Dr. Ruth Kroschewski

Research

Establishment of epithelial cell polarity

Main field of research

Organs are elaborate cell communities that perform specialized functions for the organism. In order to fulfill these functions, organs generally develop into highly specific and complex structures. Our goal is to understand the basic principles of organogenesis.

The shape of organs is precisely controlled and reproduced with high fidelity throughout generations. Structurally, organs are simply associations of cells organized into a community. Little is known how cells influence each other, and functionally and morphologically react to their neighbors during organogenesis. Therefore, my approach is to study in vitro how cells come together to form multicellular structures.

The most ancient organs developed as specialized interfaces between the organism and the outside world. As such epithelial cells are key players in these organs. Thus we are focusing on “epithelial organs” (like breast, kidney) and ask how epithelial cells communicate and assemble complex, multicellular communities.

Our model systems consist of two epithelial cell lines. MDCK kidney cells can differentiate and reproduce different steps of nephrogenesis in vitro. And MCF10A non cancerous human breast epithelial cells develop in polarized acini (cysts), suborgan structures of the breast. Our group is interested in two major aspects of in vitro organogenesis:

1. We study the events controlling how cells orient themselves in their community. Cdc42 is a small GTPase essential for cell polarity in budding yeast. I showed that Cdc42 controls basolateral polarity in MDCK cells. To understand how cells position their polarity axis, we study the spatial and temporal regulation of Cdc42 during the formation of a polarized epithelial monolayer (Figure 1).

- We use biochemistry to identify and characterize its most direct activators, particularly guanine nucleotide exchange factors.

- We analyze the regulation of Cdc42 and its regulators (like guanine nucleotide exchange factors and a Ras-like GTPase which is a tumor suppressor) in response to growth factors and cell-cell contacts.

Fig.1 Schema of comparative polarity establishment
Depicted on the left side the hierarchical cascade for the generation of a bud in S. cerevisiae (A). On the right side is a hypothetical homologous cascade for mammalian cells (B). The gradient-shaded bars between Bud1-GTP and Cdc24 or Rap1a-GTP and mammalian GEFs, respectively, represents the physical interaction leading to the production of activated Cdc42 (Cdc42-GTP).

We analyze the question of how cells communicate with their neighbors to form cysts and tubules. How is a lumen formed in a cell aggregate? Tubules forming out of epithelial layers consist of at least three morphologically different cell types. How does each of the cells know, which fate to adopt?

Fig.2 Model of cysto- and tubulogenesis
Please note the shape changes during cytogenesis and the undefined late development of the apical surface. Three cell types are present after tube formation, polarized cuboidal cells, cells at the base of the tubule and the tip cell of the tubule. Microvilli are indicated as small membrane projections into the lumina.

To address these questions, we combine the strengths of experimental investigation using inducible systems to down regulate specific proteins (siRNA) and computational simulation. We focus this synergy on the organogenetic process of mammalian epithelial cells: the differentiation of an amorphous set of cells initially to a cyst and subsequently to a tubule (Figure2).

In summary, we are addressing in mammalian epithelial cells the questions of how are plasma membrane domains determined.

Main fields of research, keywords

• Epithelial Polarity
• Plasma Membrane Domains
• Cdc42
• Guanine Nucleotide Exchange Factors
• Tumor Suppressor
• Ras-like GTPase
• In Vitro Organogenesis
• Cell-Cell Contacts
• Computational Simulation
• Forces
• Surface Generation
• Cystogenesis
• Tubulogenesis

Techniques

• RNAi
• Gene Gun
• 3D Cultures of Mammalian Epithelia Cells
• Computational Simulation
• In Vivo Live Cell Imaging,
• Protein Purification
• Threading
• Biochemical Assays for Protein-Protein Interaction
• GEF Assays
• Conformation Specific Single Chain Antibodies