The objective of this project is to understand a control mechanism that regulates the centrosome from the adjacent Golgi apparatus in mammalian cells. While the proximity of the Golgi and centrosome is unique to mammalian cells, and not observed in yeast, plant or fly cells, its functional significance has been elusive. Work in our laboratory has identified a centrosome regulatory pathway in which a Golgi protein, GM130, causes a guanine nucleotide exchange factor called Tuba to activate Cdc42 at the Golgi. This project seeks to determine how these events at the Golgi control centrosome organization and function and whether they depend on Golgi-centrosome proximity.
Aim 1 will determine whether GM130 activates Cdc42 at the Golgi by increasing the binding affinity of Tuba for Cdc42 or by recruiting Cdc42 to the Golgi.
This aim will also examine if GM130 is the major regulator of Cdc42 at the Golgi by assaying whether GM130 controls additional cellular processes at the Golgi that are known to be regulated by Cdc42, such as Golgi to ER transport and local actin assembly at the Golgi. A potential negative regulator of Golgi-associated Cdc42 will also be studied.
Aim 2 will examine how the GM130-Cdc42 pathway exerts its effect on the centrosome. A Cdc42 effector, Par6?, will be studied to determine if it is a downstream component of this regulatory pathway and if it is transported to the centrosome by the motor protein, dynein, through interactions with the dynactin subunit, p150Glued. PCM-1, a protein that recruits additional proteins to the centrosome, will be studied to determine if it acts downstream of Par6? to mediate the control of centrosome organization by the GM130-Cdc42 pathway.
Aim 3 will study the functional significance of Golgi-centrosome proximity by examining if Cdc42 can be mobilized from the Golgi to the centrosome so that it can regulate centrosome organization. The pericentriolar location of the Golgi will be disrupted as another means of testing whether Golgi centrosome proximity is necessary for regulation of centrosome organization by the GM130-Cdc42 pathway.
Our studies on the control of centrosome organization have a specific connection to cancer as centrosome organization is abnormal in the majority of human cancers, which contributes to genetic instability and to loss of tissue differentiation. These studies will also reveal novel mechanisms for the regulation of the small GTPase, Cdc42, whose uncontrolled activity may lead to cell transformation and to enhanced cell migration, tissue invasion and metastasis.
