Microtubule assembly is a long studied process because of its importance in mitotic division, and thus in cancer. Microtubules are complex polymers of tubulin, which itself is a heterodimer of &alpha and Β tubulin. The maintenance of equilibrium between tubulin monomers and polymers is essential for the proper functioning of the cell. Nucleation or the formation of oligomers of tubulin is the rate determining stage of microtubule assembly. Immense importance lies on the understanding of the nucleation stage and how it mediates the assembly process. Many antimitotic drugs influence the nucleation stage of microtubule assembly. Over the last few years, gamma tubulin (a 50kDa protein) has been identified as the nucleating agent in the cell, although the mechanism is not well known. The mechanism of action of gamma tubulin to nucleate microtubules and the controlling factors in the cell are very much important to know the process of mitosis. Study of the nucleation stage by the gamma tubulin complex as well as other nucleating agents would help us to understand the mechanism of microtubule formation through nucleation in normal cells as well as in abnormal situations like cancer, which in turn would throw light on how these agents control microtubule assembly. This would help in the improvement of their use as cancer chemotherapeutic agents

During the last decade, fluorescent proteins like Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) isolated from marine Anthozoa like species have become increasingly popular for their use as intrinsic fluorescent probes in following different cellular processes. In exception to normal proteins, which show fluorescence in the UV region, these proteins show fluorescence in the visible region. These properties arise due to the ability of the proteins to exhibit some specific conformations, which in turn come from the spatial arrangement of some amino acids. My work involves study of a protein with unique fluorescent property and also its structural aspects which give rise to this property. This provides an excellent system that different conformational constraints and requirements can be related to the spectral properties of a protein and can enrich our knowledge in the structural studies of various proteins in future. The study also intends to use this protein fused with gamma tubulin as a probe in the study of the nucleation stage of microtubule assembly in the cell.

DAT1 [4-amino-5-benzoyl-2(4-methoxyphenylamino) thiazole] was selected in our laboratory as a cytotoxic agent in a preliminary screen based on the cytotoxic activities of a series of synthetic diaminoaroylthiazole analogues of dendrodoine. It binds to the colchicine binding site of tubulin, causing mitotic arrest and disrupting the spindle morphology. It has an average cytotoxicity of 300 nM towards a number of cell lines. The apoptosis inducing activity of DAT1 and the apoptosis pathways activated have been studied. Both the intrinsic and extrinsic pathways are activated following DAT1 treatment. We will further study the effect of DAT1 on the proteins involved in the apoptotic pathway. In addition, DAT1 was found to have cytotoxicity in p53 deficient cancer cells. We would look further into this aspect and extend these studies in animal models.

Microtubule nucleation in eukaryotic cells occurs from the centrosome. The newer member of the tubulin superfamily, gamma-tubulin, is known to mediate microtubule nucleation from the centrosome with aid of some other proteins. The major amount of gamma tubulin complex is believed to be located in the centrosome before the onset of mitotic division. However, a considerable amount is found in the cytoplasm in the form of a complex, the function of which is not known. Gamma tubulin ring complex was purified from brain in our laboratory and it was found to nucleate microtubules in vitro. I am now working on the function of the component proteins in the gamma-tubulin ring complex.

Chemotherapy is being widely used in the treatment of cancer patients, yet it is limited by the development of resistance in cancer cells to multiple chemotherapeutic agents. This is due to various changes in cancer cells such as altered membrane transport involving P-glycoprotein over expression, enhanced expression of heat shock proteins, altered target, decreased drug activation, increased drug degradation and failure to apoptosis. Antimitotic drugs occupy an important place in cancer chemotherapy as some of them are quite successful in the treatment of different cancers and many are in different stages of clinical trials. However, they also face the problem of being resistant after continued use.
DAT1, a diaminothiazole, has been shown in our laboratory to have cytotoxicity towards a number of cancer cell lines. It brings about mitotic arrest and is an inhibitor of microtubule assembly. The action of DAT1 on drug resistant cancer cell-lines will be explored in comparison to other antimitotic drugs.

DAT1 binds to tubulin to a site, which is the same or overlapping to that of the colchicine-binding site. Studies have been undertaken to get a detailed insight of the binding of DAT1 to tubulin. Our studies indicate that the binding of the DAT1 to tubulin induces a conformational change. Further mechanistic studies of this interaction are being carried out to help in the development of structural analogues of DAT1