Fission yeast cells are cylindrical and grow from exactly opposite "antipodal" ends. This is achieved through delivery by cytoplasmic microtubules (MTs) of proteins that decorate cell tips, which include the proteins Tea1p, Tea2p (a MT-associated motor), and Tip1p (a MT-associated protein (MAP)). These proteins and MTs collaborate in maintenance of cell-end identity. Nevertheless, the proteins rely on each other and on MTs for their cell-end localization in vivo, and seem to affect MTs in cells. This has rendered difficult to understand in vivo how initiation of cell -end identification occurs. My aim is to develop in vitro assays to examine yeast MT assembly in order to characterize the specific interaction of each of those proteins with MTs outside the cellular context. I will use recombinantly expressed yeast proteins and assay quantitatively their effect on microtubule assembly in pure tubulin and in yeast lysates competent for MT assembly , which I will develop. Expected results of the project include:
a) Determination of the affinity of Tea1p, Tea2p and Tip1p for MTs;
b ) Determination of their specific effect on MTs (stabilization, transport);
c) Determination of their effect(s) on MT stability in yeast cytoplasmic lysates; d) Identification, among those proteins, of the initiator(s) of cell-end identification in viva. Our development of yeast in vitra assays will introduce a very powerful tool to study quantitatively morphogenesis in this organism. Training content: The project will involve training in biochemistry and genetics of fission yeast, and development of in vitro assays for quantitative analysis of yeast MT assembly by microscopy . Expected impact for applicant: I will learn a new system, the fission yeast, and acquire knowledge in biochemistry and genetics. This will nicely complement my previous training in physics and biology. My long term aim is to understand cellular events quantitatively as the outcome of the collective properties of their components. The development of quantitative assays to study yeast morphogenesis in vitro will help me reach that aim. Expected impact for host: A need for cell-free assays to study yeast MT assembly and regulation outside of the cellular context is manifesting in the field. Therefore, the prospective research will be innovative and very important to the field. In particular, my host laboratory will greatly benefit from the development of such assays.