Open University (UK)

PhD degree at Stowers Institute for Medical Research

Arupratan Das

Stowers Institute for Medical Research
An Affiliated Research Centre of the Open University

 Cdc42 recycling mechanism in dynamic maintenance of cortical polarity

Arupratan Das joined the PhD programme of Open University in October 2007 and started his PhD research in Rong Li’s lab at the Stowers Institute for Medical Research. Before coming to USA Arupratan completed his Bachelor’s degree in Chemistry (2004) and Master’s in Biochemistry (2006). After successful completion of his master’s he joined a research institute, Satyendra Nath Bose National Centre for Basic sciences, India. There he developed his theoretical and experimental knowledge in advanced spectroscopy and his research described in the journal article “Spectroscopic studies of catanionic reverse microemulsion: correlation with the superactivity of horseradish peroxidase enzyme in a restricted environment” which was published in February 2008 issue of Journal of Physical Chemistry B. Inspired with enormous opportunity of the interface of spectroscopy and Bilogical science he proceeded to use his knowledge of spectroscopy into the Biological system and joined Open University PhD programme in October 2007.

Biological processes such as development, cell motility, and immune response depend on the successful completion of cell polarization steps. Budding yeast Saccharomyces cerevisiae polarizes at the G1/S transition1. Cdc42, a conserved Rho GTPase, is one of the principle players responsible for cell polarization. Genetic studies have revealed many molecular players responsible for cell polarization. Though it is believed that Cdc42 is recycled by two complementary mechanisms, actin transport and cytosolic recycling through the Rho GDP dissociation inhibitor Rdi1, the molecular interactions controlling this recycling are not completely understood. Fluorescence correlation spectroscopy (FCS) and cross correlation spectroscopy (FCCS) are powerful techniques to obtain parameters of molecular dynamics, concentration and interactions of mobile proteins in live cells.  Here, we implement these techniques in living yeast cells to study recycling of Cdc42 at the polarized site. Experiments have shown that Rdi1 forms a stable complex with Cdc42-GDP and hypothesized that Rdi1 helps to extract Cdc42-GDP from the polar site. In support of this hypothesis, we observe interactions between wild type Cdc42 and Rdi1 in live yeast, but not with the Cdc42 mutant (R66E), that does not bind Rdi1 in vitro. Interestingly, mutants locked in GDP or GTP forms do not interact strongly with Rdi1 in the cytosol, suggesting that the GTPase cycle is necessary for Rdi1 to bind Cdc42.  FCS results suggest that mobile Cdc42 is present in two dynamic populations in the cytosol, that vary in percentage with mutation of Cdc42 or deletion of Rdi1. Fluorescence recovery after photobleaching (FRAP) of Cdc42 at the polar tip with different Cdc42 mutants , Rdi1 deletion, or treatment with a drug to inhibit actin transport revealed that multiple recycling mechanisms exist to control polarization of Cdc42.  We are now trying to decipher the molecular interactions that are responsible for the different aspects of Cdc42 recycling.

About the Stowers Institute
Housed in a 600,000 square-foot state-of-the-art facility on a 10-acre campus in the heart of Kansas City, Missouri, the Stowers Institute for Medical Research conducts basic research on fundamental processes of cellular life. Through its commitment to collaborative research and the use of cutting-edge technology, the Institute seeks more effective means of preventing and curing disease. The Institute was founded by Jim and Virginia Stowers, two cancer survivors who have created combined endowments of $2 billion in support of basic research of the highest quality.