- Examination of microvilli formation on the apical surface of small intestine and axonal elongation of neurons
- Common determinants of cell polarity in instestine, nerve, and blood cells
- Functional analysis of animal models and primary cell culture as a basis for human disease
- Analysis of fine structures by superresolution and electron microscopy
Utilizing state-of-the-art technology to explore the dynamics of cell polarity
Cell polarity plays an important role in the function of a variety of cells. For example, the orientation of the apical and basolateral membranes of epithelial cells helps to facilitate secretion. Similarly, the orientations of axons and dendrites in neurons are essential for neurotransmission and communication (Figure 1).
In polarized cells, a polarized transport is responsible for delivering proteins to their destination in an orderly process. First, proteins are localized and concentrated into transport vesicles in the Golgi apparatus. Next, the vesicles are transported to their destination. Finally, the vesicles anchor and fuse with the cell membrane for release of the proteins.
In order to investigate the mechanism of polarized transport, we generate knockout mice lacking particular genes involved in protein trafficking (e.g. SNARE, Rab, etc.). Polarized transport can be studied in knockout mice and in primary cultured epithelial and neuronal cells. Analytical techniques utilized in the laboratory include the introduction of foreign genes, visualization of intracellular transport by live superresolution microscopy and examination of cell morphology by electron microscopy. Moreover, known and novel molecules important for polarized transport can be identified by immunoprecipitation and yeast two-hybrid analyses.
In previous work, we demonstrated that mice lacking Rab8 died 3 to 4 weeks after birth owing to malnutrition. Rab8 is a low molecular weight GTP-binding protein that is highly expressed in adsorptive epithelial cells of the small intestine and is thought to be critical for protein transport to the basolateral domain. Rab8 knockout mice showed an accumulation of enzymes and transporters underneath the apical plasma membrane of adsorptive epithelial cells, and nutrient absorption was greatly reduced.We also discovered that Rab8 is also drastically decreased in human patients with microvillous atrophy — a disease in which nutrients cannot be absorbed in the small intestine.
We are currently focused on how Rab8 regulates vesicular transport to the apical domain. From a yeast two-hybrid study, a protein called EHBP1L1 was found to bind to Rab8 and is also known to interact with another protein called Bin1, which is involved in vesicle budding. The current hypothesis is that Rab8, EHBP1L1 and Bin1 work together to transport proteins to the apical plasma membrane, therefore EHBP1L1 interacting molecules are being further investigated. Furthermore, knockout mice lacking candidate genes thought to be involved in the transport of vesicles to the apical plasma membrane are also being generated for analysis.