060125 Lansbergen-GJA.Pdf

Functional Analysis of Protein Interactions at Microtubule Tips De functionele analyse van eiwit interacties aan microtubuli uiteinden Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. S.W.J. Lamberts en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 25 januari 2006 om 11:45 uur door Gideon WillebrordusGideon W.A. Adrianus Lansbergen Lansbergen geboren te Rotterdam © 2005 Gideon W.A. Lansbergen ISBN-10: 90-9020233-1 ISBN-13: 978-90-9020233-4 Thesis lay-out: Gideon Lansbergen Graphical assistance: Tom de Vries Lentsch Printed by PrintPartners Ipskamp, Enschede The studies presented in this thesis were performed in the Department of Cell Biology of the Erasmus MC in Rotterdam, The Netherlands. Research performed in this thesis was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). Printing of the thesis was partially supported by the J.E. Jurriaanse Stichting and Alzheimer Nederland. Cover: Tom de Vries Lentsch based on sculpture ‘Zonder Titel’, artist: Auke de Vries. Artwork is situated in front of Netherlands Architecture Institute near the Erasmus University Medical Center Rotterdam. Functional Analysis of Protein Interactions at Microtubule Tips De functionele analyse van eiwit interacties aan microtubuli uiteinden Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. S.W.J. Lamberts en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 25 januari 2006 om 11:45 uur door Gideon Willebrordus Adrianus Lansbergen geboren te Rotterdam Promotiecommissie Promotor: Prof.dr. F.G. Grosveld Overige leden: Prof.dr. J.G.G. Borst Dr.ir. N.J. Galjart Dr.ir. D.N. Meijer Copromotor: Dr. A.S. Akhmanova Voor Marieke en mijn ouders Index: Scope of the thesis 7 Chapter 1 Introduction into the cytoskeleton 11 Chapter 2 Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin 43 localization. Chapter 3 EB1 and EB3 control CLIP dissociation from the ends of growing microtubules. 57 Chapter 4 CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell 71 cortex. Chapter 5 CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5β and 87 ELKS. Chapter 6 Mass spectrometry analysis of membrane-bound protein complexes cross-linked with 115 formaldehyde: identiﬁcation of liprin-α1 as a component of LL5β-containing cortical clusters in HeLa cells Chapter 7 Discussion 127 Appendixes 153 Summary 160 Samenvatting 162 Curriculum Vitae & Publications 164 Dankwoord 166 Scope of the thesis Microtubules are a part of the cytoskeleton involved in many essential processes, such as intracellular transport of cargoes, cell migration, positioning of cellular organelles and formation of the mitotic spindle for chromosome segregation. The fast growing end of microtubules (the plus-end) can interact with specific microtubule plus-end binding proteins (also known as plus-end tracking proteins, or +TIPs). +TIPs participate in microtubule interactions with different cellular structures and control microtubule dynamics. This thesis describes the functional analysis of protein interactions at the microtubule plus-ends. Chapter 1 is an introduction into the cytoskeleton. It gives an overview of the three types of cytoskeletal filaments (intermediate filaments, actin filaments and microtubules) and diverse microtubule associated proteins (MAPs), including microtubule associated motors, general microtubule associated proteins and microtubule plus-end binding proteins. Chapter 2 describes the microtubule plus-end tracking protein CLIP-170 (cytoplasmic linker protein of 170 kDa), which regulates its association with microtubules by changing its conformation. CLIP-170 possesses two NH2-terminal CAP-Gly domains that are involved in the interaction with microtubules, and two COOH-terminal metal binding domains, which interact with various partners. The NH2 terminus of CLIP-170 binds directly to the COOH terminus through its first metal-binding domain. Also the dynactin subunit p150Glued and LIS1 interact with the COOH terminus of CLIP-170, but depend on the second metal-binding domain. The folded head-to-tail conformation of CLIP-170 inhibits microtubule association and also interferes with the binding of dynactin and LIS1 to the CLIP-170 COOH terminus. Chapter 3 reports the functional relationship of CLIP-170 and CLIP-115 with the three EB family members EB1, EB2 and EB3. CLIPs bind directly to the COOH terminus of the EB proteins. This interaction contributes to CLIP-170 localization and enhances its intrinsic affinity for growing microtubule ends. Chapter 4 shows that the two CLIP-associating proteins, CLASP1 and CLASP2 play redundant roles in regulating the density, stability and dynamics