Areas of Research
Our research program uses the single celled eukaryote, Saccharomyces cerevisiae, to study a fourth class of cytoskeletal proteins, termed the "septins." The septins are highly conserved across all metazoans and fungi, and display remarkable geometric properties in vivo. In budding yeast, there are five mitotically expressed septin subunits, Cdc11, Cdc12, Cdc3, Cdc10, and Shs1. Two additional subunits, Spr28, and Spr3, are exclusively expressed during sporulation and are required for meiosis. Past genetic and biochemical analyses lead to the identification of the organization of the septin subunits within the core protein complex. The subunits are arranged in a hetero-octamer with the arrangement Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1. This core structure is polymerized into paired filaments within the cell that assemble to form higher order structures (such as the hour-glass shaped collar encircling the bud neck). Our lab is interested in understanding the molecular determinants that govern these different cellular geometries and how they contribute to septin function(s).
[Crystal structure of three human septins, SEPT2, SEPT6, and SEPT7 in complex; PDB 2QAG, Sirajuddin et al., 2007, Nature 449, 311-315].
Septin octamer assembly and regulation
The septin subunits are assembled into a core octameric structure that is further polymerized into long filaments and superstructure within the yeast cell bud neck/division site. Our lab is interested in studying the molecular mechanisms regulating how septin subunits are assembled into octamers and larger structures. The core subunits are related to GTP-binding enzymes and, within the octamer, the septin subunits coordinate to bind GTP between one protein-protein interface (termed the G-interface). On the opposite interface, contacts are made between each subunit's alpha helices (termed the NC-interface). Aside from coordinate binding of GTP, the septin proteins are highly modified throughout the cell cycle by other enzymes. Our lab is interested in studying how modulation of the septin subunits can impose regulation on septin structure and function in vivo.
Information exchange at the bud neck
The core septin octamers in yeast assemble into long filaments on the inner leaflet of the plasma membrane and may also aid in promoting and/or regulating curvature of the membrane itself. Due to their positioning at the membrane, the septins can serve as a barrier/corral to regulate the passage of cellular components (such as protein complexes or entire organelles) between the divided membrane compartments (such as the mother and daughter cell). Finally, a major role of the yeast septins is to serve as a platform positioned at the division site where many other enzymes are recruited to carry out their function(s). For example, the checkpoint kinase, Hsl1, directly binds to the septin collar and initiates a signaling cascade that recruits many other components to the bud neck to allow for progression of the cell cycle from G2 to M phase. Our lab is very interested in characterizing the non-septin proteins that are found at the bud neck during various stages of the cell cycle. We will employ a number of new techniques to identify and characterize protein-protein interactions and cellular localization of the septins and their recruited components in live cells. Our long term goals include utilizing a combination of high-throughput genetic and proteomic approaches to address these questions at a detailed mechanistic level.
[Schematic of the G2/M checkpoint in budding yeast].
Evolution of biological complexity
Our lab is also interested in studying how macromolecular machines can evolve through deep time. A common trend across the tree of life is the occurrence of protein complexes with many similar components that arose through gene duplication events where both subunit(s) were maintained. We will use the septin protein family as a case study for experimentally examining the evolution of this protein complex across the metazoan and fungal kingdoms. Remarkably, both the yeast and human core septin complexes are linear hetero-octamers even though yeast have seven total subunits and humans have thirteen subunits (with different splice variants and isoforms). We are interested in studying the molecular mechanisms that allow for this protein complex to adopt and incorporate newly duplicated subunits as a way to impart novel cellular function(s) and/or as a means to impose additional regulation and control over septin superstructure in vivo.
CRISPR/Cas gene editing technology
A new gene editing technology, termed "CRISPR" (Clustered Regularly Interspaced Short Palindromic Repeats) has taken the field of molecular biology by storm and, in only a few short years, has been widely accepted by both academic and biotechnology labs across the globe. This system evolved as a simple immune system in bacteria and archaea to defend against viral infection. However, the Class II CRISPR system from S. pyogenes was recognized and repurposed to become a powerful tool for genome editing in all types of living systems. Briefly, the Cas9 endonuclease binds a short stretch of RNA containing both targeting (crRNA) and structural (tracrRNA) sequence, and is able to target the protein to a specific position within the genome of choice with incredible accuracy. The sgRNA (single guide) sequence consisting of 20 nucleotides is programmable to match any position within the genome to introduce a double strand break (DSB). Healing and repair of this DSB can be achieved by several mechanisms, including by homologous recombination from added exogenous sequence to be integrated into the chromosome. This system provides researchers the ability to manipulate genomes with greater speed, lower cost, and higher accuracy than ever before. My lab is interested in utilizing the CRISPR/Cas technology to understand (i) how to further optimize the utility and application of this system across living organisms, (ii) study novel applications relevant to biotechnology and the study of human disease, and (iii) study methods to impose additional regulation and control over the system in living cells.
[Model of the CRISPR gene editing technology: the Cas9 nuclease bound to its sgRNA at a genomic locus. Reproduced and adapted from DiCarlo et al. 2013, Nucleic Acids Research 41, 4336-4343. By permission of Oxford University Press. For commercial reuse please contact firstname.lastname@example.org].