5 Marc Hernaiz Llorens ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intelꞏlectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. 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In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. ® Thesis cover illustration: Anna Llorens Christina's World Andrew Wyeth (1917-1989) Tempera on gypsum. 1948 MUSEUM OF MODERN ART (MOMA): NEW YORK This painting shows Christina Olson, an American girl from Cushing, Maine, who suffered from poliomyelitis. Andrew Wyeth was a friend of Christina's parents and stayed at their house on several occasions. There, Wyeth met Christina and witnessed her extraordinary strenght and willpower when it came to getting about, despite that she was unable to walk due to her illness. Wyeth said of her that she was "an admirable woman". With great difficulty, in this painting Christina drags herself across the lawn to reach the house. Andrew Wyeth was born in Chadds Ford, Pennsylvania in 1917. He devoted himself above all to watercolors and tempera on gypsum. His oeuvre is realistic, depicting mainly scenes from rural American day life, in which sentiment dominate each of his paintings. "OBSERVED VULGARITY BECOMES ROMANTIC" ANDREW WYETH To Victor and Anna, ABSTRACT During brain development, UNC5 receptors play a key role in controlling axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into specific cholesterol-enriched membrane raft microdomains is of crucial importance for the efficient transduction of the recognized signal. Whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we extend the classical biochemical characterization of raft microdomains by performing confocal microscopy, live cell FRAP analysis and sptPALM of fluorescently-tagged UNC5 receptors and we reveal for the first time differences into their membrane mobility properties. Using a combination of pharmacological and genetical approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibit the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that their specific localization have functional consequences for the axonal chemorepulsion against Netrin-1. Alternative splicing is a key mechanism in which a variety of proteins is achieved from "cut and paste" mRNA. The plethora of proteins resulting from alternative splicing processes is crucial for the proper function of certain protein isoforms. In this thesis we have detected alternative splicing in the UNC5A receptor in the nervous system. The resulting product is a UNC5A isoform that lacks the first TSPI repeat, uncovered here, to be important for cell membrane targeting. We have further analyzed the implications of losing the first TSPI repeat. 1 2 TABLE OF CONTENTS ABSTRACT................................................................................................................................. 1 TABLE OF CONTENTS ................................................................................................................ 3 ABREVIATIONS ......................................................................................................................... 7 INTRODUCTION ...................................................................................................................... 11 1. DEVELOPMENT OF THE MAMMAL NERVOUS SYSTEM....................................................................... 11 1. 1. EARLY STAGES OF EMBRYONIC DEVELOPMENT ..................................................................... 11 1. 2. THE NEURAL INDUCTION................................................................................................. 12 1. 3. NEURULATION ............................................................................................................. 13 1. 4. REGIONALIZATION OF THE CENTRAL NERVOUS SYSTEM .......................................................... 14 2. CEREBELLAR DEVELOPMENT ...................................................................................................... 15 2. 1. DEFINING BOUNDARIES (CEREBELLAR PRIMORDIUM) ............................................................ 15 2. 2. CEREBELLAR HISTOGENESIS ............................................................................................. 16 2. 3. CEREBELLAR CYTOARCHITECTURE AND CONNECTIVITY ........................................................... 17 2. 3. 1. Structure of the cerebellum .......................................................................... 17 2. 3. 2. INTRINSIC CONNECTIVITY ..................................................................................... 17 2. 3. 3. AFFERENT PROJECTIONS ...................................................................................... 18 2. 3. 4. EFFERENT PROJECTIONS ...................................................................................... 18 2. 3. 5. GRANULE CELLS ................................................................................................. 19 2. 3. 5. 1. FROM THE RHOMBIC LIP TO THE EXTERNAL GRANULAR LAYER (EGL) ................... 19 2. 3. 5. 2. FROM PROLIFERATION TO DIFFERENTIATION ................................................. 20 2. 3. 5. 3. EXITING THE EXTERNAL GRANULAR LAYER (EGL) ............................................ 20 3. AXON GUIDANCE..................................................................................................................... 22 3. 1. GROWTH CONE ............................................................................................................ 22 3. 2. CYTOSKELETAL DYNAMICS ............................................................................................... 22 3. 3. GUIDANCE CUES ........................................................................................................... 24 3. 3. 1. NETRINS .......................................................................................................... 27 3. 3. 1. 1. NETRIN-1 RECEPTORS .............................................................................. 27 3. 3. 1. 1. 1. DELETED IN COLORECTAL CANCER (DCC) .......................................... 27 3. 3. 1. 1. 2. UNCOORDINATED-5 (UNC5) ......................................................... 28 3. 3. 1. 1. 3. NEOGENIN ................................................................................. 28 3. 3. 1. 1. 4. DOWN SYNDROME CELL ADHESION MOLECULE (DSCAM) ................... 28 3. 2. 1. 2. NETRIN-1 IN NERVOUS SYSTEM DEVELOPMENT .............................................. 30 4. CHOLESTEROL ......................................................................................................................... 30 4. 1. CHOLESTEROL IN THE CENTRAL NERVOUS SYSTEM ...............................................................