Investigating Pix protein function in microtubule-related processes

As multicellular organisms, humans are composed of millions of cells that are organized into the specific tissues and organs of the body. Cells are complex structures consisting of a nucleus, which contains the genetic material, embedded within a membrane-bound cytoplasm, where the majority of chemical reactions required to sustain life are carried out. However, cells come in all shapes and sizes, from almost spherical white blood cells to highly elongated nerve cells. Determination and maintenance of cell shape depends upon an internal support structure known as the cytoskeleton. There are several components to the cytoskeleton, but among the most important are large flexible rods called microtubules. Microtubules are dynamic structures, continually elongating and shrinking, that play many roles in the biology of the cell beyond simply maintaining cell shape. For example, they act as highways within the cell for trafficking of components and form specialized scaffolds that ensure the equal segregation of genetic material when cells divide. Microtubule function is largely dependent upon proteins that bind to microtubules and regulate either their dynamics or their association with and transport of other cellular structures. However, the identity, properties and function of many microtubule-associated proteins remain largely unknown. During recent work on the mechanism by which germ cells develop in the African clawed toad, Xenopus laevis, we identified two novel proteins, called Pix1 and Pix2, that we have unambiguously shown associate with microtubules. Pix proteins are found from flies to man and are present in cells from many different tissues. Pilot experiments to study the function of these proteins revealed an essential role in normal cell division, while localization studies indicated that these proteins concentrate at two particular microtubule-associated structures within the cell: the centrosome, the site from which microtubules are produced, and mitochondria, the powerhouses of the cell within which energy is generated. Interestingly, the region of cytoplasm from which germ cells develop in fertilized Xenopus eggs is rich in mitochondria and the distribution of these mitochondria is thought to be under the control of microtubules. Having (a) identified two novel microtubule-associated proteins, (b) shown them to also be present in centrosomes and mitochondria, and (c) found them to be essential for cell division, we now wish to explore their function through a series of detailed experiments which tests their requirement in different biological processes and examines their binding partners in cells and Xenopus eggs. Firstly, we will generate a series of biological reagents that will enable us to specifically interfere with the function of either Pix1 or Pix2 in human or Xenopus cells. Secondly, we will examine Pix protein function in microtubule stability and dynamics, cell division, centrosome organization and distribution of mitochondria, including in nerve cells. Thirdly, we will address the role of Pix proteins in the organization and development of germ cells in Xenopus; and, finally, we will address the mechanism by which Pix proteins associate with microtubules through identifying their interacting partners. This ambitious project will use state-of-the-art cell biology technologies including high resolution microscopy in living cells and protein identification by mass spectrometry. It will also extend the collaboration between the two midlands universities, Leicester and Warwick, that led to identification of these proteins, with the work in human and Xenopus systems being undertaken in Leicester and Warwick, respectively. Not only will this study provide a major advance in our basic understanding of the role of these proteins in cells, but it is likely to have important implications for the use of drugs, currently used as anti-cancer medicines, that act through disturbing microtubules.

Microtubules (MTs) are filamentous structures that play diverse roles in cell biology including cytoplasmic distribution of organelles and mitotic division of chromosomes. Their dynamic properties and attachment to cargo is governed by MT-binding proteins, although the cellular function of many such proteins are poorly understood. This proposal describes the functional characterization of two novel MT-binding proteins, Pix1 and Pix2, that we identified through studies of germ plasm organization in Xenopus oocytes. Germ plasm is the cytoplasmic region destined to form the primordial germ cells; it is rich in RNAs and mitochondria and organized by MTs, although how remains unclear. We isolated Pix proteins through interaction with Xpat, a known protein component of germ plasm. Pix proteins are conserved from Drosophila to humans and comprise an N-terminal WD40 domain and a C-terminal coiled-coil. Importantly, human Pix proteins bind MTs in vitro and localize to MTs and MT-related structures, including the centrosome, in interphase and mitosis. Immuno-EM revealed that Pix is located within the centriole itself. Moreover, injection of Pix antibodies interfered with cell division consistent with a role in mitotic MT organization. Intriguingly, Pix proteins also localize to mitochondria both in cultured cells and within the germ plasm of Xenopus oocytes, leading us to hypothesize that Pix proteins regulate attachment of complexes and organelles, such as mitochondria, to MTs. Our aim is to test this hypothesis with the main objectives being to use RNAi, protein overexpression, antibody injection and immunodepletion to examine Pix function in human cultured cells and Xenopus oocytes, embryos and egg extracts. In particular, we will examine MT dynamics, cell division, centrosome organization and mitochondrial distribution, including in neurite extensions, and determine the mechanism of MT binding through candidate coimmunoprecipitation and mass spectrometry approaches.