of interphase microtubules. CLASPs stabilize microtubules at the cell periphery, possibly through forming a complex with EB1 at microtubule tips. The association of CLASPs with the cortex, which is microtubule-independent, mediates interactions between microtubule plus- ends and the cell cortex. Chapter 5 describes the identification of two novel binding partners of CLASP1 and CLASP2 using pull down assays coupled to mass spectrometry. These partners, LL5β and ELKS, are both required for the normal cortical CLASP accumulation and microtubule organisation. Since LL5β contains a PIP3-binding pleckstrin homology (PH) domain it is 7 Scope proposed that LL5β and ELKS can form a PIP3-regulated cortical platform, to which CLASPs attach distal microtubule ends. Chapter 6 describes an optimised procedure for using mass spectrometry to identify the components of membrane-bound protein complexes. For the cortical protein LL5β, which is involved in attaching microtubules to the membrane, it is shown that at least one of the isolated potential partners, liprin-α1, is indeed a component of the LL5β clusters at the margin of HeLa cells. 8 Chapter 1 Introduction into the cytoskeleton Introduction into the cytoskeleton 1.1 The cytoskeleton Living cells are highly complex systems. Eukaryotic cells, such as human cells, have specialized organelles and structures with specific vital functions. For instance, mitochondria take care of the energy metabolism, the cell nucleus is the place of DNA storage and RNA transcription, the endoplasmic reticulum and the Golgi apparatus harbour the machinery for synthesis of glycoproteins and lipids and the cytoskeleton is necessary for maintaining cell morphology and intracellular transport (Fig. 1). This thesis will focuss on the last structure, the cytoskeleton. All eukaryotic cells have a cytoskeleton, from unicellular organisms such as yeasts to multicellular organisms such as plants, flies and mammals. Many cytoskeletal components are highly conserved in evolution, indicating that they are essential for the normal cell functioning. The cytoskeleton is involved in specialized functions of diverse cell types. For example, cell migration during embryonic development, sperm cell motility, chromosome segregation during cell division, muscle contraction, intracellular vesicle transport, cell morphology and strength all depend on the cytoskeleton (Janmey 1991). Because the cytoskeleton is involved in so many essential processes, it is not surprising that its malfunctioning causes cell disorders and human diseases. Today, many diseases, such as cardiovascular syndromes, liver cirrhosis, neurodegeneration, pulmonary fibrosis, blistering skin diseases and cancer have been associated with abnormalities in cytoskeletal proteins. Research into the function, control and maintenance of the cytoskeleton is therefore of great interest. For instance, frequently used cancer therapies are based on application of anti- cytoskeletal drugs that inhibit cell division. In future, the basic knowledge about the cytoskeleton might lead to more specific drugs (Fuchs and Cleveland 1998; Benitez-King et al. 2004; Ramaekers and Bosman 2004). Figure 1: The major features of eukaryotic cells. The drawing depicts an intestinal animal cell with its major organelles and structures. Microvilli and cilia, for instance, are cell structures dense with cytoskeleton filaments, especially abundant on the absorptive surface of intestinal epithelial cells. (Fundamentals of General, Organic, and Biological Chemistry; McMurry and Castellion; 1999) 11 Chapter 1 There are three types of cytoskeletal structures or filaments: the intermediate filaments, the microfilaments (or actin) and the microtubules (Fig. 2). All these filaments, which are constructed from smaller building blocks, play various roles within the cell. What makes them all highly important are their dynamics, their multi-functionality and their ability to cooperate with each other in different parts of the cell. Interplay between different cytoskeletal elements is regulated by a variety of proteins, protein complexes, organelles and other specific targets within the cell. Next, the cytoskeletal structures will be discussed in more detail. A B Figure 2: Components of the cytoskeleton. (A) The cytoskeleton of a cell is constructed from three major networks: actin filaments, intermediate filaments and microtubules. Each one has its own specific function and localization within cells. (B) Schematic representation with electron micrographs of the three types of filaments. Microtubules have

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