| Septins proteins function in cortical organization and cell division. Septins have two defining characteristics: the ability to bind and hydrolyze guanine nucleotide, and the ability to form filaments. These characteristics are believed to be the key to septin function, yet their in vivo relevance is unknown. The work presented in this thesis is towards an understanding of how these defining characteristics contribute to the functions of the septin proteins.;To understand the relevance of septin bound guanine nucleotide, in vitro (32P based) and in vivo (mass spectrometry of pulse-chase isotope labeled samples) nucleotide exchange assays were designed for use with the yeast Saccharomyces cerevisiae . It was found that the majority of the septin bound nucleotides do not undergo significant turnover either in vitro or in vivo. This suggests that the guanine nucleotide is unexchangeable and probably plays a structural role in the assembly of the septin complex. This is in contrast to the standard nucleotide-binding cytoskeletal systems, such as actin and microtubules, which use GTP in an energetic role.;Polarized fluorescence microscopy of septin-GFP fusion proteins was used to study the organization of septin filaments in living yeast. In yeast these filaments localize at the isthmus between the mother and the daughter cells. They transition from an hourglass-shaped assembly to two separate rings at the onset of cytokinesis. The two septin structures (hourglass and rings) were found to be highly ordered. Specifically, the hourglass is made of septin filaments aligned along the yeast bud-neck, most likely arranged in an antiparallel twisted array with sub-resolution C2 symmetry. During the hourglass to rings transition the filaments rotate 90-degrees in the membrane plane and assemble into two rings with opposite handedness around the mother-daughter axis. This complex re-organization may play a fundamental role in cell division. |
