The biochemical basis of PAR polarization

The development of multicellular organisms, such as human beings, depends on the differentiation of cells into many different cell types, which administrate different, specialized functions. One important mechanism of cell differentiation is asymmetric cell division, by which cells of different sizes, contents and developmental potentials are formed. This process depends on a prior polarization of the cell, where molecules inside of the cell are asymmetrically distributed. One of the best-established model organisms to study cell polarity is the nematode Caenorhabditis elegans. Here, cell polarity is determined by the Partitioning defective (PAR) proteins, which form two distinct domains in the single cell embryo, the anterior and the posterior domain. More than 25 years of research helped to shed identify the core molecular players of the PAR protein network and to shed light onto the mechanism cell polarization, PAR proteins form two sets of transient protein complexes, the anterior and posterior PAR complex, each of which covers the membrane of about one half of the cell. This distribution is thought to emerge due to reciprocal biochemical regulation of the PAR proteins, a phenomenon, which is termed mutual exclusion. However, the biochemical basis of this process is poorly understood and how exactly PAR proteins regulate each other is still not known. In order to fill this gap, a precise characterization of the PAR proteins and their molecular properties is required. The aim of our research proposal is to purify the components of the PAR polarity network and to rebuild this system outside of the living cell. We will use fluorescence microscopy to study how PAR proteins interact with a lipid membrane and with each other, and how they lead to large-scale protein patterns similar as in the cell. With the help of this approach, we will bridge the gap between what we know about the individual proteins and how they organize the C. elegans embryo. We believe that this in vitro approach represents the most powerful way to test our current understanding of the PAR system. This work will address one of the most important questions of cell and developmental biology, namely how proteins interact to give rise to a living cell and eventually a whole organism.

In the course of this project we established the biochemical basis to anayse the PAR proteins which mediate the first asymmetric cell division in C. elegans. This work achieved to describe a workflow for the purification of 6 periphal membrane proteins. These proteins play a key role in the self-organization of two mutually exlusive membrane domains within the C.elegans zygote. The zygote reads out polarity from the two established membrane domains and divides in an asymmetric manner along the polarity axis, creating two daughter cells of different fate. How those membrane domains organize themselves is poorly understood. The aim of this study was the reconstitution of central PAR polarity feedback circuits in vitro. By reconstituting the major biochemical reactions underlying PAR domain self-organisation we aimed to understand the biochemical cues of each PAR component to the overall process of symmetry braking and establishment. While it is a challenging task to purify peripheral membrane proteins as they are prone to aggregation, we achieved to establish condition for the purification of full-length versions of 6 PAR proteins and tested their functionality. Liposomes were used to mimick the cell membrane to which the polarity proteins can bind in a dynamic manner. This constitutes a minimal reconsitution system of the C.elegans cortex and the main polarity proteins. In addition, we began to characherize the biophsyical properties of PAR proteins in regards to membrane binding kinetics and how lipid composition affects membrane binding.In the course of this project, we purified the main players of PAR polarity in satisfying yield with high purity. This required several optimization rounds in respect to expression organism, expression conditions and purification conditions. Most proteins were overexpressed in insect cells and extacted from the membrane using detergents, as this proved to be the most efficient way to ensure solubility of the purified proteins. Subsequently, proteins were analyzed by mass spectrometry and subjected to functional assays, for example kinase assays and lipsosome (membrane) binding assays. We observed membrane binding events and phosphorylation events (for details see part II) and concluded that the established biochemical protocols yielded the required components to reconstitute PAR polarity circuits in vitro.