Abstract of dissertation

Re-evaluating the Functional Significance of Golgi Ribbon Structure and Pericentrosomal Positioning

By

Kati Tormanen

Doctor of Philosophy in Biological Sciences, University of California, Irvine

Professor Christine Sütterlin, Chair

This study examines the significance of Golgi ribbon structure and pericentrosomal positioning for microtubule organization and higher cellular functions such as protein transport, directional migration and primary cilia formation. Because the centrosome and Golgi are both microtubule organizing centers (MTOCs), and the structure and protein composition of both organelles is dependent on microtubules, it has been challenging to determine the exact role of each organelle. We took two approaches to dissect the relationship between microtubules and Golgi ribbon structure and positioning. In the first approach we attempted to determine if Golgi ribbon structure is necessary for microtubule organization and function by disrupting the Golgi ribbon. This work identified a novel component in the microtubule nucleation and organization pathway. In the second approach, we selectively disconnected the close association between the Golgi and the centrosome without disrupting Golgi or microtubule organization to determine the significance of this unique association in mammalian cells. We found that cells with a loss of Golgi-centrosome proximity had apparently normal microtubule organization, were able migrate directionally and form a primary cilium. This suggests that Golgi-centrosome proximity is not necessary for cell homeostasis.

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.

Chlamydia are obligate intracellular bacterial pathogens with a significant impact on human health. Chlamydia infect eukaryotic cells, and a wide range of effects on the host cell have been attributed to a chlamydial protease, CPAF, through cleavage or degradation of numerous host proteins. We discovered that this reported proteolysis was an in vitro phenomenon that occurred during preparation of infected cells for protein analysis. We showed that CPAF activity was induced by detachment of Chlamydia-infected cells from a cell culture monolayer and that this protease remained active in cell lysates resulting in artifactual proteolysis. Chlamydial phenotypes attributed to CPAF were still observed when we took precautions to prevent in vitro proteolysis. Our findings challenged the prevailing model about the function of CPAF during the chlamydial infection and its intracellular targets. In a second project, we studied the unusual chlamydial developmental cycle with a novel three-dimensional electron microscopy (3D-EM) method. During the intracellular infection, the bacterium converts within the chlamydial inclusion from an infectious, but non-dividing, elementary body (EB) into a reticulate body (RB) that divides repeatedly by binary fission before converting back into EBs to infect new cells. We analyzed each Chlamydia-infected cell with several hundred two dimensional EM slices and used computational methods to reconstruct the three-dimensional images of the chlamydial inclusions. The results provided the first comprehensive analysis of the developmental cycle, including the number, size, and location of each EB, RB, and intermediates of RB replication and RB-to-EB conversion. We described for the first time that RB volume progressively decreases as RB number increases, indicating that RBs divide before doubling in size. However, we did not observe RBs below a minimum size, leading us to postulate that RBs below a size threshold convert into an EB. We propose a new model of chlamydial development in which RB-to-EB conversion is regulated by RB size because several rounds of RB replication are required for the RB to become small enough to convert into an EB. In this model, RB size acts as a timer and provides a mechanism to delay RB-to-EB conversion until the RB pool has expanded.