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New technologies for a better understanding of the Golgi: FLIM-FRET and click chemistry

  • Author(s): Herrington, Kari Anne
  • Advisor(s): Sütterlin, Christine
  • et al.
Creative Commons 'BY-ND' version 4.0 license

Abstract of dissertation

New technologies for a better understanding of the Golgi:

FLIM-FRET and click chemistry

By Kari Herrington

Doctor of Philosophy in Biological Sciences

University of California, Irvine 2016

Professor Christine Sütterlin, Chair

The Golgi is the central component of the secretory system and essential to cell homeostasis, but many mechanisms that regulate Golgi structure and function are incompletely understood. In this dissertation I pursued two projects with the overall goal of applying cutting-edge technologies to answer specific questions about Golgi regulation. In the first project, I investigated the regulation of the Rho GTPase Cdc42 at the Golgi. Cdc42 is critical for Golgi transport and is proposed to be regulated by the Golgi protein GM130 to control centrosome organization. It is not known how GM130 or other Cdc42 regulators control Cdc42 activity at the Golgi. I used the phasor approach to FLIM-FRET with a Cdc42 biosensor, Cdc42-FLARE, to detect localized Cdc42 activity. Using this method I investigated Golgi-associated Cdc42 regulators, FGD1, Tuba, and ARHGAP10, and structure proteins, GM130 and Golgin-84. I found Tuba and FGD1 did not regulate Cdc42 equally, as loss of Tuba, but not FGD1, led to a decrease in Cdc42 activity at the Golgi. However, both were required for Cdc42 activity at the plasma membrane (PM). In addition, I found neither GM130 nor Golgin-84 depletion reduced Cdc42 activity at the Golgi, but both led to a decrease in Cdc42 activity at the PM. It is likely this reduction in Cdc42 activity is due to the disruption of Golgi organization or transport. My results suggest a new model in which GM130 controls centrosome organization through regulating Cdc42 activity at the PM. My second project focused on using click chemistry to identify the mechanism through which the small molecule MacE induces extensive Golgi fragmentation. I observed that MacE bound to numerous proteins in cells and in lysates. Surprisingly, I found non-specific lysine binders with similar structures as MacE analogs produced a MacE-like phenotype. MacE is known to bind lysines, and this result suggests that MacE functions through modifying lysines.

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