Proteins at the cell cortex link the cell interior and exterior by transmitting and responding to a variety of signals. Thus, the cell cortex is an information processing center where cells can receive and react to diverse stimuli in their environment or within the cell. A conserved family of proteins called septins organizes and regulates structures and processes at the cortex of eukaryotic cells. In addition to performing essential functions specific to certain cell types, septins are also important for normal cell division; they can act as "molecular fences" in membranes to keep proteins in specific locations and they are thought to act as protein "scaffolds" to assemble specific groups of proteins at the cell cortex. In most cells, septin proteins self-assemble into filaments that further assemble into complex higher-order structures such as rings and fibers. Despite the widespread distribution and varied functions of septins, little is known about the molecular mechanisms that direct septin assembly into filaments and the higher order organization of these filaments. A major goal of this project is to determine how septins assemble into an array of morphologically different forms within a single cell. The PI and her colleagues will investigate how septin structures are assembled, reorganized and regulated in the multinucleated, filamentous fungus, Ashbya gossypii. In this filamentous fungus, septins assemble into many morphologically distinct and dynamic structures, making it an excellent model for understanding the molecular controls of complex septin organization. The simple lifestyle and small genome of the fungus facilitates generation and analysis of mutants in regulatory proteins. Additionally, septin proteins can be visualized in living cells by linking the septin proteins to fluorescent proteins that are visible under the microscope. The specific goals of this project are to: 1. Identify the composition and dynamics of different septin complexes that coexist in one A. gossypii cell. 2. Establish the regulatory pathways that control the assembly of different septin rings. 3. Determine if the nuclear division cycle directs changes in the septin cortex in a multinucleated cell. These goals will be achieved using a combination of time-lapse fluorescence microscopy, electron microscopy and biochemistry for mutant analysis. This work is significant because the septins are a nearly ubiquitous, highly conserved protein family, yet major gaps exist in our knowledge of how they assemble and function in complexes in living cells. Furthermore, little is known about how internal or external signals lead to the assembly of elaborate types of septin structures. Knowledge gained from this work will provide a mechanistic foundation for understanding septin organization in other eukaryotic cells. The project will have substantial broader impact on education and training. The PI will mentor graduate and undergraduate students from multiple programs in research projects; undergraduate students will perform experiments, attend weekly lab meetings and present their data at Dartmouth's undergraduate research symposia.
Cells can control and change their shape using proteins that form filaments called the cytoskeleton. This supported work focused on a part of the cytoskeleton called septin proteins. The septins are found throughout the living world from fungi to humans, where they are highly expressed in the brain. They are important for serving as "scaffolds" to support cell membranes and bring together many different kinds of proteins to a single location in the cell such as the site where cells divide. Very little was known about how cells determine where to build septin scaffolds, how they control the size of the scaffold and how the filaments are organized to make a functional structure. This is critical information to know how cells use these highly conserved proteins. These can be thought of as cell architectural engineering questions and in essence we aim to understand what rules determine where and how a cytoskeletal structures forms in cells. In this project, we developed new microscopy approaches that take advantage of polarized light to study how septin filaments are arranged in the cell and found that all cells from yeast to mammals create highly ordered bundles of septin filaments. Using genetics and biochemistry, we discovered some key ways that cells control the size of the scaffolds using just one part of a septin protein that is highly flexible. We also found that one novel function of the septin scaffold is to help cells respond to environmental stresses. A network of control proteins was identified that determines where septin filaments form at particular times in the cell. With these findings, we now understand the basic organization principals for the septin cytoskeleton that we are using in our next set of studies. We developed new publically available software to analyze microscopy images that is useful to anyone implementing polarized light microscopy. We communicated our findings in seven journal articles and through posters and presentations at national and international conferences. Additionally, we spoke to the general public about the work at local schools and hosted high school teachers at Dartmouth to learn about innovations in light microscopy.
