Function and regulation of the CLASP-dependent microtubule array at the Golgi

Abstract

This project provides a detailed characterization of the CLASP-dependent microtubule array at the Golgi in terms of function and regulation. First, I briefly outline the process of MT nucleation at the Golgi and highlight my efforts that contributed to identifying the Golgi as a microtubule organizing center. Second, I discuss principles whereby motile cells establish microtubule array asymmetry, specifically highlighting the role of Golgi-derived microtubules in this process. We further extended functional studies by siRNA targeting of CLASPs, which specifically removes the Golgi-derived microtubule array. Utilizing this approach we have determined that Golgi-derived microtubules are required for proper Golgi assembly and organization as well as polarized post-Golgi trafficking to the leading edge of motile cells. We propose a model whereby Golgi-derived microtubules polarize post-Golgi trafficking by properly organizing an asymmetric Golgi ribbon as well as providing a direct link between the Golgi and the cell front. Both of these Golgi-derived microtubule functions contribute to establishing polarity that is required for cell migration by organizing an asymmetric microtubule array that regulates directional delivery of cargos and organelles to distinct cellular domains. In cells lacking Golgi-derived microtubules, the Golgi is highly fragmented and disorganized and the process of post-Golgi trafficking becomes chaotic. Cells lacking Golgi-derived microtubules also lose the ability to maintain directionally persistent migration patterns. Finally, I report our initial findings that GSK3β regulates microtubule formation at the Golgi by modulating the turnover rate of Golgi-associated CLASPs. In control cells, CLASPs exchange rapidly at the Golgi and coat newly formed Golgi-derived microtubules. In contrast, when CLASPs are dephosphorylated by GSK3β knockdown or inhibition, CLASPs exchange significantly slower at the Golgi and the Golgi-derived microtubule array is absent. Overall, this thesis details the functional significance of Golgi-derived microtubules as well as provides insight as to how this particular microtubule subset is regulated.