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. The microtubule plus-end tracking protein CLIP-170 (cytoplasmic linker protein of 170 kDa) regulates its association with microtubules by changing its conformation. 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. The functional relationship of CLIP family members with three EB family members EB1, EB2 and EB3 is described. CLIPs bind directly to the COOH terminus of the EB proteins. Furthermore, 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 and mediate interactions between microtubule plus- ends and the cell cortex. Two novel binding partners of CLASP1 and CLASP2 are identified 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. An optimised procedure is described for 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 a component of the LL5β clusters at the margin of HeLa cells.

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F.G. Grosveld (Frank)
Erasmus University Rotterdam
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
Erasmus MC: University Medical Center Rotterdam

Lansbergen, G.W.A. (2006, January 25). Functional Analysis of Protein Interactions at Microtubule Tips. Erasmus University Rotterdam. Retrieved from http://hdl.handle.net/1765/7254